Type VII secretion — mycobacteria show the way

PROTEIN SECRETION AND VIRULENCE IN

PATHOGENIC MYCOBACTERIA

Abdallah M. Abdallah

Title: Protein Secretion and Virulence in Pathogenic Mycobacteria

Abdallah Musa Abdallah

Thesis Vrije Universiteit Amsterdam, with summary in Dutch Cover:

Front: Mycobacterium marinum supplemented with DsRed

Back: The Arab-Islamic Origins of Modern Science: Copy of Avicenna’s five-volume Canon of Medicine, which is the most famous of all medical books in history. It was the final authority in medical matters in Europe for nearly six centuries. Avicenna {Ibn Sina} was a universal genius, who was called "the prince of physicians" in the West and considered the father of modern medicine, and regarded as one of the greatest thinkers and medical scholars in history. An Islamic Astrolabe; the Astrolabe {Astrology and Astronomy} was highly developed in the Islamic world and was introduced to Europe from Islamic Spain {Andalusia} in the early 12th century.

Photograph: W. Bitter, Vrije Universiteit Medical Centre, Amsterdam

W. Müller, Utrecht Uiversity, Utrecht, The Netherlands

Cover design: ACSMedia

Printed by: PrintPartner Ipskamp, Enschede, The Netherlands ISBN: 978-90-9022779-5 Publication of this thesis was financially supported by KNCV Tuberculosis Foundation, Wyeth Pharmaceuticals B.V., Pfizer B.V., Bayer HealthCare, and Promega Benelux B.V. Copyright © 2008 by A.M. Abdallah All rights reserved. No part of the material protected by this copyright notice may be reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording or by any information storage and retrival system, without written permission from the author, or from publishers of the publications.

VRIJE UNIVERSITEIT

PROTEIN SECRETION AND VIRULENCE IN

PATHOGENIC MYCOBACTERIA

ACADEMISCH PROEFSCHRIFT

ter verkrijging van de graad Doctor aan

de Vrije Universiteit Amsterdam, op gezag van de rector magnificus

prof.dr. L.M. Bouter, in het openbaar te verdedigen

ten overstaan van de promotiecommissie van de faculteit der Geneeskunde

op dinsdag 11 maart 2008 om 10.45 uur in de aula van de universiteit,

De Boelelaan 1105

door

Abdallah Musa Abdallah

geboren te Elobeid, Sudan

promotor prof.dr. C.M.J.E. Vandenbroucke-Grauls

copromotor dr. W. Bitter

“Science knows no country, because knowledge belongs to humanity, and it is the

torch, which illuminates the world”

Louis Pasteur (1822-1895)

To my family and friends

Doctoral Committee

Other members:

Prof. dr. Stewart Cole Global Health Institute, Lausanne, Switzerland

Prof. dr. T.H.M. Ottenhoff Leiden University Medical Center, Leiden, the Netherlands Prof. dr. P. Peters Netherlands Cancer Institute, Amsterdam, the Netherlands Prof. dr. Y. van Kooyk VU University Medical Centre, Amsterdam, the Netherlands Dr. J. Luirink Vrije Universiteit, Amsterdam, the Netherlands The studeis described in this thesis were performed at the Department of Medical Microbiology and Infection Control of the VU University Medical Centre, van der Boechorstraat 7, 1081 BT Amsterdam, the Netherlands.

CONTENTS

Chapter 1 Introduction 9

Chapter 2 Type VII--mycobacteria show the way 19 (Nat. Rev. Microbiol. 5:883-891, 2007)

Chapter 3 Mycobacterium marinum strains can be divided into two 47 distinct types based on genetic diversity and virulence (Infect. Immun. 72:6306-6312,2004)

Chapter 4 A specific secretion system mediates PPE41 transport in 63 pathogenic mycobacteria (Mol. Microbiol. 62:667-679, 2006 )

Chapter 5 Mycobacterial PPE and PE_PGRS proteins are transported 87 via a type VII secretion system (Submitted for puplication)

Chapter 6 The ESX-5 secretion system of Mycobacterium Marinum 109 modulates the macrophage response (Manuscript in preparation)

Chapter 7 General Discussion 129

Chapter 8 Summary and Samenvatting 139

Acknowledgements 145

Curriculum Vitae 148

List of publications 149

Ch

apter 1

Introduction and

Outline of the thesis

Chapter 1: Introduction and outline of the thesis 10

Tuberculosis

Tuberculosis (TB) is a devastating disease that has already been present in the human population since prehistoric times. Tuberculosis is primarily caused by the high GC-gram positive bacterium Mycobacterium tuberculosis, but it can also be caused by several closely related mycobacterial species belonging to the so-called M. tuberculosis complex. The earliest detection of a member of the M. tuberculosis complex is in the remains of a North American bison dated 17,000 years before the present (BP)1. Detection of M. tuberculosis complex DNA in humans dates back to the iron age, approximately 2,000-2,200 years BP2,3, but skeletal malformations indicate that tuberculosis has been a human disease before this time. For instance, tubercular decay has been found in the fragments of the spinal column of Egyptian mummies from 3,000-2,400 BP4. Interestingly, ancient cases of TB were also present in South America for almost 2,000 years5, which means long before the arrival of Columbus or the Vikings. These results indicate that tuberculosis was probably already introduced in the Americas during the first waves of human migration. The first description of tuberculosis is by Hippocrates in ancient Greece, who used the term “phthisis” for the disease. He describes phthisis as a widespread disease and that patients had fever and were coughing up blood and noted that it was almost always fatal. Today, tuberculosis is still one of the most devastating infectious diseases. Globally, about one-third of the world population is believed to be infected with the tuberculosis bacillus and new carriers occur at a rate of one per second6. Only a small minority of people infected with tuberculosis bacillus (5-10%) develops active disease, whereas the vast majority of infected healthy individuals neither develop disease nor become infectious. However, they are usually unable to kill all bacilli, leading to a latent tuberculosis infection7. 5-10% of people with latent tuberculosis develop full-blown disease years or decades after the initial infection8. In total more than 8 million people become sick with tuberculosis every year, and nearly two million die of the disease. This is the highest rate claimed by a single infectious bacterial agent6. Recently, Stop Tuberculosis Partnership initiated an ambitious and intensive eradication programme, the Global Plan to Stop Tuberculosis with the long-term target of eliminating tuberculosis as a global public health problem9. With a combination of Bacille Calmette–Guerin (BCG) vaccination and extensive treatment of tuberculosis cases with anti-mycobacterial drugs, one hopes to be able to deal with the tuberculosis problem once and for all. However, since a few decades developing countries are dealing with a strong resurgence of tuberculosis, such that more people are now suffering from tuberculosis than ever before. Different factors have contributed to this resurgence and precarious global situation, including (i) the variable efficacy of the BCG vaccine, (ii) the emergence of multi-drug resistant M. tuberculosis strains, (iii) the co-infection with another deadly pathogen, HIV; and (iv) factors that affect the public-health system, such as continuing political instability and war, poverty, natural disasters and more frequent transmission due to an increased human


population. Apart from the human suffering, tuberculosis is responsible for significant economic burden, which results in losses somewhere near $12 billion from the global economic growth annually10.

Mycobacterium marinum

Development of novel interventions for tuberculosis and other mycobacterial diseases requires detailed knowledge of the disease and of the pathogen itself. Therefore, understanding the mechanisms of latent tuberculosis infection and the mode of resistance of the causative bacterium to both innate and adaptive immune responses are important. However, our knowledge of the mycobacteria is lagging behind compared to other bacterial pathogens. This lack of knowledge is mainly due to the fact that the study of M. tuberculosis is inherently difficult: (i) experiments are lengthy and expensive because of the extremely slow growth rate and high pathogenicity (ii) there is a lack of molecular tools; and (iii) M. tuberculosis is not closely related to well-known bacteria, such as Escherichia coli and Bacillus subtilis. To explain this last point in more detail, M. tuberculosis belongs to the mycolata, which are characterised by a unique and complex second hydrophobic layer. In practice this means that mycobacterial virulence factors cannot be inferred from work on other pathogens and that molecular tools have to be specifically developed for M. tuberculosis. Despite these difficulties important progress has been made in tuberculosis research in the last decennium, especially in the development of genetic tools that have facilitated the identification and characterization of virulence determinants. However, M. tuberculosis is still mainly a human-specific pathogen. Despite the fact that some laboratory animals can be experimentally used as infection models, these animal models do not mimic certain important aspects of human infection and disease. Therefore, alternative models for tuberculosis have been developed in the past years. One of these models is the use of the relatively rapidly growing animal pathogen M. marinum, the causative agent of fish tuberculosis. The genus Mycobacterium comprises more than 80 species, which can be divided into slow and fast growing, depending on the time needed to form visible colonies (i.e. within 7 days for the fast growers). This seemingly arbitrary separation nicely corresponds with molecular data on mycobacterial phylogeny, which shows that the fast growers are the more ancient mycobacterial species, whereas the slow-growers belong to a more recently evolved clade11. Furthermore, most pathogens are present in the group of slow-growing mycobacteria. Apart from M. tuberculosis, the best known mycobacterial pathogens are Mycobacterium leprae, which causes leprosy and Mycobacterium ulcerans, the causative agent of Buruli ulcer. In addition, there are several species causing mainly diseases in animals, such as M. bovis and Mycobacterium avium, which cause tuberculosis in cattle and birds respectively, and of course M. marinum.


M. marinum is one of the closest relatives of the M. tuberculosis complex organisms12, and offers the advantage that a number of its natural hosts, such as fish and frogs, are also convenient laboratory animals13. M. marinum is only mildly pathogenic for humans, it causes superficial lesions called fish tank-granuloma or swimming pool-granulomas, which are clinically and pathologically indistinguishable from dermal M. tuberculosis lesions14,15. M. marinum has a preference for reduced temperatures, its optimal temperature for growth is 30–33°C, which most likely explains the limitation of the infections to the cooler surface areas of the human body, such as the extremities. In this respect it resembles somewhat M. leprae. In humans M. marinum rarely disseminates to systemic organs even in severely immunocompromised patients, making it a less formidable pathogen than M. tuberculosis. In addition, there are no definitive reports of person-to-person transmission of M. marinum by aerosol infection or otherwise, although M. marinum infections have been described without direct contact of contaminated water16. In cold-blooded vertebrate species, such as fish (both salt- and fresh-water), reptiles and amphibians, M. marinum causes a chronic wasting disease that affects all parts of the body.

Mycobacterial cell wall and protein secretion

The cell envelope of Mycobacterium species is rather complex and differs substantially from the cell wall structures of both gram-negative and gram-positive bacteria. The most striking feature is the extraordinarily high lipid content of the mycobacterial cell envelope, which constitutes up to 40% of their dry weight. In addition to conferring unique tinctorial properties, which are exploited for diagnostic purposes, the cellular envelope of Mycobacterium is essential for the interacting of the host with Mycobacterium species17,18. The cell envelope of Mycobacterium consists of three layers: the cytoplasmic membrane, the outer layer or mycomembrane and the capsule. The outer layer or mycomembrane is the most remarkable of these structures and consists mainly of one large complex, consisting of three different covalently linked structures: peptidoglycan, arabinogalactan and mycolic acids. Mycolic acids are extremely large hydroxylated branched-chained fatty acids (C30-C90) and their covalent linkage results in a hydrophobic layer of extremely low fluidity. The mycolic acids are most likely orientated perpendicular to the cytoplasmic membrane17,19. In addition, free lipids, most of which are specific for mycobacteria, are intercalated with the mycolic acids. These lipids include phenolic glycolipids, phthiocerol dimycocerosates, cord factor/dimycolyltrehalose, sulfolipids and phosphatidylinositol mannosides. Together, these lipids form a pseudo-bilayer, which is analogous to the outer membrane of gram-negative bacteria20. The capsule mainly contains polysaccharides (glucan and arabinomannan). This cell wall forms an extremely hydrophobic layer that confers to mycobacteria the properties of resistance to solutes including many antibiotic and therapeutic agents. In addition, some of


these lipids also display strong biological activities and therefore may be considered as essential virulence factors21,22. The specialised cell wall of mycobacteria protects them against different extracellular harmful agents, but also imposes a new problem: how to secrete proteins? Secretory protein pathways play and perform a variety of important "remote-control" functions for bacterial pathogens, and mycobacteria are most likely no exception. The outcome of mycobacterial infection is dependent on both effector molecules secreted by the bacteria and host factors. Therefore, the secreted mycobacterial proteins and, accordingly, the respective systems responsible for their export, are critical for virulence and not to be neglected. Microbial proteins presented on the bacterial surface or released extracellularly often depend on specialized secretion systems. The diversity of these molecular secretion systems correlates with the complexity of the bacterial cell wall. For instance, a multitude of protein secretion systems has been identified in gram-negative bacteria (brief description is provided in Chapter 2), which function to deliver bacterial effector proteins into the surrounding or directly into the host cell. These effector molecules often mediate crucial interactions during infection, in order to manipulate the host immune response and to allow pathogen survival in the hostile environment of the host23-25. In contrast, the number of and the knowledge about protein secretion systems in mycobacteria is limited and homologues of these systems are absent in Mycobacterium species. However, mycobacteria do release proteins into the extracellular environment and they are renowned for manipulation of the host immune system, which will be (partially) accomplished by the secretion of effector proteins. For instance, the inability of the live vaccine strain BCG to cause diseases is (partially) due to the absence of the secreted proteins ESAT-6 and CFP-1026-29. Bioinformatic analysis and secretome studies of the extracellular proteome of M. tuberculosis cultures showed that this bacterium secretes a large number of different proteins, albeit in relatively small amounts30,31. However, most of these proteins lack a classical N-terminal signal sequence31,32, suggesting that alternative protein secretion systems must be present in these bacteria.

PE and PPE gene families

Two interesting families of proteins that might be surface exposed or secreted are PE and PPE. The PE and PPE gene families were one of the major surprises of the M. tuberculosis genome-sequencing project, because these gene families together make up almost 10% of the genome of this pathogen33. The PE and PPE families are named after the conserved Proline and Glutamic acid (PE) and Pro–Pro–Glu (PPE) motifs near the N terminus of their gene-products33,34. However, in fact the protein family members share homologous N-terminal domains of approximately 110 amino acids for the PEs and 180 amino acids for PPE proteins, and can be divided into subfamilies on the basis of their C-terminal domains.


These two families are unique for mycobacteria, and both are considerably extended in pathogenic mycobacteria11,34. Although the exact function of these protein families is still unknown, their vast abundance in the genome of pathogenic mycobacteria is intriguing and indicates that these proteins might have an essential biological role yet to be discovered. It has for instance been speculated that the PE and PPE protein families play a crucial role in immune evasion and antigenic variation33,35-37, and as immunodominant proteins38. Members of these families also have been found to associate with the cell surface26,39-41, and some members have been linked to virulence42,43.

***

Scope of the thesis

The objective of the studies described in this thesis was to investigate protein secretion in M. marinum, with specific emphasis to the secretion system of the mysterious PE and PPE proteins. Subsequently, the role of this secretion system and its substrates in virulence would be analysed. In Chapter 2, the current knowledge is described of a novel secretion system identified in mycobacteria, type VII secretion. The striking similarities between the type VII secretion systems in mycobacteria and related secretion systems in other Gram-positive bacteria are also discussed. Finally, this chapter also contains a description of the role of these secretion systems in virulence. Initial experiments were directed towards the evaluation of a novel infection model for M. marinum using the zebrafish (Danio rerio). In Chapter 3, the genetic variation between different isolates of M. marinum and the effect of strain variation on pathogenicity in adult zebrafish are described. This study shows that M. marinum isolates can be grouped into two distinct clusters, designated cluster I and cluster II, based on genetic analysis. This study also correlates specific M. marinum genotypes to virulence in humans and increased virulence in zebrafish. In Chapter 4, the identification of a novel protein secretion system in M. marinum is described. A single gene was identified, which is required for the transport of the PPE41 substrate from the cell. This gene is located in a gene cluster whose predicted proteins encode components of a type VII secretion system, which is designated ESX-5. This secretion system is conserved in slow-growing pathogenic mycobacteria, but not in the fast-growing species. In chapter 4 and in a paper by Nico Gey van Pittius11 it has been hypothesized that the ESX-5 pathway of mycobacteria may be responsible for the transport of all recently evolved PE and PPE proteins. Genome comparison of the genus Mycobacterium revealed that recent expansion of the PE and PPE gene families,


which resulted in the emergence of the PE_PGRS and PPE-MPTR protein subfamilies, is linked to the evolution of the ESX-5 cluster. In Chapter 5, the secretome of ESX-5 pathway of M. marinum is described, which indeed shows that ESX-5 is a major secretion pathway for mycobacteria, and is responsible for the transport of probably all recently evolved PE and PPE proteins. Aspects of the virulence and pathogenesis of the M. marinum ESX-5 pathway are described in Chapter 6. Here we show that ESX-5 effector molecules subvert the normal macrophage innate immune response by inhibiting the macrophage inflammatory responses. Furthermore, the ESX-5 system seems to be involved in macrophage cell death. This study provided the first evidence that ESX-5 is a major determinant of mycobacterial virulence. Finally, the overall results of the thesis project are summarized and discussed in Chapter 7.

References

1. Rothschild,B.M. et al. Mycobacterium tuberculosis complex DNA from an extinct bison dated 17,000 years before the present. Clin. Infect. Dis. 33, 305-311 (2001).

2. Taylor,G.M., Young,D.B. & Mays,S.A. Genotypic analysis of the earliest known prehistoric case of tuberculosis in Britain. J. Clin. Microbiol. 43, 2236-2240 (2005).

3. Taylor,G.M., Murphy,E., Hopkins,R., Rutland,P. & Chistov,Y. First report of Mycobacterium bovis DNA in human remains from the Iron Age. Microbiology 153, 1243-1249 (2007).

4. Zink,A.R. et al. Characterization of Mycobacterium tuberculosis complex DNAs from Egyptian mummies by spoligotyping. J. Clin. Microbiol. 41, 359-367 (2003).

5. Konomi,N., Lebwohl,E., Mowbray,K., Tattersall,I. & Zhang,D. Detection of mycobacterial DNA in Andean mummies. J. Clin. Microbiol. 40, 4738-4740 (2002).

6. World Health Organization. Tuberculosis. Infection and transmission fact sheet 104. [WWW document]. URL http://www.who.int/mediacentre/factsheets/fs104/en/ (2007).

7. Manabe,Y.C. & Bishai,W.R. Latent Mycobacterium tuberculosis-persistence, patience, and winning by waiting. Nat. Med. 6, 1327-1329 (2000).

8. Kaufmann,S.H. How can immunology contribute to the control of tuberculosis? Nat. Rev. Immunol. 1, 20-30 (2001).

9. Stop T.B.Partnership and WHO. Stop T.B. Partnership and WHO (World Health Organization), The Global Plan to Stop TB, 2006-2015, WHO/HTM/STB/2006.35. World Health Organization, Geneva (2006).

10. Kim,J.Y., Shakow,A., Castro,A., Vande,C. & Farmer,P. Tuberculosis Control. World Health Organization. Available at: http://www.who.int/trade/distance_learning /gpgh/gpgh3/en/print.html Accessed April 17, 2007., (2007).


11. Gey van Pittius,N.C. et al. Evolution and expansion of the Mycobacterium

tuberculosis PE and PPE multigene families and their association with the duplication of the ESAT-6 (esx) gene cluster regions. BMC Evol. Biol. 6, 95 (2006).

12. Tonjum,T., Welty,D.B., Jantzen,E. & Small,P.L. Differentiation of Mycobacterium ulcerans, M. marinum, and M. haemophilum: mapping of their relationships to M. tuberculosis by fatty acid profile analysis, DNA-DNA hybridization, and 16S rRNA gene sequence analysis. J. Clin. Microbiol. 36, 918-925 (1998).

13. Clark,H.F. & Shepard,C.C. Effect of environmental temperatures on infection with Mycobacterium marinum (Balnei) of mice and a number of poikilothermic species. J. Bacteriol. 86, 1057-1069 (1963).

14. Travis,W.D., Travis,L.B., Roberts,G.D., Su,D.W. & Weiland,L.W. The histopathologic spectrum in Mycobacterium marinum infection. Arch. Pathol. Lab Med. 109, 1109-1113 (1985).

15. MacGregor,R.R. Cutaneous tuberculosis. Clin. Dermatol. 13, 245-255 (1995). 16. doedens,R.A., van der Sar,A.M., Bitter,W. & Scholvinck,E.H. Transmission of

Mycobacterium marinum From Fish to a Very Young Child. Pediatr. Infect. Dis. J. 27, 81-83 (2008).

17. Brennan,P.J. & Nikaido,H. The envelope of mycobacteria. Annu. Rev. Biochem. 64, 29-63 (1995).

18. Daffe,M. & Draper,P. The envelope layers of mycobacteria with reference to their pathogenicity. Adv. Microb. Physiol 39, 131-203 (1998).

19. Minnikin,D.E. Chemical principles in the organization of lipid components in the mycobacterial cell envelope. Res. Microbiol. 142, 423-427 (1991).

20. Liu,J., Rosenberg,E.Y. & Nikaido,H. Fluidity of the lipid domain of cell wall from Mycobacterium chelonae. Proc. Natl. Acad. Sci. U. S. A 92, 11254-11258 (1995).

21. Cox,J.S., Chen,B., McNeil,M. & Jacobs,W.R., Jr. Complex lipid determines tissue-specific replication of Mycobacterium tuberculosis in mice. Nature 402, 79-83 (1999).

22. Camacho,L.R. et al. Analysis of the phthiocerol dimycocerosate locus of Mycobacterium tuberculosis. Evidence that this lipid is involved in the cell wall permeability barrier. J. Biol. Chem. 276, 19845-19854 (2001).

23. Finlay,B.B. & Falkow,S. Common themes in microbial pathogenicity revisited. Microbiol. Mol. Biol. Rev. 61, 136-169 (1997).

24. Thanassi,D.G. & Hultgren,S.J. Multiple pathways allow protein secretion across the bacterial outer membrane. Curr. Opin. Cell Biol. 12, 420-430 (2000).

25. Lee,V.T. & Schneewind,O. Protein secretion and the pathogenesis of bacterial infections. Genes Dev. 15, 1725-1752 (2001).

26. Pym,A.S., Brodin,P., Brosch,R., Huerre,M. & Cole,S.T. Loss of RD1 contributed to the attenuation of the live tuberculosis vaccines Mycobacterium bovis BCG and Mycobacterium microti. Molecular Microbiology 46, 709-717 (2002).

27. Lewis,K.N. et al. Deletion of RD1 from Mycobacterium tuberculosis mimics bacille Calmette-Guerin attenuation. J. Infect. Dis. 187, 117-123 (2003).

28. Pym,A.S. et al. Recombinant BCG exporting ESAT-6 confers enhanced protection against tuberculosis. Nat. Med. 9, 533-539 (2003).

29. Hsu,T. et al. The primary mechanism of attenuation of bacillus Calmette-Guerin is a loss of secreted lytic function required for invasion of lung interstitial tissue. Proc. Natl. Acad. Sci. U. S. A 100, 12420-12425 (2003).


30. Jungblut,P.R. et al. Comparative proteome analysis of Mycobacterium tuberculosis

and Mycobacterium bovis BCG strains: towards functional genomics of microbial pathogens. Mol. Microbiol. 33, 1103-1117 (1999).

31. Rosenkrands,I. et al. Mapping and identification of Mycobacterium tuberculosis proteins by two-dimensional gel electrophoresis, microsequencing and immunodetection. Electrophoresis 21, 935-948 (2000).

32. Gomez,M., Johnson,S. & Gennaro,M.L. Identification of secreted proteins of Mycobacterium tuberculosis by a bioinformatic approach. Infect. Immun. 68, 2323-2327 (2000).

33. Cole,S.T. et al. Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393, 537-544 (1998).

34. Brennan,M.J.E.C.a.G.v.P.N. The PE and PPE multigene families of Mycobacterium tuberculosis. Tuberculosis, 2nd Edition 513-525 (2004).

35. Delogu,G. & Brennan,M.J. Comparative immune response to PE and PE_PGRS antigens of Mycobacterium tuberculosis. Infection and Immunity 69, 5606-5611 (2001).

36. Banu,S. et al. Are the PE-PGRS proteins of Mycobacterium tuberculosis variable surface antigens? Mol. Microbiol. 44, 9-19 (2002).

37. Brennan,M.J. & Delogu,G. The PE multigene family: a 'molecular mantra' for mycobacteria. Trends Microbiol. 10, 246-249 (2002).

38. Choudhary,R.K. et al. PPE antigen Rv2430c of Mycobacterium tuberculosis induces a strong B-cell response. Infect. Immun. 71, 6338-6343 (2003).

39. Brennan,M.J. et al. Evidence that mycobacterial PE_PGRS proteins are cell surface constituents that influence interactions with other cells. Infection and Immunity 69, 7326-7333 (2001).

40. Sampson,S.L. et al. Expression, characterization and subcellular localization of the Mycobacterium tuberculosis PPE gene Rv1917c. Tuberculosis. (Edinb. ) 81, 305-317 (2001).

41. Delogu,G. et al. Rv1818c-encoded PE_PGRS protein of Mycobacterium tuberculosis is surface exposed and influences bacterial cell structure. Molecular Microbiology 52, 725-733 (2004).

42. Ramakrishnan,L., Federspiel,N.A. & Falkow,S. Granuloma-specific expression of Mycobacterium virulence proteins from the glycine-rich PE-PGRS family. Science 288, 1436-1439 (2000).

43. Li,Y.J., Miltner,E., Wu,M., Petrofsky,M. & Bermudez,L.E. A Mycobacterium avium PPE gene is associated with the ability of the bacterium to grow in macrophages and virulence in mice. Cellular Microbiology 7, 539-548 (2005).

Type VII secretion — mycobacteria show the way

Abdallah M. Abdallah*, Nicolaas C. Gey van Pittius‡, Patricia A. DiGiuseppe Champion§, Jeffery Cox§, Joen Luirink¶, Christina M. J. E. Vandenbroucke-Grauls*, Ben J. Appelmelk* and Wilbert Bitter*

*Department of Medical Microbiology, VU medical center, Amsterdam, The Netherlands. ‡Division of Molecular Biology and Human Genetics, Department of Biomedical Sciences, Stellenbosch University, Tygerberg, South Africa. §Department of Microbiology and Immunology, University of California, San Francisco, USA. ¶Department of Molecular Microbiology, Vrije Universiteit, Amsterdam, The Netherlands.

Ch

apter 2

Nature Review Microbiology 5: 883-891, 2007

Chapter 2: Type VII secretion — mycobacteria show the way 20

Abstract

Recent evidence shows that mycobacteria have developed a novel and specialized secretion system for the transport of extracellular proteins across their hydrophobic, and highly impermeable, cell wall. Strikingly, mycobacterial genomes encode up to five of these transport systems. Two of these systems, ESX-1 and ESX-5, are involved in virulence — they both affect the cell-to-cell migration of pathogenic mycobacteria. Here, we review this novel secretion pathway and discuss variants of this pathway that are present in various Gram-positive bacteria. Given the unique composition of this secretion system and its general importance, we propose to call it, in line with the accepted nomenclature, type VII secretion. Introduction

Bacterial pathogenicity depends on the ability to secrete virulence factors, which can be displayed on the bacterial cell surface, secreted into the extracellular milieu or injected directly into a host cell1. Historically, the mechanisms of protein secretion have been most extensively investigated in Gram-negative bacteria, which resulted in the identification of different specialized secretion systems, designated type I–VI (Box 1). Protein secretion in Gram-negative bacteria is particularly complex because these bacteria are surrounded by two membranes that secreted proteins must pass through to enter the extracellular environment or host cell. In contrast to Gram-negative bacteria, Gram-positive bacteria are regarded as simpler in structure as they lack a second membrane; secretory proteins of Gram-positive bacteria therefore only need to traverse the cytoplasmic membrane and the peptidoglycan layer to enter the extracellular environment2. However, recent studies have provided evidence that there is an alternative protein-secretion system in Gram-positive bacteria3-6. Perhaps not surprisingly, this specialized secretion system was identified in Mycobacterium tuberculosis, a Gram-positive bacterium with an extremely complex cell envelope. Phylogenetically, this bacterium belongs to the class of Actinobacteria (high G+C Gram-positive bacteria). Within this class they are placed in a distinct taxon of organisms, the mycolata, which are characterized by the presence of large hydroxylated branched-chain fatty acids called mycolic acids7,8. The mycolic acids are covalently linked to the cell wall matrix and form a second hydrophobic barrier, called the mycomembrane9 (Fig. 1). Proteins that are secreted across the mycomembrane probably need a specialized secretion system. The identification of this specialized secretion system began with the isolation of the tuberculosis vaccine strain Mycobacterium bovis Bacille Calmette-Guérin (BCG) at the Pasteur Institute (Lille, France) in 1921. This strain was isolated following prolonged serial passage of a virulent strain of M. bovis, during which attenuating genetic alterations occurred10. This weakened strain was shown to protect animals when they were challenged with a lethal dose of virulent tubercle bacilli and has since been used as a vaccine to prevent human tuberculosis, although its efficacy


for preventing tuberculosis in adults is questionable11. During continuous in vitro passage, BCG lost 38 open reading frames10,12. The deleted regions included region of difference 1 (RD1), which was shown to be crucial for the attenuated virulence of BCG13-15. RD1 (Fig. 3) is 9.5-kb in length and comprises 9 genes, including the genes that encode the secreted proteins ‘early secreted antigenic target of 6 kDa’ (ESAT-6) and ‘culture filtrate protein of 10 kDa’ (CFP-10). Both of these proteins are important T-cell antigenic targets and are essential for the virulence of M. tuberculosis (discussed below). However, both lack a distinguishable Sec-signal sequence, which suggested the existence of a specialized secretion pathway. Recently, several independent studies have demonstrated that the genes surrounding the ESAT-6- and CFP-10-encoding genes are involved in the production of such a specialized secretion system. Perhaps even more intriguing was the finding that M. tuberculosis contains four additional gene clusters that are homologous to the RD1 secretion locus16-18. In this Review, we first discuss the secretion system that is encoded by RD1, the so-called ESX-1 system, and propose that this system is an example of type VII secretion, named in line with the conventional nomenclature. Next, we describe the additional type VII secretion systems (T7SSs) in mycobacteria and related secretion systems in other Gram-positive bacteria. Last, we discuss the role of these secretion systems in virulence.

Box 1 | Type I–VI secretion systems

Secretion in Gram-negative bacteria involves transport across a multipart cell envelope that consists of two membranes (the inner membrane (IM) and the outer membrane (OM)) and the periplasm in between. Gram-negative bacteria, and pathogenic species in particular, have developed strategies to get substrates into the extracellular milieu or directly into a host cell. Generally, secretion involves either a one-step mechanism, in which the cell envelope is crossed in one go, or two-step mechanisms, in which the OM is crossed using a specific machinery (see the figure). In the one-step type I secretion pathway, proteins are secreted by a simple machinery that spans the entire cell envelope19. The translocon consists of an IM ATP-ase binding cassette (ABC) transporter, a membrane-fusion protein and an OM pore. Substrates of this pathway, such as α-hemolysin, possess an uncleaved C-terminal signal sequence.


Substrates of the two-step type II system contain a normal amino-terminal signal sequence to mediate translocation across the IM via the general Sec- or Tat-translocons20. The proteins fold in the periplasm before translocation across the OM, which is mediated by a complex structure known as the secreton. The secreton consists of a conserved OM pore (the secretin) and a pilus-like structure in the IM that might act as a piston to push substrates through the secretin. The biogenesis of the type II system is closely related to that of type IV pili. The type III system is characterized by the ‘injectisome’, a needle-like structure that forms a channel that crosses the entire cell envelope and extends to contact host cells21. The architecture of the structure allows direct injection of virulence factors from the bacterial cytoplasm into host cells. Like the type III system, the type IV secretion system transports substrates directly into host cells through complex trans-envelope structures that culminate in a pilus structure at the bacterial cell surface22. This versatile system can transport DNA and protein via one-step or two-step mechanisms. The type V system seems to use a simple two-step mechanism in which the Sec-translocon is used for translocation across the IM23. A β-barrel translocator domain that is either contiguous with the secreted protein (the autotransporter variant) or expressed as a separate entity (the two-partner secretion pathway) is needed for translocation across the OM. The translocator might act as a cognate pore, but recent evidence has questioned this model24. The recently discovered type VI system25,26 has not yet been studied in detail. The system is required for secretion of certain virulence factors in Vibrio cholerae26 and Pseudomonas aeruginos25. The substrates are synthesized without an amino-terminal Sec-type signal sequence, which suggests that the trans-envelope translocation machinery is independent of the Sec or Tat pathway. The term type VI might also describe the secretion of cell material by the budding of vesicles from the OM. Periplasmic cargo proteins may be included in this process, but the specificity of recruitment remains elusive and the typing is therefore premature. The grey channel in the IM represents signal-sequence-dependent transport of proteins through the Sec and/or Tat system.

The ESX-1 system

The existence of the ESX-1 secretion system had been predicted by several in silico analyses prior to its identification16-18. These predictions were based on the clustering of the genes that encode ESAT-6 and CFP-10 with genes that encode membrane-associated proteins and putative ATPases. The first experimental evidence for such a system was obtained when the BCG vaccine strain was complemented with the RD1 locus. Secretion of ESAT-6 was only restored when a complete RD1 region was introduced3. In a separate approach, individual genes in the RD1 locus were identified as virulence factors for M. tuberculosis4-6. The disruption of these individual genes (specifically, Rv3870, Rv3871 and Rv3877 – for consistency we will use the M. tuberculosis H37Rv gene nomenclature throughout this Review) also prevented the secretion of ESAT-6 and CFP-10. This system, which is now called ESX-1, was subsequently analysed in more detail in M.


tuberculosis, the fish pathogen Mycobacterium marinum27 and the non-pathogenic species Mycobacterium smegmatis28. ESX-1 is involved in virulence and haemolysis In M. marinum27 and in conjugation in M. smegmatis29. Together, these experiments have shown that in addition to Rv3870, Rv3871 and Rv3877 (for consistency we will use the M. tuberculosis H37Rv gene nomenclature throughout this review), the RD1 gene cluster contains many other components, possibly more than 14, that are essential for the functioning of the ESX-1 secretion system. However, the exact number of components involved in ESX-1 secretion is still debated and seems to vary between different mycobacterial species (Fig. 3). When we take a closer look at the components that seem to be unambiguously involved in ESAT-6 secretion in at least one Mycobacterium species, a number of proteins with known functional domains can be identified: Rv3868, a putative cytoplasmic chaperone with an AAA+ ATPase domain; Rv3883c (MycP1), a subtilisin-like serine protease; and Rv3870 and Rv3871, which together probably form an FtsK/SpoIIIE-like ATPase. The other proteins involved in ESAT-6 secretion (such as Rv3869, Rv3877, Rv3881c and Rv3882) have no homology with proteins with known functions, but most are predicted to be located in the cytoplasmic membrane (Fig. 3). For instance, Rv3877 is a multitransmembrane spanning protein that could be part of the translocation pore in the cytosolic membrane. Recently, a second gene cluster, the Rv3614c–Rv3616c locus, was also found to be involved in ESAT-6 secretion30,31 (Fig. 3).

How does type VII secretion work?

The proteins of the ESX-1 system are dissimilar to those of other secretion systems (Box 1) and the secretion process itself has some unusual and novel characteristics (discussed below) that would justify a special name for this system. Now that additional ESX-1-like secretion systems have been identified, both within the mycobacteria32 and in other Gram-positive species33, there is a pressing need for a more universal nomenclature. Therefore we propose to call this system type VII secretion. Secretion of ESAT-6 and other substrates by ESX-1. The ESX-1 components probably form a multisubunit cell-envelope spanning structure, similar to those of the type I–IV secretion systems (Box 1), although structural data are lacking. However, protein–protein interaction studies have provided some insight into the working of ESX-1. First, the secreted proteins ESAT-6 and CFP-10 are dependent on each other for stability and form a tight dimer4,37,38. The solution structure of the ESAT-6--CFP-10 pair revealed that each protein forms a 2-helix hairpin and that they are held together by extensive hydrophobic interactions (Fig. 2). The C-terminal tail of CFP-10, which is unstructured, does not participate in dimer formation37. Both proteins are also dependent on each other for secretion, although these data are more difficult to interpret owing to their interdependent expression and stability of the proteins.


Figure 1 | Schematic representation of the cell envelope of Mycobacterium tuberculosis. Depicted here is one of the current views of the mycobacterial cell wall. The cell wall is mainly composed of a large cell-wall core or complex that contains three different covalently linked structures (peptidoglycan (grey), arabinogalactan (blue) and mycolic acids (green)). The covalent linkage of mycolic acids results in a hydrophobic layer of extremely low fluidity. This layer is also referred to as mycomembrane. The outer part of the mycomembrane contains various free lipids, such as phenolic glycolipids, phthiocerol dimycocerosates, cord factor or dimycolyltrehalose, sulfolipids and phsophatidylinositol mannosides, that are intercalated with the mycolic acids. Most of these lipids are specific for mycobacteria. The outer layer, which is generally called capsule, mainly contains polysaccharides (glucan and arabinomannan). Signal sequence

ESA

T-6

CFP-10

C terminus

Figure 2 | Structure of the ESAT-6--CFP-10 dimer. ESAT-6 (light blue) and CFP-10 (dark blue) form a tight 1:1 complex37. The subunit interface mainly contains hydrophobic residues. The carboxy (C)-terminal tail of CFP-10 is indicated and the 7 amino acids that are involved in the secretion signal are shown in pink. The location of the tryptophan-variable-glycine (WXG) motif is indicated for both proteins in red. This figure is modified, with permission, from Ref37.

25

Figure 3 | Genes involved in the ESX-1 secretion system. The ESX-1 secretion system is encoded by two different loci, the ESX-1 locus and the EspA operon (shown in the top left corner). Homologies between these two loci are indicated by arrows. Plus or minus signs below the coding sequences show the involvement of each gene in: ESAT-6 secretion in species belonging to the Mycobacterium tuberculosis complex (Mtb)4-

6,30,31,34; haemolysis and ESAT-6 secretion in Mycobacterium marinum (Mm)27,35; ESAT-6 secretion in Mycobacterium smegmatis (Msm secr)28; and conjugation in M. smegmatis (Msm conj)29. A question mark indicates conflicting results, and a blank means that no data is available. Gene families that are also present in other ESX gene clusters are shown in different colours, whereas ESX-1 specific genes are shown in dark grey. Different deletions in the ESX-1 region that affect ESAT-6 secretion in the vaccine strain Mycobacterium bovis Bacille Calmette-Guérin (BCG)--the RD1 deletion--and in the natural mycobacterial species Mycobaterium microti, Mycobacterium ulcerans, Mycobacterium avium and the Dassie bacillus36 are also indicated. All undeleted genes of the ESX region in M. ulcerans are pseudogenes. The arrows represent the different coding sequences and the direction of their transcription.


In yeast two-hybrid assays, it was shown that Rv3870 probably interacts with Rv38714. Furthermore, Rv3870 and Rv3871 homologues in other T7SSs are fused and form a single gene (discussed below)16-18. Because the results of yeast two-hybrid experiments also indicated that Rv3871 interacts with CFP-10, it was hypothesized that Rv3871 recognizes the CFP-10/ESAT-6 substrate pair, and delivers it in an ATP-dependent manner to Rv3870 and thereby to the secretion machinery at the cell membrane4. The interaction of Rv3871 with CFP-10 in yeast two-hybrid assays was used to identify the interacting domain in CFP-1039. Single amino acid changes in the C-terminal 7 amino acids of CFP-10 were shown to be crucial for the interaction with Rv3871 and for the secretion of both ESAT-6 and CFP-1039. As mentioned above, this region of CFP-10 is unstructured and is not required for interaction with ESAT-6. Therefore, Rv3871 can readily bind this region and thereby target the ESAT-6--CFP-10 dimer for secretion. Importantly, this ESX-1 signal sequence is portable: the last 7 amino acids of CFP-10 are sufficient for an unrelated soluble protein to be secreted by ESX-139. The situation became more complex with the realization that a second locus is involved in ESX-1 secretion (the Rv3614c–Rv3616c locus)30,31. First, Rv3616c (also called EspA) was shown to be secreted into the culture filtrate in an ESX-1-dependent manner31. EspA probably forms an operon together with Rv3614c and Rv3615c and, as Rv3614c–Rv3616c are homologous to Rv3864–Rv3867 from RD1 (Fig. 3), this locus is probably a product of a gene-duplication event. Recently, it was shown that another protein encoded by the espA operon, Rv3615c, is also a secreted protein (MacGurn and Cox, unpublished observations). Furthermore, like CFP-10, the C-terminus of Rv3615c is required for secretion (Champion and Cox, unpublished observations). Interestingly, because EspA seems to lack a secretion signal, it is possible that EspA, like ESAT-6, ‘piggybacks’ on another protein for targeting. The most surprising finding from these studies was that the ESAT-6--CFP-10 dimer is produced efficiently but is retained in the bacterial cell in the absence of EspA or Rv3615c30,31. This means that all ESX-1 substrates (EspA, Rv3615c, ESAT-6 and CFP-10) are dependent on each other for secretion30,31. Understanding the as-yet-unidentified molecular basis for this phenomenon will be crucial for understanding the mechanism of substrate recognition and secretion by ESX-1. For example, it may indicate that these type VII substrates are only secreted as multimeric complexes. Alternatively, these four proteins may actually be components of the secretion machine and form some sort of pilus or extracellular structure40. This surface structure might occasionally be lost by shearing and end up in the supernatant. This line of reasoning suggests that the true substrates of ESX-1 have not yet been identified. This hypothesis is strengthened by a recent report that showed that, although ESX-1 is required to arrest phagosome maturation, the known ESX-1 substrates are not involved in this process41. A candidate for a new ESX-1 substrate is Rv3881c (also known as MTB48), which is encoded by RD1 and is a secreted antigen42.


Building a working model of type VII secretion. Although there are no sequence homologies with other secretion systems, there are some interesting functional parallels between ESX-1 secretion and type IV secretion systems. Like CFP-10, type IV substrates are targeted for secretion by an unstructured C-terminal signal sequence22,43,44. In type IV secretion, this signal sequence is recognized by coupling proteins, which are integral membrane proteins with two transmembrane domains and a large cytoplasmic domain. These coupling proteins are also members of the FtsK/SpoIIIE family, an obvious parallel with Rv3870 and Rv387139. On the basis of protein–protein interaction data (Fig. 4A), a working model for type VII secretion can be generated (Fig. 4B). First, the ESAT-6--CFP-10 dimer is targeted for secretion through the recognition of the C-terminal signal sequence by the cytoplasmic protein Rv3871. Rv3871 then interacts with Rv3870 at the cell membrane to form an active ATPase. The Rv3781/Rv3780 complex could, by analogy to ATPases involved in type II or type IV secretion, form a hexameric ring structure with a central cavity that propels ESX-1 substrates through the secretion channel. One of the major candidates to constitute (part of) the inner membrane secretion channel is the multitransmembrane protein Rv3877. Other ESX-1 components. The functions of the other ESX-1 components are more difficult to predict, and the available data on their protein–protein interactions are difficult to interpret. For example, Rv3614c, which is predicted to be a cytosolic protein, interacts in yeast two-hybrid experiments with Rv3882c, a membrane protein that is predicted to face the periplasm30. If these proteins indeed interact, either one of the predictions is wrong or one of these proteins has a dynamic topology. It was recently shown using a new protein–protein interaction assay that CFP-10 interacts with Rv068645. Because Rv0686 is a member of the signal-recognition particle (SRP) family of GTP-binding proteins, it has been suggested that ESAT-6 secretion might be linked to the Sec system45. However, these results must be treated carefully because Rv2916c, not Rv0686, is the most probable SRP protein of M. tuberculosis, and there is no other evidence that the Sec system is directly involved in the translocation of ESAT-6 or CFP-10. Clearly, many questions still remain about the working mechanism of ESX-1 remain unanswered (see Box 2). For example, what is the function of the subtilisin-like MycP1 protease in ESX-1 secretion? Although this protein is essential for the function of ESX-128, secreted substrates that have been processed by MycP1 have not yet been identified46. Another major question is: how are ESX-1 substrates exported across the mycomembrane? This layer is exceptionally thick (9–10 nm) and no components of the ESX-1 system are predicted to be located in it. However, knowledge of mycomembrane proteins is limited. In fact, structural information is only available for one transmycomembrane protein of M. smegmatis, MspA47,48, for which there are no homologues in M. tuberculosis. It is possible that one of the identified ESX-1 components forms the channel across the mycomembrane. Alternatively, as the genetic screens that have been used have not


been comprehensive and some components might be essential (and therefore cannot be mutated), the mycomembrane component (or components) might not yet have been detected. Figure 4 | Working model for the ESX-1 secretion system. Known interactions (a) and predicted localizations (b) of the ESAT-6--CFP-10 heterodimeric complex. The secretion of Rv3616c (also known as EspA) is co-dependent on the presence of the ESAT-6--CFP-10 complex. However, there is no formal evidence that these proteins form a larger complex. The ESAT-6--CFP-10 complex is recognized by the FtsK/SpoIIIE-like protein Rv3871, which binds the carboxy (C)-terminal tail of CFP-10. Rv3871 itself is associated with the inner membrane (IM) by its interaction with Rv3870. The translocation channel in the IM is probably formed by Rv3877, which has many transmembrane domains, although it is unknown which protein (or proteins) forms the channel in the mycomembrane (MM). The AAA+ chaperone-like protein Rv3868 could be involved in the biogenesis of the secretion machinery. The function of the subtilisin-like protease MycP1 is essential, but it is not known why, as no protein has been identified that is cleaved upon secretion by ESX-1. Gene families that are also present in other ESX gene clusters are shown in colours, whereas ESX-1 specific genes are shown in dark grey. A question mark indicates that the mycomembrane channel has not yet been identified.

29

Actinobacterial progenitor (ESX-4 only)

Fast-growing mycobacteria

Slow-growing mycobacteria

M. sp. KMSr

M. smegmatisM. gilvum M. vanbaalenii M. sp. MCS and JLS

ESX-4 onlyNo PE/PPE Duplication of ESX-1

First pair of PE/PPE

Duplication of ESX-3 Second pair of PE/PPE ESX-4

ESX-1 + PE/PPE ESX-3 + PE/PPE

Duplication of ESX-2 Third pair of PE/PPE

ESX-4ESX-1 + PE/PPE ESX-3 + PE/PPE ESX-2 + PE/PPE

Duplication of ESX-5 Fourth pair of PE/PPE

Expansion of the PE and PPE families M. avium*

M. leprae* M. marinum M. tuberculosis complex

ESX-4ESX-1 + PE/PPE ESX-3 + PE/PPE ESX-2 + PE/PPE ESX-5 + PE/PPE Other PE/PPE’s

Nocardia farcinicaCorynebacterium sp. Streptomyces coelicolor

Type VII secretion systems species

* Some mycobacteria have lost ESX systems owing to secondary deletions, for instance M. avium has deleted part of the ESX-

1 system whereas M. leprae has large deletions in ESX-2 and ESX-4 Figure 5 | Proposed evolutionary scenario of mycobacteria with respect to the different Type VII secretion systems (ESX-1 to ESX-5) and the related PE and PPE gene families.


Type VII secretion in mycobacteria

The five ESX systems. Although the identification of a specialized secretion system in M. tuberculosis was expected because of the nature of the cell wall7,8, it was unexpected that M. tuberculosis would encode five such secretion systems. The M. tuberculosis genome contains 11 or 12 loci (depending on the strain) that comprise tandem pairs of genes that encode ESAT-6 family members52,53 (Fig.5). Four of these gene clusters are part of larger loci, which contain more homologues of the ESX-1 cluster; these clusters have been named ESX-2--5. A comparison of the different ESX systems resulted in the identification of a set of six genes that are present in all of these clusters and therefore probably encode the core components of the mycobacterial T7SSs16,17. These proteins are: two members of the ESAT-6 family; a member of the FtsK/SpoIIIE family (although this is sometimes encoded by two genes, for example, Rv3870 and Rv3871 of ESX-1); a subtilisin-like protease (MycP1 in ESX-1); an integral membrane protein with 10–11 transmembrane domains (Rv3877 in ESX-1); and a member of another membrane-protein family (Rv3869 in ESX-1). In addition, some genes are shared by most, but not all, ESX systems, such as the PE and PPE genes (discussed below). Finally, some ESX clusters have genes that are unique for their system (the dark-grey arrows in FIGS 3, 7). These genes could encode secreted substrates or specific components.

Box 2 | Important Questions

• Are type VII secretion systems (T7SSs) only involved in protein transport, or are they also involved in DNA, glycolipid and carbohydrate transport?

• Do T7SSs form injectosomes that can directly inject secreted proteins into eukaryotic host cells, similarly to type III secretion systems (T3SSs)?

• Is secretion through T7SSs inducible, as is the case for T3SSs? If it is, this could mean that we have missed a large number of substrates.

• Are ESX substrates secreted across the cell envelope in a one-step mechanism? • Why are T7SS substrates binary complexes of proteins (for example, ESAT-6--

CFP-10 and PE--PPE)? Is this finding merely coincidental? • Does the interdependence of EspA, Rv3515c, ESAT-6 and CFP-10 mean that

multi-component protein complexes are the real units of secretion? • Which protein (or proteins) forms the channel in the mycomembrane? • Are the recently identified mycobacterial pili49 functionally linked to one of the

ESX secretion systems? • Why is the ESX-1 locus deleted in a number of mycobacterial pathogens? • Is ESAT-6 an effector protein, a structural protein or both? • Why is ESX-3 essential for Mycobacterium tuberculosis viability? • Does ESX-5 secrete all the recently evolved PE and PPE proteins? • Why are there so many PE and PPE proteins, and why are they not

redundant50,51?


Phylogenetic analyses and comparative genomics have revealed that the five different ESX systems in the genus Mycobacterium probably evolved through gene duplication, in the order ESX-4, ESX-1, ESX-3, ESX-2 and, most recently, ESX-517 (Fig. 5). ESX-4 is therefore, the most archaic T7SS in the mycobacteria17. It is also the smallest (9,870 base pairs in M. tuberculosis compared with 14–22 kb of other ESX loci) and has the fewest genes (7 genes in M. tuberculosis compared with the 11–18 genes of other ESX loci). Interestingly, the most recently evolved ESX system, ESX-5, separates two groups of mycobacterial species. The genus Mycobacterium can be roughly divided into fast-growing (colonies that form within 7 days) and slow-growing species, and the duplication of ESX-5 seems to coincide with the emergence of the slow-growing species54 (Fig. 5, table 1).

Rv3619c LTASDF--WGG-AGSAACQGFITQLGRNFQVIYEQA Rv1037c LTASDF--WGG-AGSAACQGFITQLGRNFQVIYEQA Rv1198 LTASDF--WGG-AGSAACQGFITQLGRNFQVIYEQA Rv2346c LAAGDF--WGG-AGSVACQEFITQLGRNFQVIYEQA Rv1793 (ESX-5) LAAGDF--WGG-AGSVACQEFITQLGRNFQVIYEQA Rv0288 (ESX-3) AALQSA--WQG-DTGITYQAWQAQWNQAMEDLVRAY Rv3019c AVLSSA--WQG-DTGITYQGWQTQWNQALEDLVRAY Rv3017c TAPSRA--CQG-DLGMSHQDWQAQWNQAMEALARAY Rv0287 (ESX-3) MSAQAF--HQG-ESSAAFQAAHARFVAAAAKVNTLL Rv3020c MSAQAF--HQG-ESAAAFQGAHARFVAAAAKVNTLL Rv1038c QNISGAG-WSG-MAEATSLDTMTQMNQAFRNIVNML Rv1792 (ESX-5) QNISGAG-WSG-MAEATSLDTMT-MNQAFRNIVNML Rv3620c QNISGAG-WSG-MAEATSLDTMTQMNQAFRNIVNML Rv1197 QNISGAG-WSG-MAEATSLDTMAQMNQAFRNIVNML Rv2347c QNISGAG-WSG-MAEATSLDTMAQMNQAFRNIVNML CFP-10 (ESX-1) GSLQGQ--WRG-AAGTAAQAAVVRFQEAANKQKQEL Rv3890c(ESX-2) NALQEF--FAG-HGAQGFFDAQAQMLSGLQGLIETV Rv3891c(ESX-2) NVMNPAT-WSG-TGVVASHMTATEITNELNKVLTGG Rv3905c GQMLGG--WRG-ASGSAYGSAWELWHRGAGEVQLGL Rv3904c TRLHVT--WTG-EGAAAHAEAQRHWAAGEAMMRQAL ESAT-6 (ESX-1) TKLAAA--WGG-SGSEAYQGVQQKWDATATELNNAL Rv3444c(ESX-4) APLQQL--WTR-EAAAAYHAEQLKWHQAASALNEIL Rv3445c(ESX-4) SGVPPSV-WGG-LAAARFQDVVDRWNAESTRLYHVL

Figure 6 | Partial sequence alignment of the members of the ESAT-6/WXG100 protein family in M. tuberculosis. Amino acid residues in bold indicates potential family-specific motif [W]-x-[G]. Nearly identical ESAT-6/WXG100 members related to the ESX-3 cluster and ESX-5 cluster are shown in red and blue, respectively.

32

Figure 7 | Comparison of different gene clusters that encode type VII or type VIIb secretion systems. The colour coding for the figure is presented in the key. Type VIIb secretion systems are indicated and all the region-specific genes are shown in dark grey. Members of the FtsK/SpoIIIE family and the ESAT-6/WXG100 family, which are both present in all of the different type VII secretion systems, are shown in purple and blue, respectively. The physical gaps between the different coding sequences do not reflect the relative length of these gaps. They have been chosen to clearly show the conservation between the different regions.


All the mycobacterial ESX clusters contain genes that encode ESAT-6 family members and these proteins are, of course, the major substrate candidates for the various putative secretion systems. Different studies have shown that (in addition to ESAT-6 and CFP-10) the ESAT-6 family members encoded by ESX-3 and ESX-5 as well as the ESAT-6 family members that are highly homologous to the ESX-3 and ESX-5 clusters (Fig. 6) are indeed present in the culture supernatant of M. tuberculosis30,39,55,56. Furthermore, an intact ESX-5 region is essential for the secretion of the ESX-5 encoded ESAT-6 family member EsxN by M. marinum (A.M.A. and W.B., unpublished observations). Together, these data indicate that ESX-3 and ESX-5 are indeed functional secretion systems. By contrast, the ESAT-6 homologues of ESX-2 and ESX-4 have not yet been detected extracellularly.

PE and PPE proteins in the ESX systems. PE and PPE are two gene families that are unique to mycobacteria, and they are significantly expanded in slow-growing pathogenic mycobacteria; almost 9% of the coding capacity of M. tuberculosis is dedicated to these gene families52. The most archaic PE and PPE genes were probably inserted into the first duplication of the ESX system (ESX-1) and were subsequently co-duplicated with the ESX gene clusters, until their recent expansion54. This co-evolution suggests that these proteins are functionally associated with the ESX secretion systems. PE and PPE proteins also share a number of characteristics with ESAT-6 and CFP-10: they are secreted proteins that do not have a classical secretion signal; the ancestral PE- and PPE- encoding genes often form gene pairs (and are adjacent to ESAT-6--CFP-10 gene pairs)17; and they form a tight 1:1 complex57. Evidence for the association between PE–PPE proteins and ESX secretion systems has been accumulating. First, the RD1-encoded PPE protein Rv3873 probably interacts with CFP-10 and/or ESAT-6, as shown by overlay experiments and yeast two-hybrid experiments58,59. This could mean that Rv3873 is secreted together with the ESAT-6--CFP-10 complex. Second, a transposon insertion of the M. smegmatis ESX-1 locus in the PE gene showed the same phenotype as other ESX-1 mutations — increased conjugation frequency29. Third, in a recent study that investigated M. marinum, it was shown that PPE41 is secreted both in culture and in infected macrophages. It was also determined that this secretion is dependent both on the presence of PE25, which forms a complex with PPE4157, and on an intact ESX-5 cluster. Reconstitution of the entire ESX-5 cluster in the fast-growing M. smegmatis, which does not contain an endogenous copy of ESX-5, enabled M. smegmatis to secrete heterologously expressed PPE4132. Thus, the ESX-5 locus is both necessary and sufficient to produce a functional secretion machinery for PPE41. To what extent the PE and PPE genes are functionally linked to the ESX clusters remains unknown, but recent data suggest that many more PE and PPE proteins are secreted by ESX-5 (A.M.A. and W.B., unpublished observations). The analysis of ESX-5 mutants might shed some light on the function of these mysterious PE and PPE proteins.


ESX systems do not complement one another. The presence of five T7SSs raises the question of why several secretion systems exist and why they cannot complement each other. This is highlighted by the inability of the four ESX systems (ESX-2–5) to complement the loss of virulence caused by deletions in ESX-1. This is especially intriguing for ESX-5 as both ESX-5 and ESX-1 seem to be involved in macrophage escape and in cell-to-cell spread16,27,32. This might mean that these two secretion systems have independent roles in successive steps of the cellular infection cycle. The inability of the various ESX systems to complement each other could be due to divergent evolution of their secretion signals39 and also their differential regulation. For example, ESX-1 genes are down-regulated upon starvation, whereas genes from ESX-2 are up-regulated under these conditions60. Furthermore, ESX-3 is regulated by the availability of iron and zinc, as part of the ideR and Fur/Zur regulons61,62, whereas ESX-4 is regulated by the alternative sigma factor SigM63. Apart from the differences in regulation, there are also some other apparent differences. For example, high-density transposon mutagenesis studies have shown that whereas genes from ESX-1, 2 and 4 could all be disrupted, most genes from ESX-3 and some from ESX-5 cannot be disrupted64,65. This suggests that these systems are essential for growth in culture. This hypothesis has been proven for ESX-3 in M. tuberculosis (Lawrence and Jacobs, personal communication), although ESX-3 is not essential for the viability of M. smegmatis. The fact that knockouts of specialized secretion systems are usually not essential for growth in culture medium probably means that either ESX-3 secretes components that are involved in essential function (or functions) or that the cytoplasmic accumulation of substrates is toxic for the cell. Although all five ESX secretion systems seem to function independently, there may be a certain level of cross-talk. For example, the deletion of the cfp10--esat-6 operon resulted in the increased secretion of the ESX-5 substrate PPE4132, whereas the ESX-5 mutant itself showed an increase in the secretion of certain ESX-1 substrates (A.M.A. and colleagues, unpublished observations). Partial complementation between ESX secretion systems could also explain why different mycobacterial species require different ESX-1 components for protein secretion (Fig.2).

Other Gram-positive T7SSs

T7SSs seem to be involved in the transport of proteins across the mycobacterial cell envelope, which includes the mycomembrane. Therefore it is unsurprising that similar secretion systems have been identified in other mycolata species, such as Corynebacterium (Fig. 7) and Nocardia17. However, it is surprising that some high G+C Gram-positive bacteria that do not have a mycomembrane, such as Streptomyces spp., also contain a putative T7SS (Fig. 7). As T7SSs in high G+C Gram-positive bacteria are most related to the archaic ESX-4 system, this might


indicate that ESX-4 was not designed to transport proteins across the mycomembrane and that this function was only introduced upon the evolution of ESX-1. The WXG100 motif. Initially, no T7SSs were identified other than in the high G+C Gram-positive bacteria. However, when the different homologues of the ESAT-6 family where systematically compared18, the sequence similarity of these proteins was found to be low and the members were characterized by a central tryptophan-variable-glycine (WXG) motif. This WXG motif is located in the loop that connects the two main α-helices of ESAT-6 and CFP-1037 (Fig. 2). Replacing the conserved tryptophan residue of ESAT-6 with arginine did not affect ESAT-6 secretion, but did result in severe attenuation38. As well as the conserved WXG motif, all family members identified were composed of ~100 amino acids, and so these family members are also called WXG100 proteins. Subsequently, other members of the ESAT-6/WXG100 family that shared the same characteristics were identified using PSI-BLAST, which resulted in the identification of dozens of distantly related members18. Surprisingly, these proteins were not restricted to the Actinobacteria, but were also found in members of the Firmicutes (low G+C Gram-positive bacteria), such as Bacillus and Clostridium spp., Staphylococcus aureus, Streptococcus agalactiae and Listeria monocytogenes18 (Fig. 7). An analysis of genomic loci from these species showed that, in addition to the ESAT-6/WXG100 family members, they also contain a gene encoding an FtsK/SpoIIIE family protein that is homologous to the one found in T7SSs18. However, other genes that are homologous to those of the T7SSs of the high G+C Gram-positive bacteria are missing (Fig. 7). Instead, these loci contain a variable number (between 3 and 10) of additional genes, 3 of which are present in all loci in the Firmicutes (Fig. 7), but not in the T7SSs of the Actinobacteria. Together, these data strongly suggest that the T7SSs of Firmicutes belong to a specific and distant subfamily, which we propose should be designated type VIIb secretion systems. The existence of a subfamily is comparable to the T4SSs, which are subdivided in three different groups22. S. aureus type VII secretion. Recently, Burts and colleagues33 showed that the human pathogen S. aureus secretes ESAT-6/WXG100 family members, which is the first report of a functional T7SS outside the mycobacteria. The secreted proteins, EsxA and EsxB, are encoded by genes of the ess locus. This locus contains 12 coding sequences (CDSs) (Fig. 7), including the FtsK/SpoIIIE family member essC, which is essential for the secretion of EsxA--EsxB33. Furthermore, two other CDSs (specifically, essA and essB) that encode transmembrane proteins are involved in secretion (Fig. 7). Finally, secretion of EsxA is dependent on the presence of EsxB, and vice versa. This co-dependence is similar to that of ESAT-6 and CFP-10 in M. tuberculosis. Interestingly, S. aureus mutants that fail to secrete EsxA and EsxB display significantly reduced virulence and are defective in dissemination and colonization33.


Bacillus species type VII secretion. Various Bacillus species also contain a gene cluster that is homologous to the S. aureus ess cluster. In Bacillus subtilis this cluster is known as the yuk locus18,66. The yuk locus contains five CDSs (yukAB, yukC, yukD, yukE and yueB), of which yukE encodes an ESAT-6/WXG100 family protein. At present it is not clear whether YukE is secreted, but an ESAT-6/WXG100 family member of Bacillus anthracis is present in the culture supernatant (as shown by secretome analysis)67. An unexpected finding was that one protein encoded by the Yuk regulon of B. subtilis, YueB, is a phage receptor66,68. Homologues of this phage receptor are also present in the S. aureus ess cluster and in the other type VIIb clusters. Overview of type VII secretion in Gram-positive bacteria. The fact that putative T7SSs are present in several non-pathogenic and non-related bacterial species indicates that this system is not designed to act as a secretion system for virulence per se, and the virulence function was probably acquired through evolution of function. For example, the putative T7SS of L. monocytogenes does not seem to be involved in virulence69. It remains to be elucidated why Gram-positive species without a mycolic acid layer need special secretion systems. Perhaps T7SSs in these bacteria are involved in the biosynthesis of specialized surface structures or appendages. Investigations of the function of the archetypal T7SS ESX-4 and of the Bacillus Yuk system are key to understanding the evolution, as well as the original function, of these systems.

Type VII secretion in virulence

Although T7SSs are not virulence systems per se (as discussed above), it is clear that some T7SSs have important roles in virulence: the ESX-5 system of M. marinum seems to be crucial for cell-to-cell spread32 and the S. aureus Ess--Esx system is involved in dissemination and colonization33. The role of the ESX-1 system in pathogenic mycobacteria has been studied in most detail. The lack of virulence of M. bovis BCG as compared with M. bovis and M. tuberculosis indicated that ESX-1 is essential for virulence10,12. This notion was confirmed by the finding that if M. tuberculosis lacking a functional ESX-1 system it is reduced in virulence5,13, whereas if M. bovis BCG is complemented with an intact ESX-1 locus partially regains virulence14,15. Mycobacterium leprae, which has a strongly degenerated genome owing to reductive evolution, also seems to have an active ESX-1 secretion system, as T cells of patients with leprosy react strongly to M. leprae ESAT-670. However, it should be realized that a functional ESX-1 system is not unique to pathogenic species (Table 1). Furthermore, among the few mycobacterial species that lack part of the ESX-1 region, and therefore lack ESAT-6 secretion, are some pathogens, including Mycobacterium ulceran71, Mycobacterium microti72 and Mycobacterium avium17 (Fig. 3). So, what are the roles of the secreted proteins in virulence, and why does the ESX-1 secretion system only contribute to virulence in certain species? Two possibilities can be envisaged: ESAT-6 and/or

37

Table 1 | Experimental evidence for the presence of the ESX gene cluster regions in the mycobacteria*

Species and Strain Pathogenicity (a) ESX Gene Cluster Region (b)

Region 1 Region 2 Region 3 Region 5

M.africanum Pathogenic Present (d) (f) (g) M.asiaticum Pathogenic Not detected (d) M.avium Pathogenic Not detected (d) (f) (g) (h) (j) Present (i) Present (h) Not detected (e)M.bovis ATCC19210 Pathogenic Present (j) M.bovis Branch Pathogenic Present (d) M.bovis KML Pathogenic Present (i) M.bovis MNC 27 Pathogenic Present (g) (h) Present (h) M.bovis NADL Pathogenic Present (d) M.bovis Ravenel Pathogenic Present (d) M.bovis BCG Brazil Non-pathogenic (c) Not detected (j) M.bovis BCG Connaught Non-pathogenic (c) Not detected (d) (j) M.bovis BCG Danish 1331 Non-pathogenic (c) Not detected (f) (g) (h) Present (h) M.bovis BCG Glaxo 1077 Non-pathogenic (c) Not detected (g) M.bovis BCG Japan 172 Non-pathogenic (c) Not detected (d) M.bovis BCG Montreal Non-pathogenic (c) Not detected (d) M.bovis BCG Moreau Non-pathogenic (c) Not detected (g) M.bovis BCG Pasteur 1173P2 Non-pathogenic (c) Not detected (d) (g) (j) Present (i) M.bovis BCG Tice Non-pathogenic (c) Not detected (g) M.bovis BCG Tokyo Non-pathogenic (c) Not detected (g) (h) Present (h) M.bovis BCG Russia Non-pathogenic (c) Not detected (d) (g)

38



Region 1 Region 2 Region 3 Region 5 M.chelonae Pathogenic Not detected (e)M.flavescens Non-pathogenic Present (g) M.fortuitum Pathogenic Not detected (d) (f) (g) (h) Not detected (i) Not detected (h) Not detected (e)M.gastri Non-pathogenic Present (d) M.gordonae Non-pathogenic Not detected (i) Not detected (e)M.heamophilum Pathogenic Not detected (d) M.intracellulare Pathogenic Not detected (f) (g) (h) Present (i) Present (h) M.kansasii Pathogenic Present (d) (f) (g) (h) Present (i) Present (h) M.leprae Pathogenic Not detected (g) Not detected (e)M.malmoense Pathogenic Not detected (d) M.marinum Pathogenic Present (f) (g) (h) Not detected (i) Present (h) M.paratuberculosis Pathogenic Present (i) M.phlei Non-pathogenic Not detected (d) Not detected (i) M.scrofulaceum Pathogenic Not detected (d) (f) (g) (h) Present (i) Not detected (h) Not detected (e)M.simiae Pathogenic Not detected (d) M.smegmatis Non-pathogenic Not detected (j) Not detected (i) Not detected (e)M.szulgai Pathogenic Present (f) (g) (h) Not detected (h) M.terrae Non-pathogenic Not detected (d) Not detected (i) M.triviale Non-pathogenic Not detected (d) M.tuberculosis CSU#93 Pathogenic Present (d) M.tuberculosis Erdman Pathogenic Present (g) (j) Present (e)

39



Region 1 Region 2 Region 3 Region 5 M.tuberculosis H37Ra Non-pathogenic (c) Present (d) (f) (g) (j) Present (i) Present (e) M.tuberculosis H37Rv Pathogenic Present (d) (f) (g) (h) (j) Present (i) Present (h) Present (e) M.tuberculosis R1609 Pathogenic Present (f) M.tuberculosis W Pathogenic Present (d) M.ulcerans Pathogenic Not detected (d) M.vaccae Non-pathogenic Not detected (e)M.xenopi Pathogenic Not detected (f) (g) (h) Not detected (h)

* Based on previously published Southern blotting, Western blotting and PCR data of selected genes and regions within the gene clusters (a) = Shinnick and Good, 1994; (b) = No work has been done on any of the genes within region 4; (c) = Attenuated strains, potentially hazardous; (d) = Southern blotting data using mtsa-10 (cfp-10) and esat-6 as probes (Colangeli et al., 2000); (e) = Southern blotting data using mtb9.9a as probe (Alderson et al., 2000); (f) = Western blotting data using monoclonal anti-ESAT-6 antibodies (HYB 76-8) as probe (Sorenson et al., 1995); (g) = Southern blotting and PCR data using esat-6 as probe (Harboe et al., 1996); (h) Southern blotting data usingtb10.4 and cfp-10 as probes (Skjøt et al., 2000); (i) = Southern blotting data using Pan promoter sequence as probe (Gormley et al., 1997);(j) = Southern blotting data using RD1 deletion region specific probe (Mahairas et al., 1996).


CFP-10 of pathogenic mycobacteria have an additional function compared with non-pathogenic species, or additional proteins are secreted through ESX-1 by mycobacterial pathogens. Therefore, the key questions are: which secreted proteins are crucial for virulence and with which host components do they interact? At present, the available knowledge is mostly phenomenological and fragmentary. This is illustrated in table 2, which summarizes the literature on the role of ESX-1 and its substrates in mycobacterial virulence. A complicating factor is the co-dependence of the various ESX-1 substrates for secretion, which makes it difficult to pinpoint the exact component responsible for virulence. Interestingly, the lack of in vivo persistence of mycobacteria with a defective ESX-1 system seems to run in parallel with their delay in forming granulomas73. Recent data provide an important clue to how this defect might arise: both M. tuberculosis and M. marinum can, at some stage in the macrophage-infection cycle, escape the phagolysosome compartment and be translocated into the cytoplasm74,75. A functional ESX-1 systems seems to be crucial for this transition75. Apparently, the presence of mycobacteria in the cytosol of infected macrophages is needed for the attraction of other macrophages. Which of the ESX-1-secreted polypeptides is causing this lytic effect, and how lysis occurs is not known, but the principal suspect for this function is ESAT-6, which has been found to cause apoptosis76 and membrane perturbation5,77. The detailed characterization of ESAT-6 point-mutations that do not affect secretion but lead to an attenuated phenotype38 will have to prove this hypothesis. Table 2 | Liquorice all sorts: the diverse roles of ESX-1 secretion and its substrates in mycobacteria

Role References

ESX-1 is required for survival of mycobacteria in vivo (M. tuberculosis) 13 ESX-1 is required for (initiation of) granuloma formation (M. marinum) 73 ESX-1 is essential for escape from phagosomes into cytosol (M. tuberculosis).

75

ESX-1 is essential for cell lysis and cell-to-cell spreading (M. tuberculosis and M. marinum)

5,27

ESX-1 is required for phagosome maturation (M. marinum) 35,41 ESX-1 essential for induction of immunosuppressive type I interferons (M. tuberculosis)

78

ESX-1 represses conjugation in donor cells (M. smegmatis) 29 ESX-1 secreted proteins escape into cytosol, are processed by proteosome and loaded onto MHC I (M. tuberculosis)

79

Purified CFP-10 down-regulates IFNγ production in dendritic cells 80 Purified CFP-10 modifies macrophage intracellular signaling through affecting protein phosphorylation.

81,82

Purified ESAT-6 induces IFNγ in NK cells 83 Purified ESAT-6 causes apoptosis of macrophages through caspase activation ESAT-6 has membrane lysing activity

76

77


Conclusion

The mycobacterial ESX secretion systems form the paradigm for a new secretion pathway -- type VII secretion. This pathway is regarded as novel because: the T7SS is composed of a unique set of proteins; the main secreted proteins all belong to the same unique protein family (the ESAT-6/WXG100 family); and this pathway is mechanistically unlike any other secretion system that has been detected, as all the secreted proteins seem to be co-dependent on each other for secretion. The T7SS is also different from type I–VI secretion as, unlike these other system, T7SSs seems to be present only in Gram-positive bacteria. Therefore, it could be argued that this system should not form part of the sequential secretion-system nomenclature system. However, this is a Gram-negative-centered view, and type I–VI secretion systems are also present outside Gram-negative bacteria. For example, flagellar biosynthesis in Archaea and type IV pili of Clostridia belong to the class of type II secretion38,84, and genes that encode T4SSs have been identified on plasmids from different Gram-positive bacteria85,86. T7SSs are probably complicated machineries that are involved in the secretion of protein complexes and possibly in the assembly of extracellular structures. The most important questions on T7SSs that must be answered are listed in Box 2. Answering these questions and understanding T7SSs will change our way of thinking about the virulence of M. tuberculosis and hopefully will give us new opportunities to combat this old foe.

Notes:

Supporting Online Material (SOM) accompanies the paper on the Nature website (http://www.nature.com/nrmicro/) and includes SOM Figures S1 and S2, and SOM Tables S1 and S2. References

1. Finlay,B.B. & Falkow,S. Common themes in microbial pathogenicity revisited. Microbiol. Mol. Biol. Rev. 61, 136-169 (1997).

2. van Wely,K.H., Swaving,J., Freudl,R. & Driessen,A.J. Translocation of proteins across the cell envelope of Gram-positive bacteria. FEMS Microbiol. Rev. 25, 437-454 (2001).

3. Pym,A.S. et al. Recombinant BCG exporting ESAT-6 confers enhanced protection against tuberculosis. Nat. Med. 9, 533-539 (2003).

4. Stanley,S.A., Raghavan,S., Hwang,W.W. & Cox,J.S. Acute infection and macrophage subversion by Mycobacterium tuberculosis require a specialized secretion system. Proceedings of the National Academy of Sciences of the United States of America 100, 13001-13006 (2003).

http://www.nature.com/nrmicro/

https://www.researchgate.net/publication/9051531_Acute_infection_and_macrophage_subversion_by_Mycobacterium_tuberculosis_require_a_specialized_secretion_system?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=




https://www.researchgate.net/publication/11821991_Translocation_of_proteins_across_the_cell_envelope_of_Gram-positive_bacteria_FEMS_Microbiol_Rev?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=



https://www.researchgate.net/publication/20587332_Common_Themes_in_Microbial_Pathogenicity?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=

https://www.researchgate.net/publication/20587332_Common_Themes_in_Microbial_Pathogenicity?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=


5. Hsu,T. et al. The primary mechanism of attenuation of bacillus Calmette-Guerin is

a loss of secreted lytic function required for invasion of lung interstitial tissue. Proceedings of the National Academy of Sciences of the United States of America 100, 12420-12425 (2003).

6. Guinn,K.M. et al. Individual RD1-region genes are required for export of ESAT-6/CFP-10 and for virulence of Mycobacterium tuberculosis. Molecular Microbiology 51, 359-370 (2004).

7. Sutcliffe,I.C. Cell envelope composition and organisation in the genus Rhodococcus. Antonie Van Leeuwenhoek 74, 49-58 (1998).

8. Minnikin,D.E., Kremer,L., Dover,L.G. & Besra,G.S. The methyl-branched fortifications of Mycobacterium tuberculosis. Chem. Biol. 9, 545-553 (2002).

9. Bayan,N., Houssin,C., Chami,M. & Leblon,G. Mycomembrane and S-layer: two important structures of Corynebacterium glutamicum cell envelope with promising biotechnology applications. J. Biotechnol. 104, 55-67 (2003).

10. Mahairas,G.G., Sabo,P.J., Hickey,M.J., Singh,D.C. & Stover,C.K. Molecular analysis of genetic differences between Mycobacterium bovis BCG and virulent M. bovis. J. Bacteriol. 178, 1274-1282 (1996).

11. Andersen,P. & Doherty,T.M. The success and failure of BCG - implications for a novel tuberculosis vaccine. Nature Reviews Microbiology 3, 656-662 (2005).

12. Gordon,S.V. et al. Identification of variable regions in the genomes of tubercle bacilli using bacterial artificial chromosome arrays. Mol. Microbiol. 32, 643-655 (1999).

13. Lewis,K.N. et al. Deletion of RD1 from Mycobacterium tuberculosis mimics bacille Calmette-Guerin attenuation. Journal of Infectious Diseases 187, 117-123 (2003).

14. Majlessi,L. et al. Influence of ESAT-6 secretion system 1 (RD1) of Mycobacterium tuberculosis on the interaction between mycobacteria and the host immune system. J. Immunol. 174, 3570-3579 (2005).

15. Pym,A.S., Brodin,P., Brosch,R., Huerre,M. & Cole,S.T. Loss of RD1 contributed to the attenuation of the live tuberculosis vaccines Mycobacterium bovis BCG and Mycobacterium microti. Mol. Microbiol. 46, 709-717 (2002).

16. Tekaia,F. et al. Analysis of the proteome of Mycobacterium tuberculosis in silico. Tuber. Lung Dis. 79, 329-342 (1999).

17. Gey van Pittius,N.C. et al. The ESAT-6 gene cluster of Mycobacterium tuberculosis and other high G+C Gram-positive bacteria. Genome Biol. 2, RESEARCH0044 (2001).

18. Pallen,M.J. The ESAT-6/WXG100 superfamily -- and a new Gram-positive secretion system? Trends Microbiol. 10, 209-212 (2002).

19. Holland,I.B., Schmitt,L. & Young,J. Type 1 protein secretion in bacteria, the ABC-transporter dependent pathway (review). Mol. Membr. Biol. 22, 29-39 (2005).

20. Johnson,T.L., Abendroth,J., Hol,W.G. & Sandkvist,M. Type II secretion: from structure to function. FEMS Microbiol. Lett. 255, 175-186 (2006).

21. Cornelis,G.R. The type III secretion injectisome. Nat. Rev. Microbiol. 4, 811-825 (2006).

22. Christie,P.J., Atmakuri,K., Krishnamoorthy,V., Jakubowski,S. & Cascales,E. Biogenesis, architecture, and function of bacterial type IV secretion systems. Annu. Rev. Microbiol. 59, 451-485 (2005).

https://www.researchgate.net/publication/11340191_The_Methyl-Branched_Fortifications_of_Mycobacterium_tuberculosis?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=

https://www.researchgate.net/publication/11340191_The_Methyl-Branched_Fortifications_of_Mycobacterium_tuberculosis?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=

https://www.researchgate.net/publication/null?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=


https://www.researchgate.net/publication/13221588_Cell_envelope_composition_and_organisation_in_the_genus_Rhodococcus?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=

https://www.researchgate.net/publication/13221588_Cell_envelope_composition_and_organisation_in_the_genus_Rhodococcus?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=




https://www.researchgate.net/publication/14568263_Mahairas_G_G_et_al_Molecular_analysis_of_genetic_differences_between_Mycobacterium_bovis_BCG_and_virulent_M_bovis_J_Bacteriol_178_1274-1282?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=



https://www.researchgate.net/publication/11053902_Pym_A_S_Brodin_P_Brosch_R_Huerre_M_Cole_S_T_Loss_of_RD1_contributed_to_the_attenuation_of_the_live_tuberculosis_vaccines_Mycobacterium_bovis_BCG_and_Mycobacterium_microti_Mol_Microbiol_46_709-717?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=





https://www.researchgate.net/publication/10588271_Bayan_N_Houssin_C_Chami_M_Leblon_G_Mycomembrane_and_S-layer_two_important_structures_of_Corynebacterium_glutamicum_cell_envelope_with_promising_biotechnology_applications_J_Biotechnol_104_55-67?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=



https://www.researchgate.net/publication/295218116_The_type-III_secretion_injectisome?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=

https://www.researchgate.net/publication/295218116_The_type-III_secretion_injectisome?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=


23. Henderson,I.R., Navarro-Garcia,F., Desvaux,M., Fernandez,R.C. & Ala'Aldeen,D.

Type V protein secretion pathway: the autotransporter story. Microbiol. Mol. Biol. Rev. 68, 692-744 (2004).

24. Oomen,C.J. et al. Structure of the translocator domain of a bacterial autotransporter. EMBO J. 23, 1257-1266 (2004).

25. Mougous,J.D. et al. A virulence locus of Pseudomonas aeruginosa encodes a protein secretion apparatus. Science 312, 1526-1530 (2006).

26. Pukatzki,S. et al. Identification of a conserved bacterial protein secretion system in Vibrio cholerae using the Dictyostelium host model system. Proc. Natl. Acad. Sci. U. S. A 103, 1528-1533 (2006).

27. Gao,L.Y. et al. A mycobacterial virulence gene cluster extending RD1 is required for cytolysis, bacterial spreading and ESAT-6 secretion. Molecular Microbiology 53, 1677-1693 (2004).

28. Converse,S.E. & Cox,J.S. A protein secretion pathway critical for Mycobacterium tuberculosis virulence is conserved and functional in Mycobacterium smegmatis. J. Bacteriol. 187, 1238-1245 (2005).

29. Flint,J.L., Kowalski,J.C., Karnati,P.K. & Derbyshire,K.M. The RD1 virulence locus of Mycobacterium tuberculosis regulates DNA transfer in Mycobacterium smegmatis. Proc. Natl. Acad. Sci. U. S. A 101, 12598-12603 (2004).

30. MacGurn,J.A., Raghavan,S., Stanley,S.A. & Cox,J.S. A non-RD1 gene cluster is required for Snm secretion in Mycobacterium tuberculosis. Mol. Microbiol. 57, 1653-1663 (2005).

31. Fortune,S.M. et al. Mutually dependent secretion of proteins required for mycobacterial virulence. Proceedings of the National Academy of Sciences of the United States of America 102, 10676-10681 (2005).

32. Abdallah,A.M. et al. A specific secretion system mediates PPE41 transport in pathogenic mycobacteria. Molecular Microbiology 62, 667-679 (2006).

33. Burts,M.L., Williams,W.A., DeBord,K. & Missiakas,D.M. EsxA and EsxB are secreted by an ESAT-6-like system that is required for the pathogenesis of Staphylococcus aureus infections. Proc. Natl. Acad. Sci. U. S. A 102, 1169-1174 (2005).

34. Brodin,P. et al. Dissection of ESAT-6 system 1 of Mycobacterium tuberculosis and impact on immunogenicity and virulence. Infect. Immun. 74, 88-98 (2006).

35. Tan,T., Lee,W.L., Alexander,D.C., Grinstein,S. & Liu,J. The ESAT-6/CFP-10 secretion system of Mycobacterium marinum modulates phagosome maturation. Cell Microbiol. 8, 1417-1429 (2006).

36. Mostowy,S., Cousins,D. & Behr,M.A. Genomic interrogation of the dassie bacillus reveals it as a unique RD1 mutant within the Mycobacterium tuberculosis complex. J. Bacteriol. 186, 104-109 (2004).

37. Renshaw,P.S. et al. Structure and function of the complex formed by the tuberculosis virulence factors CFP-10 and ESAT-6. EMBO J. 24, 2491-2498 (2005).

38. Brodin,P. et al. Functional analysis of early secreted antigenic target-6, the dominant T-cell antigen of Mycobacterium tuberculosis, reveals key residues involved in secretion, complex formation, virulence, and immunogenicity. J. Biol. Chem. 280, 33953-33959 (2005).

39. Champion,P.A., Stanley,S.A., Champion,M.M., Brown,E.J. & Cox,J.S. C-terminal signal sequence promotes virulence factor secretion in Mycobacterium tuberculosis. Science 313, 1632-1636 (2006).




https://www.researchgate.net/publication/8956596_Genomic_Interrogation_of_the_Dassie_Bacillus_Reveals_It_as_a_Unique_RD1_Mutant_within_the_Mycobacterium_tuberculosis_Complex?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=









https://www.researchgate.net/publication/8136811_Type_V_Protein_Secretion_Pathway_the_Autotransporter_Story?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=

https://www.researchgate.net/publication/8136811_Type_V_Protein_Secretion_Pathway_the_Autotransporter_Story?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=

https://www.researchgate.net/publication/8397675_The_RD1_virulence_locus_of_Mycobacterium_tuberculosis_regulates_DNA_transfer_in_Mycobacterium_smegmatis?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=






https://www.researchgate.net/publication/8045699_A_Protein_Secretion_Pathway_Critical_for_Mycobacterium_tuberculosis_Virulence_Is_Conserved_and_Functional_in_Mycobacterium_smegmatis?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=




40. Ize,B. & Palmer,T. Microbiology. Mycobacteria's export strategy. Science 313,

1583-1584 (2006). 41. MacGurn,J.A. & Cox,J.S. A genetic screen for Mycobacterium tuberculosis

mutants defective for phagosome maturation arrest identifies components of the ESX-1 secretion system. Infect. Immun. 75, 2668-2678 (2007).

42. Lodes,M.J. et al. Serological expression cloning and immunological evaluation of MTB48, a novel Mycobacterium tuberculosis antigen. Journal of Clinical Microbiology 39, 2485-2493 (2001).

43. Nagai,H. et al. A C-terminal translocation signal required for Dot/Icm-dependent delivery of the Legionella RalF protein to host cells. Proc. Natl. Acad. Sci. U. S. A 102, 826-831 (2005).

44. Vergunst,A.C. et al. Positive charge is an important feature of the C-terminal transport signal of the VirB/D4-translocated proteins of Agrobacterium. Proc. Natl. Acad. Sci. U. S. A 102, 832-837 (2005).

45. Singh,A., Mai,D., Kumar,A. & Steyn,A.J. Dissecting virulence pathways of Mycobacterium tuberculosis through protein-protein association. Proc. Natl. Acad. Sci. U. S. A 103, 11346-11351 (2006).

46. Dave,J.A., Gey van Pittius,N.C., Beyers,A.D., Ehlers,M.R. & Brown,G.D. Mycosin-1, a subtilisin-like serine protease of Mycobacterium tuberculosis, is cell wall-associated and expressed during infection of macrophages. BMC. Microbiol. 2, 30 (2002).

47. Faller,M., Niederweis,M. & Schulz,G.E. The structure of a mycobacterial outer-membrane channel. Science 303, 1189-1192 (2004).

48. Niederweis,M. Mycobacterial porins--new channel proteins in unique outer membranes. Mol. Microbiol. 49, 1167-1177 (2003).

49. Alteri,C.J. et al. Mycobacterium tuberculosis produces pili during human infection. Proc. Natl. Acad. Sci. U. S. A 104, 5145-5150 (2007).



52. Cole,S.T. et al. Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393, 537-+ (1998).

53. Brodin,P., Rosenkrands,I., Andersen,P., Cole,S.T. & Brosch,R. ESAT-6 proteins: protective antigens and virulence factors? Trends Microbiol. 12, 500-508 (2004).

54. Gey van Pittius,N.C. et al. Evolution and expansion of the Mycobacterium tuberculosis PE and PPE multigene families and their association with the duplication of the ESAT-6 (esx) gene cluster regions. Bmc Evolutionary Biology 6, (2006).

55. Skjot,R.L. et al. Comparative evaluation of low-molecular-mass proteins from Mycobacterium tuberculosis identifies members of the ESAT-6 family as immunodominant T-cell antigens. Infect. Immun. 68, 214-220 (2000).

56. Alderson,M.R. et al. Expression cloning of an immunodominant family of Mycobacterium tuberculosis antigens using human CD4(+) T cells. Journal of Experimental Medicine 191, 551-559 (2000).

https://www.researchgate.net/publication/12490061_Ramakrishnan_L_Federspiel_NA_Falkow_S_Granuloma-specific_expression_of_Mycobacterium_virulence_proteins_from_the_glycine-rich_PE-PGRS_family_Science_288_1436-1439?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=



https://www.researchgate.net/publication/13650817_Deciphering_the_Biology_of_Mycobacterium_tuberculosis_from_the_Complete_Genomic_Sequence?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=

https://www.researchgate.net/publication/13650817_Deciphering_the_Biology_of_Mycobacterium_tuberculosis_from_the_Complete_Genomic_Sequence?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=

https://www.researchgate.net/publication/11092244_Dave_J_A_Gey_Van_Pittius_N_C_Beyers_A_D_Ehlers_M_R_Brown_G_D_Mycosin-1_a_subtilisin-like_serine_protease_of_Mycobacterium_tuberculosis_is_cell_wall-associated_and_expressed_during_infection_of_macroph?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=





https://www.researchgate.net/publication/8687669_The_Structure_of_a_Mycobacterial_Outer-Membrane_Channel?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=

https://www.researchgate.net/publication/8687669_The_Structure_of_a_Mycobacterial_Outer-Membrane_Channel?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=







https://www.researchgate.net/publication/10595767_Niederweis_M_Mycobacterial_porins_-_new_channel_proteins_in_unique_outer_membranes_Mol_Microbiol_49_1167-1177?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=

https://www.researchgate.net/publication/10595767_Niederweis_M_Mycobacterial_porins_-_new_channel_proteins_in_unique_outer_membranes_Mol_Microbiol_49_1167-1177?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=

https://www.researchgate.net/publication/276942612_Expression_Cloning_of_an_Immunodominant_Family_of_Mycobacterium_tuberculosis_Antigens_Using_Human_CD4_T_Cells?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=




57. Strong,M. et al. Toward the structural genomics of complexes: crystal structure of

a PE/PPE protein complex from Mycobacterium tuberculosis. Proc. Natl. Acad. Sci. U. S. A 103, 8060-8065 (2006).

58. Okkels,L.M. & Andersen,P. Protein-protein interactions of proteins from the ESAT-6 family of Mycobacterium tuberculosis. J. Bacteriol. 186, 2487-2491 (2004).

59. Teutschbein,J. et al. A protein linkage map of the ESAT-6 secretion system 1 (ESX-1) of Mycobacterium tuberculosis. Microbiol. Res. (2007).

60. Betts,J.C., Lukey,P.T., Robb,L.C., McAdam,R.A. & Duncan,K. Evaluation of a nutrient starvation model of Mycobacterium tuberculosis persistence by gene and protein expression profiling. Mol. Microbiol. 43, 717-731 (2002).

61. Maciag,A. et al. Global analysis of the Mycobacterium tuberculosis Zur (FurB) regulon. J. Bacteriol. 189, 730-740 (2007).

62. Rodriguez,G.M. & Smith,I. Mechanisms of iron regulation in mycobacteria: role in physiology and virulence. Mol. Microbiol. 47, 1485-1494 (2003).

63. Agarwal,N., Woolwine,S.C., Tyagi,S. & Bishai,W.R. Characterization of the Mycobacterium tuberculosis sigma factor SigM by assessment of virulence and identification of SigM-dependent genes. Infect. Immun. 75, 452-461 (2007).

64. Lamichhane,G. et al. A postgenomic method for predicting essential genes at subsaturation levels of mutagenesis: application to Mycobacterium tuberculosis. Proc. Natl. Acad. Sci. U. S. A 100, 7213-7218 (2003).

65. Sassetti,C.M., Boyd,D.H. & Rubin,E.J. Genes required for mycobacterial growth defined by high density mutagenesis. Molecular Microbiology 48, 77-84 (2003).

66. Sao-Jose,C., Baptista,C. & Santos,M.A. Bacillus subtilis operon encoding a membrane receptor for bacteriophage SPP1. J. Bacteriol. 186, 8337-8346 (2004).

67. Francis,A.W. et al. Proteomic analysis of Bacillus anthracis Sterne vegetative cells. Biochim. Biophys. Acta 1748, 191-200 (2005).

68. Sao-Jose,C. et al. The ectodomain of the viral receptor YueB forms a fiber that triggers ejection of bacteriophage SPP1 DNA. J. Biol. Chem. 281, 11464-11470 (2006).

69. Way,S.S. & Wilson,C.B. The Mycobacterium tuberculosis ESAT-6 homologue in Listeria monocytogenes is dispensable for growth in vitro and in vivo. Infect. Immun. 73, 6151-6153 (2005).

70. Geluk,A. et al. Identification and characterization of the ESAT-6 homologue of Mycobacterium leprae and T-cell cross-reactivity with Mycobacterium tuberculosis. Infect. Immun. 70, 2544-2548 (2002).

71. Stinear,T.P. et al. Reductive evolution and niche adaptation inferred from the genome of Mycobacterium ulcerans, the causative agent of Buruli ulcer. Genome Res. 17, 192-200 (2007).

72. Brodin,P. et al. Bacterial artificial chromosome-based comparative genomic analysis identifies Mycobacterium microti as a natural ESAT-6 deletion mutant. Infect. Immun. 70, 5568-5578 (2002).

73. Volkman,H.E. et al. Tuberculous granuloma formation is enhanced by a mycobacterium virulence determinant. PLoS. Biol. 2, e367 (2004).

74. Stamm,L.M. et al. Mycobacterium marinum escapes from phagosomes and is propelled by actin-based motility. Journal of Experimental Medicine 198, 1361-1368 (2003).

75. van der Wel,N. et al. M. tuberculosis and M. leprae translocate from the phagolysosome to the cytosol in myeloid cells. Cell 129, 1287-1298 (2007).

https://www.researchgate.net/publication/8150466_Bacillus_subtilis_Operon_Encoding_a_Membrane_Receptor_for_Bacteriophage_SPP1?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=

https://www.researchgate.net/publication/8150466_Bacillus_subtilis_Operon_Encoding_a_Membrane_Receptor_for_Bacteriophage_SPP1?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=




https://www.researchgate.net/publication/10868187_Rodriguez_G_M_Smith_I_Mechanisms_of_iron_regulation_in_mycobacteria_role_in_physiology_and_virulence_Mol_Microbiol_47_1485-1494?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=

https://www.researchgate.net/publication/10868187_Rodriguez_G_M_Smith_I_Mechanisms_of_iron_regulation_in_mycobacteria_role_in_physiology_and_virulence_Mol_Microbiol_47_1485-1494?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=




https://www.researchgate.net/publication/11435054_Betts_JC_Lukey_PT_Robb_LC_McAdam_RA_Duncan_K_Evaluation_of_a_nutrient_starvation_model_of_Mycobacterium_tuberculosis_persistence_by_gene_and_protein_expression_profiling_Mol_Microbiol_43_717-731?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=



https://www.researchgate.net/publication/292767534_Genes_required_for_mycobacterial_growth_defined_by_high_density_mutagenesis?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=

https://www.researchgate.net/publication/292767534_Genes_required_for_mycobacterial_growth_defined_by_high_density_mutagenesis?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=


76. Derrick,S.C. & Morris,S.L. The ESAT6 protein of Mycobacterium tuberculosis

induces apoptosis of macrophages by activating caspase expression. Cell Microbiol. 9, 1547-1555 (2007).

77. de Jonge,M.I. et al. ESAT-6 from Mycobacterium tuberculosis dissociates from its putative chaperone CFP-10 under acidic conditions and exhibits membrane-lysing activity. J. Bacteriol. 189, 6028-6034 (2007).

78. Stanley,S.A., Johndrow,J.E., Manzanillo,P. & Cox,J.S. The Type I IFN response to infection with Mycobacterium tuberculosis requires ESX-1-mediated secretion and contributes to pathogenesis. J. Immunol. 178, 3143-3152 (2007).

79. Lewinsohn,D.M. et al. Secreted proteins from Mycobacterium tuberculosis gain access to the cytosolic MHC class-I antigen-processing pathway. J. Immunol. 177, 437-442 (2006).

80. Natarajan,K., Latchumanan,V.K., Singh,B., Singh,S. & Sharma,P. Down-regulation of T helper 1 responses to mycobacterial antigens due to maturation of dendritic cells by 10-kDa mycobacterium tuberculosis secretory antigen. J. Infect. Dis. 187, 914-928 (2003).

81. Basu,S.K. et al. Mycobacterium tuberculosis secreted antigen (MTSA-10) modulates macrophage function by redox regulation of phosphatases. FEBS J. 273, 5517-5534 (2006).

82. Trajkovic,V., Singh,G., Singh,B., Singh,S. & Sharma,P. Effect of Mycobacterium tuberculosis-specific 10-kilodalton antigen on macrophage release of tumor necrosis factor alpha and nitric oxide. Infect. Immun. 70, 6558-6566 (2002).

83. Olsen,I. et al. Bovine NK cells can produce gamma interferon in response to the secreted mycobacterial proteins ESAT-6 and MPP14 but not in response to MPB70. Infect. Immun. 73, 5628-5635 (2005).

84. Varga,J.J. et al. Type IV pili-dependent gliding motility in the Gram-positive pathogen Clostridium perfringens and other Clostridia. Mol. Microbiol. 62, 680-694 (2006).

85. Van der Auwera,G.A., Andrup,L. & Mahillon,J. Conjugative plasmid pAW63 brings new insights into the genesis of the Bacillus anthracis virulence plasmid pXO2 and of the Bacillus thuringiensis plasmid pBT9727. BMC. Genomics 6, 103 (2005).

86. Abajy,M.Y. et al. A type IV-secretion-like system is required for conjugative DNA transport of broad-host-range plasmid pIP501 in gram-positive bacteria. J. Bacteriol. 189, 2487-2496 (2007).

Mycobacterium marinum can be divided into two distinct types based on genetic diversity and virulence

Astrid M. van der Sar*, Abdallah M. Abdallah*, Marion Sparrius*, Erik Reinders*, Christina M.J.E. Vandenbroucke-Grauls*,‡ and Wilbert Bitter*

*Department of Medical Microbiology and Infection Control, VUmc (Vrije Universiteit Medical Centre), van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands. ‡Department of Medical Microbiology, Academic Medical Center, The Netherlands.

Ch

apter 3

Infection & Immunity 72: 6306-6312, 2004

Chapter 3: Mycobacterium marinum strain variation and virulence 48

Abstract

Mycobacterium marinum causes a systemic tuberculosis-like disease in a large number of poikilothermic animals and is used as a model for mycobacterial pathogenesis. In the present study, we infected zebrafish (Danio rerio) with different strains of M. marinum to determine the variation in pathogenicity. Depending on the M. marinum isolate, the fish developed an acute or chronic disease. Acute disease was characterised by uncontrolled growth of the pathogen and death of all animals within 16 days, whereas chronic disease was characterised by granuloma formation in different organs and survival of the animals for at least 4-8 weeks. Genetic analysis of the isolates by Amplified Fragment Length Polymorphism (AFLP), showed that M. marinum strains could be divided in two clusters. Cluster I contained predominantly strains isolated from humans with fish tank granuloma, whereas the majority of the cluster II strains were isolated from poikilothermic species. Acute disease progression was only noted with strains belonging to cluster I, whereas all chronic disease causing isolates belonged to cluster II. This difference in virulence was also observed in vitro: cluster I isolate Mma20 was able to infect and survive more efficiently in the human macrophage THP-1 and in the carp leukocyte CLC cell line, as compared to the cluster II isolate Mma11. We conclude that strain characteristics play an important role in the pathogenicity of M. marinum. In addition, the correlation between genetic variation and host-origin suggests that cluster I isolates are more pathogenic for humans.

Introduction

Bacterial strains belonging to the Mycobacterium tuberculosis complex show an unusual high degree of conservation, not only in housekeeping genes1 but also in genes encoding (putative) targets of the immune system2. In fact, most genes in strains isolated worldwide show a negligible variation. These data are best explained by assuming that contemporary strains have all originated from a recent common ancestor, which would have lived 10,000 to 20,000 years ago. However, genetic analysis by DNA fingerprinting has revealed that there is genetic variation in M. tuberculosis, and recently it was shown that this diversity correlates markedly with pathogenicity3. The M. tuberculosis Beijing strain, which is highly prevalent in Asia and the former USSR, causes a significantly higher and earlier mortality in mice as compared to the prototype H37Rv strain. This effect was correlated with a non-protective immune response. These results show that also for highly homogeneous strains, genetic variation plays a role in disease development. In the past years, Mycobacterium marinum, the causative agent of fish tuberculosis, was adopted as a model to study mycobacterial infections. There are good reasons for this approach: M. marinum is the mycobacterial species most closely related to members of the M. tuberculosis complex4, it has a relatively short generation time of 4-6 h, as compared to 20 h for M. tuberculosis, and grows optimally at 30 °C and hardly at 37 °C5,6. Because of this optimal growth temperature, M. marinum


infections of humans are found almost exclusively as superficial lesions on the extremities7-9. The histopathology of these M. marinum infections, generally called fish tank granuloma or swimming pool granuloma, show the formation of granulomas that resemble those associated with tuberculosis9-11. Another advantage of M. marinum is that this bacterium is a natural pathogen of poikilothermic species, which provides the opportunity to study infection in a natural host5. Different infection models have been described that use the leopard frog (Rana pipiens)12, the goldfish (Carassius auratus)13 and recently also the genetically tractable zebrafish (Danio rerio)14-16 as a host have been described. Those studies use either the M. marinum M strain, originally isolated from an infected patient, or the M. marinum ATCC927 strain, isolated from fish. In the present study, we analysed the genetic variation between different isolates of M. marinum and their pathogenicity for zebrafish. We observed that strains of M. marinum can be grouped into two clusters based on genetic analysis (amplified fragment length polymorphism 17). Interestingly, representative strains of the first cluster, which consists almost exclusively of M. marinum strains isolated from humans, induced an acutely lethal disease in the zebrafish, whereas strains of the second cluster induced a chronic progressive disease in the zebrafish, characterized by granuloma formation.

Materials and Methods

Bacterial strains and growth conditions

The wild type M. marinum isolates included in this study (table 1) were obtained from the collections of the National Institute of Public Health and the Environment (RIVM) in Bilthoven (The Netherlands), the Institute of Aquaculture in Stirling (United Kingdom), and the Institute for Animal Science and Health (CIDC) in Lelystad (the Netherlands) and were assigned to the species M. marinum by 16S sequencing and growth characteristics, such as photochromogenic behavior. We also included reference strain M, a human isolate13. Bacteria were grown at 30°C in Middlebrook 7H9 medium supplemented with Middlebrook OADC (BD, Biosciences), 0.2% Tween and 1 mg/ml D-arabinose18 to decrease clumping of cells. Prior to inoculation in zebrafish, bacteria were washed three times with phosphate-buffered saline (PBS); bacterial numbers were determined by measuring the optical density at 600 nm and by plating and CFU determination.

Animals

Male zebrafish, Danio rerio, (approximately 1 g, 1 year old), were chosen from our breeding facilities, acclimated for 1 week to their new environment in the infection room and kept at 28°C on a 14/10 hr light/dark rhythm. The infected zebrafish were housed in 10 1itre separate tanks with separate pumps and filter systems (Ecco, Eheim).


Table 1 | Source and origin of the M. marinum strains used in this study.

Strain (original name)

Strain (name present

study)

Source

Reference or source

E6 Sciaenops ocellatus (red drum) 19 E7 Mma7 Chaetodon fasciatus (butterfly fish) 19 E11 Mma11 Dicentrarchus labrax (sea bass) 19 E12 Dicentrarchus labrax (sea bass) 19 E15 Siganus rivulatus (marbled spinefoot) 19 E16 Dicentrarchus labrax (sea bass) 19 420472-4 Mma42 Snake (unknown spp.) CIDCa

551962 Shinisaurus crocodiluris (crocodile lizard) CIDC M strain Human 13 Mis 14 Human RIVMb

14641 Human RIVM 18347 Human RIVM 9800607 Mma98 Human RIVM 9801756 Human RIVM 9900036 Human RIVM 2000-01053 Mma20 Human RIVM 2001-00796 Mma21 Human RIVM

a collection of the Institute for Animal Science and Health (CIDC), Lelystad, the Netherlands. b collection of the National Institute of Public Health and the Environment (RIVM), Bilthoven, The Netherlands.

Infection of the zebrafish

The zebrafish were anaesthetized in a 0.02% aqueous solution of Ethyl 3-aminobenzoate methanesulfonate salt (MS-222) (Sigma) and inoculated intraperitoneally (IP) with 10 µl M. marinum suspension in PBS. Ten zebrafish per group were inoculated with 104 CFU of the M. marinum strains Mma98 and Mma7, 8 zebrafish per group with Mma21 and Mma42 and 15 zebrafish per group were inoculated with the M. marinum strains Mma20 and Mma11. In addition, 10 control fish were injected with 10 µl phosphate-buffered saline (PBS). Viable bacterial counts present in the liver and kidney of three fish inoculated with Mma11 and Mma20 were determined by plating serial 10-fold dilutions of organ homogenates, decontaminated with BBL™ MycoPrep™ (BD, The Netherlands), on Middlebrook 7H10 agar. The colonies were identified as mycobacterial species by morphology and photochromogenic behavior. Counts were performed at 1 day postinfection (dpi), 1 week post infection (1 wpi), 4 wpi and 8 wpi. Two fish inoculated with Mma20, Mma11 or Mma7 were used for histological examination and Ziehl-Neelsen staining at 10 dpi (Mma20), 4 wpi and 8 wpi. All animal


experiments were approved by the local Animal Welfare Committee, under protocol number MM 01-02 and MM 03-02. Zebrafish pathology and tissue processing

The zebrafish were observed for gross signs of infection and were sacrificed upon moribund behavior. For histological examination, zebrafish were sacrificed by incubation in an overdose of MS-222 (A-5040, Sigma), fixed in Bouin (5 ml Formaldehyde 40%, 15 ml water saturated Picric acid, 1 ml Acetic acid), and processed for paraffin embedding. Frontal sections (4-7 µm) were stained with hematoxylin and eosin (HE) or according to the method of Ziehl-Neelsen and observed under a Zeiss Axioskop light microscopy. Photographs were taken with a Nikon Coolpix 900 camera and processed using Adobe Photoshop software, version 6.0. Amplified Fragment Length Polymorphism (AFLP) analysis

Bacterial strains were incubated with protein K for 60 min at room temperature prior to DNA isolation with the DNeasy® Tissue Kit (Qiagen). AFLP was performed essentially as described previously, with EcoRI and MseI as restriction enzymes and the primers EcoA and MseC 17,20,21. AFLP fragments were separated on an ABI Prism Genetic Analyser 3100 (Applied Biosystems). Data were analysed by Pearson correlation and clustered by Unweighted Pair Group Matrix Analysis (UPGMA) with Bionumerics software, version 3.0 (Applied Maths).

crtB sequence analysis

In order to determine the nucleotide sequence of the crtB gene, this gene was first amplified by PCR on the various chromosomal DNA preparations and crtB specific primers (wbcrtBF: TCGACCTGAAAGCACAGTTG and wbcrtBR: AGTCTTCAATCGGGATGTCG). Subsequently, the PCR product was purified with a PCR purification column (Qiagen) and used in a sequence reaction with one of the above primers. The different elongation products were separated on an ABI Prism Genetic Analyser 3100 (Applied Biosystems).

Cell lines and culture conditions

The human acute monocytic leukemia cell line (THP-1) was cultured in RPMI-1640 media (GIBCO, BRL) with 10% fetal calf serum (FCS) at 37°C in 5% CO2. The adherent carp monocytic /macrophage cell line (CLC) was obtained through the European Collection of Cell Culture, Salisbury, United Kingdom (ECACC NO: 95070628;) and was maintained at 28°C and 5% CO2 in RPMI-1640 media supplemented with 10% FCS.


Intracellular Survival Assays

Cellular infection assays were carried out in 24-well tissue culture plates (Costar) as previously described22,23. To differentiate the THP-1 cells into macrophage-like cells, the cells were treated with phorbol myristate acetate (PMA) (Sigma). THP-1 cells were harvested by centrifugation for 9 minutes at 1200 rpm, and the pellet was suspended in 1 ng PMA/ml RPMI-1640/10% FCS to a cell density of approximately 106 THP-1 cells/ml. One ml of cell suspension was added to each well of a 24 well plate. The plate was incubated for 24 hours at 37°C in 5% CO2. The medium was removed from each well, adherent cells were washed once with RPMI-1640/10% FCS, refreshed with new RPMI-1640/10% FCS and incubated for an additional 24 hours. CLC cells were seeded at a density of 106 cells per well 24 hours prior to use. Immediately before infection, cells were washed once with fresh RPMI-1640/10% FCS. Bacteria were harvested by centrifugation for 5 minutes at 5000 rpm and washed twice with RPMI-1640/10% FCS medium. The bacteria were suspended in RPMI-1640/10% FCS at a concentration to achieve a multiplicity of infection (MOI) of 10 for Mma11 and 1 for Mma20. Bacteria and cells were incubated for 1 hour at 33°C for THP-1 and at 28°C for CLC. Cells were then washed twice with RPMI-1640/10% FCS medium to remove free bacteria and incubated in fresh medium with amikacin (200 µg/ml; Sigma Chemical) at the appropriate temperature. After 2 hrs, the medium was replaced by medium with 30 µg/ml amikacin. The cells were incubated at the appropriate temperature and then lysed at different time points with 1 ml of 0.1% (v/v) Triton X-100 in phosphate-buffer saline (PBS). One well was processed immediately (time = 0) for determination of initial bacterial counts. Each lysate was diluted as necessary, and portions were plated on 7H10 agar plates. Survival was expressed as the percentage of CFU at each time point, with the number of CFU at time zero as reference.

Results

Different M. marinum strains cause different forms of disease

To study possible differences in pathogenicity between different M. marinum strains in zebrafish, groups of 10 or 15 zebrafish were inoculated intraperitoneally with 104 CFU of M. marinum. Four different strains were used: Mma7, Mma11, Mma20 and Mma98. The latter two strains were originally isolated from skin lesions of patients with fish tank granuloma, whereas Mma11 and Mma7 were isolated from infected fish. Almost all zebrafish inoculated with Mma7 or Mma11 survived up to the end of the experiment, at 56 days post infection (Fig. 1). However, differences were observed in external signs of disease. The zebrafish inoculated with Mma7 showed no signs of infection for 8 weeks, while three of the zebrafish infected with Mma11 showed skin lesions (ulcerations) after approximately 45 days (Fig. 2). Notably, these skin lesions were not at the site of the injection, which shows that the infection was disseminated. Furthermore, these


overtly ill animals also showed buoyancy problems for a short period of time (1-2 days) before they were sacrificed.

Days Post Infection

% S

urv

ival

Figure 1 | Survival curve of zebrafish infected intraperitoneally with 104 CFU of Mma7 (□), Mma11 (∆), Mma98 (▲) and Mma20 (■), Mma21 (○) and Mma42 (●) or treated with PBS (x). All the zebrafish inoculated with 104 bacteria of Mma20 and Mma98 developed acute symptoms, including haemorrhages and inflammation at the site of infection, and all infected fish died or were sacrificed in a moribund state between 5 to 16 days post infection (Fig. 1). To examine whether this effect was dose dependent, six zebrafish were infected with 102 CFU of Mma20. Half of these zebrafish also displayed acute disease symptoms and had to be sacrificed within 16 days (results not shown), which might indicate that the acute disease symptoms are not highly dose dependent. Together, these results show that M. marinum causes an acute or chronic infection in a strain dependent manner. A B

Figure 2 | Macroscopic characteristics of M. marinum chronic (A) and acute (B) infections. (A) Intraperitoneal infection with strain Mma11 induces skin ulcerations at 7 wpi (arrows). These ulcerations were usually not located at the site of infection. (B) Intraperitoneal infection with strain Mma20 causes a swelling of the abdomen and severe haemorrhages within 2 wpi (arrow and white dashed line). No ulcerations were observed in control animals.


M. marinum recovery from zebrafish organs

To assess the ability of the different M. marinum strains to persist and replicate in host tissue, fish were inoculated with 104 CFU of Mma11 or Mma20. Three fish were sacrificed at 1 and 7 dpi, 4 and 8 wpi and the liver and the posterior kidney were collected for bacteriological examination. From 7 dpi onwards all tested organs of both groups were colonized, which showed that the bacteria were disseminated. However, a significant difference in the bacterial load between organs derived from zebrafish infected with Mma20 and Mma11 was observed. Whereas livers from zebrafish inoculated with Mma11 contained only a small number of bacteria at 7dpi, livers from zebrafish inoculated with Mma20 contained as much as 105 CFU (Fig. 3). Since the growth rates of both strains in vitro are similar, these data suggest that Mma20 is able to survive and/or replicate better in the zebrafish than Mma11. Probably the high bacterial load caused the early death of the zebrafish. Upon prolonged infection, the number of Mma11 bacteria increased steadily and reached numbers of the same order of magnitude as the numbers of Mma20 bacteria at 7 dpi (Fig. 3). As can be seen in Figure 3 the bacterial numbers recovered from fish infected with Mma11 varied markedly, but the numbers of CFU recovered from liver and posterior kidney of the same fish were always comparable. This probably means that the onset and progression of disease in Mma11 was highly variable. We observed the same phenomenon upon prolonged incubation (6 months) of zebrafish infected with Mma11. The onset of overt signs of disease varied between 7 weeks and 6 months.

Figure 3 | Total CFU counts per homogenised liver and posterior kidney from zebrafish inoculated with 104 bacilli of M. marinum strain Mma11 or strain Mma20. The mean of three samples per time point per group is shown. The error bars represent the standard errors of the means. Bacterial numbers isolated from Mma20 liver (▲), Mma20 posterior kidney (∆), Mma11 liver (■) and Mma11 posterior kidney (□) are shown.


Histology of M. marinum infection

To determine the nature of the pathological response to M. marinum, zebrafish infected with Mma20, Mma11 and Mma7 were sacrificed and fixed in Bouin. Paraffin sections were made and slides were alternately stained with Ziehl-Neelsen or HE. Microscopic examination of the sections revealed that moribund fish infected with Mma20 contained high numbers of mycobacteria at 5 dpi (Fig. 4A, D). The peritoneum and the surrounding tissues showed severe necrosis, loss of structural organization and the influx of large numbers of inflammatory cells. This finding also suggests that the early death of the zebrafish was caused by the outgrowth of bacteria to very high numbers, which caused severe peritonitis. Also high numbers of bacteria other than mycobacteria could be seen in infected fish tissue.

D E F

CBA

Figure 4 | ZN-stained sections of zebrafish infected with Mma20 (A and D)and E), or Mma7 (C and F). (A and D) Section of Mma20 infected zebrafisConsiderable tissue damage and many mycobacteria can be observed. The myconot organized in granuloma. (D) A 1,000x magnification showing themycobacteria. (B and E) Section of Mma11- infected zebrafish at 8 wpi. organized granuloma with a necrotic centre can be observed in the pancreas. magnification, some mycobacteria in the centre of the granuloma can be seenSection of Mma7-infected zebrafish at 8 wpi. (C) Less-well organized gramycobacteria not organized in granulomas can be seen. (F) At high mmycobacteria are found in the centers of the granulomas. (A, B, and C) Ba100µm; (D, E, and F) bars represent 10µm. In contrast to Mma20 infected fish, zebrafish infected with Mma11 contorganized granulomas in the liver, pancreas, kidney and intestines Sometimes granulomas were located in the connective tissues. H

, Mh 5ba i(B)(E. (

nulagnrs

ainatis

ma11 (B dpi. (A) cteria are ndividual A well-) At high C and F) oma and ification, represent

ed well- 4 wpi. tological


examination showed that most granulomas consisted of an outer layer of tightly packed epithelial cells and a central region containing mainly macrophages and possibly some granulocytes and lymphocytes. In addition, the central region of most granulomas showed necrosis and the presence of mycobacteria. The number of granulomas and their size seemed to increase over time, as observed at 8 wpi (Fig. 4B, E). In contrast to Mma11 infected fish, only one of the two zebrafish infected with Mma7 displayed granulomas at 4 wpi and 8 wpi (Fig. 4C, E). The morphology of the granulomas was slightly different from that of the granulomas in Mma11-infected zebrafish. Mma7 induced only fewer and less organized granulomas, sometimes they contained an outer layer of epithelial cells and a necrotic center. In addition, single M. marinum cells of Mma7 were observed outside granuloma, free in the tissues (Fig. 4C). Neither bacteria nor granulomas were observed in sections of the second zebrafish infected with Mma7 at 4 wpi, but sections of the zebrafish at 8 wpi contained individual mycobacteria (i.e. not grouped in clusters).

AFLP analysis of M. marinum; two large clusters correlated with pathogenic properties

Large differences in disease progression were observed for the 4 M. marinum strains analyzed. To determine the genetic relationships between the acute and chronic disease causing strains, DNA fingerprint by AFLP were performed. For this analysis we used chromosomal DNA of 17 different M. marinum isolates, including the strains used in the infection experiments (Table 1, Fig. 5). Following analysis by Pearson correlation coefficient and Unweighted Pair Group Matrix Analyses, the 17 strains were grouped in two AFLP clusters at a delineation level of 60% (Fig. 5). Interestingly, the composition of these two clusters roughly corresponded to the origin of isolation of the M. marinum strains, i.e. cluster I contained 7 out of the 9 human isolates, whereas cluster II contained 6 of the 8 isolates from poikilothermic animals, respectively. This could indicate that cluster I isolates are more pathogenic for humans. To substantiate this result, the AFLP analysis was extended with an extra 8 human isolates, which all clustered in cluster I (results not shown). Both the M. marinum strains responsible for acute disease in zebrafish, i.e. Mma20 and Mma98, also fell in cluster I, whereas both chronic disease-causing strains fell in cluster II. This could mean that M. marinum strains belonging to cluster I provoke an acute disease in zebrafish, or that M. marinum strains that cause disease in humans are responsible for this. To test these assumptions, zebrafish were inoculated with Mma21, a strain that was isolated from a human infection case, but that fell in cluster II upon AFLP fingerprinting, and Mma42, a strain that clustered in group I but which was isolated from a reptile. Infection with Mma21 resulted in a more chronic infection while Mma42 caused an acute infection (Fig. 1), which suggests that M. marinum strains belonging to cluster I, irrespective of their origin, give rise to acute lethal infections in zebrafish.

57 Chapter 3: Mycobacterium marinum strain variation and virulence

II

I

20

40

60

80

100

M-strain 551962 Mis14 1484Mma20

Mma41834Mma990003E 12 E 15 E 16 Mma11Mma2E 6 Mma7 M. tub

Figure 5 | AFLP-DNA fingerprint. Numerical analysis of normalized AFLP band patterns generated from chromosomal DNA of M. marinum isolates and M. tuberculosis H37Rv as outgroup. M. marinum strains isolated from humans are printed in bold italics. The dendrogram was constructed using the Unweighted Pair Group Matrix Analyses. The clusters representing the human isolates (cluster I) and the poikilothermic animal isolates (cluster II) were defined at a delineation level of 60%. The grey error flags at each branch shows the standard deviation of the average similarity at this position.

By multilocus sequence typing it has been shown previously24 that M. marinum strains can be divided in two clusters. The same study also showed that one of these clusters is in fact closely linked to the human pathogen Mycobacterium ulcerans, the causative agent of Buruli ulcer. To determine if the observed subdivision of our set of M. marinum strains, based on AFLP analysis, is consistent with that of Stinear et al. (2000), we sequenced the crtB gene. This gene is the most differentiating gene in the multilocus sequence typing. We analyzed 10 strains and found consistent clustering of the strains by AFLP analysis and by crtB sequence analysis. Furthermore, the AFLP cluster I, which contained most human isolates, was identical to the cluster closely linked with M. ulcerans in the study by Stinear and colleagues24. Cell culture

From the in vivo infection studies it is clear that at 7 dpi there is a major difference between the bacterial load of fish infected with Mma20 or Mma11 (Fig. 3). To test whether this might be attributed to differences in intracellular survival and/or persistence of the M. marinum strains, the human THP-1 cell line was infected with Mma20 or Mma11 and the intracellular survival of the bacteria was monitored over time (Fig. 6). Already at 4 hours of incubation with THP-1 cells a decrease in Mma11 CFU was observed. This number showed a steady decline, and at 120 h postinfection hardly any Mma11 could be cultured from these cells. In contrast, bacteria of strain Mma20 were able to survive intracellularly for a long

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time in THP-1 cells, since the number of cells did not change substantially over 120 hours. M. marinum strain M, which is used to determine the genome sequence, belongs to cluster I (Fig. 5) and is also able to maintain itself in the human macrophage cell line THP-1, similar to Mma20 (results not shown). In a second experiment, the in vitro growth of these two M. marinum strains in carp leukocytes (CLC line) was determined. This cell line can be maintained at 28°C, the optimal growth temperature of M. marinum, and has been shown previously to be a useful model for M. marinum intracellular growth and survival 20. In contrast to the results obtained with the THP-1 cells, Mma11 was able to maintain itself in these CLCs (Fig. 6). However, also in this experiment there was a clear difference in outgrowth of Mma20 as compared to Mma11 (Fig. 6). This showed that strain Mma20 is able to survive and/or replicate more efficiently in macrophages, which might explain the increased virulence of this strain.

Figure 6 | Survival of Mma11 (♦) and Mma20 (■) in the human macrophage cell line THP-1 (A) and the carp leukocyte cell line CLC (B). Bacteria and cells were incubated at 33°C for THP-1 and at 28°C for CLC. After a 1-h coincubation of cells and bacteria, the cells were washed, treated with 200 µg of amikacin per ml, and incubated in medium with 30 µg of amikacin per ml at the appropriate temperature. At different time points, samples were taken for CFU determination. Survival was expressed as the percentage of CFU at each time point, with the number of CFU at time zero as a reference. The means from three experiments are shown.


Discussion

Since the 1920-ties, M. marinum has been known as the causative agent of fish tuberculosis in poikilothermic species and fish tank granuloma or swimming pool granuloma in humans. Strain variation with respect to virulence was, however, never investigated. In the present study we demonstrate that different M. marinum strains showed marked differences in pathogenicity. These differences correlated with the origin of the isolates (from infected humans or infected fish) and with genetic clustering. Indeed, by AFLP fingerprinting, two clusters, designated cluster I and II, could be recognized, with human isolates falling predominantly in cluster I and isolates from poikilothermic species falling predominantly in cluster II. In addition, we confirm that the zebrafish is a useful animal model to study mycobacterial infection15. Depending on the M. marinum strain used, the zebrafish developed an acute or a chronic infection. The acute symptoms were not expected, since the relative inoculum was known to result in a chronic disease in goldfish13. Zebrafish with an acute diseased suffered from loss of equilibrium, swelling and haemorrhage at the site of infection, hung at the bottom of the tank and did not eat. Histological examination showed a massive amount of acid-fast rods at the site of infection and a severe peritonitis. The fish with chronic infections showed signs typical of fish tuberculosis, i.e. a systemic spread of the infection, granuloma formation in different organs, shedding of scales and skin lesions. The differences in disease progression that we observed in fish inoculated with strain Mma20 (a human isolate) compared to those inoculated with strain Mma11 (a fish isolate) were not merely an effect of inoculum size, since strain Mma20 also caused acute infection in 50% of the fish when a 100 times lower inoculum was used (102 CFU versus 104 CFU) (data not shown). Since the zebrafish infected with the human isolates had to be sacrificed earlier than originally planned, due to the unexpected fast progression of the infection, we could isolate organs of these zebrafish only at 1dpi and 7 dpi. At day 7 only the fish infected with the human isolate Mma20 showed a large increase in bacterial numbers in the liver and the posterior kidney. These results were substantiated in experiments with both human THP-1 and carp CLC cells: cluster I isolate Mma20 infected and survived better in macrophages than cluster II strain Mma11. The striking difference in disease characteristics between the different M. marinum isolates correlated with genetic differences, as was determined by AFLP analysis. This analysis showed that all isolates grouped in two clusters, I and II. All strains causing acute disease belonged to cluster I. Surprisingly; most strains of this cluster were human isolates, which raised the hypothesis whether passage through a human host would result in an increase in poikilothermic virulence. However, also the cluster I snake isolate Mma42 induced an acute lethal disease, which indicates that the genetic background is important and not the human passage. Of course human infection is caused by isolates that are transmitted to humans from fish or other poikilothermic species, but our results suggests that strains that


have the potential also to induce infection in humans, differ genetically from strains that only cause infection in fish. The human isolates were collected during the last decade from patients admitted to Dutch hospitals, therefore these strains were isolated from patients with severe infections, in need of medical attention. The finding that these human isolates are strongly over-represented in cluster I indicates that strains of cluster I more frequently give rise to more severe and persistent human infections. This observation, combined with the evidence that these strains show enhanced survival both in human and in fish cell lines, suggests that cluster I forms a subspecies of M. marinum with increased pathogenicity for humans and zebrafish. Cluster I is genetically more closely related to the human pathogen M. ulcerans as compared to cluster II24. However, the implication of this relationship is at present unclear. The observed differences in M. marinum virulence are in contrast with previous studies, which mention briefly that there were no differences in disease outcome when different strains of M. marinum were used to infect the leopard frog or the goldfish12,13. The difference between these studies and our report is probably not due to the use of different M. marinum strains, since these studies also report the use of several strains, derived both from humans and from fish and frogs. The observed difference might be due to the choice of host organism; the leopard frog has been shown to be relatively resistant to M. marinum, with stable bacterial loads and non-caseous granulomas12. On the other hand, the link with the human isolates indicates that the difference in virulence can be seen in widely different species. The finding that M. marinum strains cluster in two major groups, with one cluster containing strains pathogenic for humans and zebrafish, allows us to use the zebrafish model to identify mycobacterial virulence factors important for survival and persistence in fish and in humans.

Acknowledgements

The authors thank Monique Raats and Wim Schouten for their expert technical assistance, Ben Appelmelk for helpful discussions and Sandrine Florquin (AMC, Amsterdam, The Netherlands) for her assistance with the interpretation of the histopathology. We thank Peter Willemsen (CIDC, Lelystad, the -Netherlands), Kim Thompson (The Institute of Aquaculture, Stirling, United Kingdom), Dick van Soolingen and Tridia van der Laan (RIVM, Bilthoven, The Netherlands) for the field isolates of Mycobacterium marinum. This research was supported in part by grant 050-71-001 from the Netherlands Genomics Initiative (NROG).


References

1. Sreevatsan,S. et al. Restricted structural gene polymorphism in the Mycobacterium tuberculosis complex indicates evolutionarily recent global dissemination. Proc. Natl. Acad. Sci. U. S. A 94, 9869-9874 (1997).

2. Musser,J.M., Amin,A. & Ramaswamy,S. Negligible genetic diversity of mycobacterium tuberculosis host immune system protein targets: evidence of limited selective pressure. Genetics 155, 7-16 (2000).

3. Lopez,B. et al. A marked difference in pathogenesis and immune response induced by different Mycobacterium tuberculosis genotypes. Clin. Exp. Immunol. 133, 30-37 (2003).

4. Rogall,T., Wolters,J., Flohr,T. & Bottger,E.C. Towards a phylogeny and definition of species at the molecular level within the genus Mycobacterium. Int J. Syst. Bacteriol. 40, 323-330 (1990).


6. Aronson,J.D. Spontaneous tuberculosis in saltwater fish. J. of Infect. Dis. 39, 315-320 (1926).

7. Bailey,J.P., Jr. et al. Mycobacterium marinum infection. A fishy story. JAMA 247, 1314 (1982).

8. Clark,R.B. et al. Osteomyelitis and synovitis produced by Mycobacterium marinum in a fisherman. J. Clin. Microbiol. 28, 2570-2572 (1990).

9. Collins,C.H., Grange,J.M., Noble,W.C. & Yates,M.D. Mycobacterium marinum infections in man. J. Hyg. (Lond) 94, 135-149 (1985).

10. Huminer,D. et al. Aquarium-borne Mycobacterium marinum skin infection. Report of a case and review of the literature. Arch. Dermatol. 122, 698-703 (1986).

11. Vanduijn,C. Tuberculosis in fishes. Small Animal Prac. 22, 391-411 (1981). 12. Ramakrishnan,L., Valdivia,R.H., McKerrow,J.H. & Falkow,S. Mycobacterium

marinum causes both long-term subclinical infection and acute disease in the leopard frog (Rana pipiens). Infect. Immun. 65, 767-773 (1997).

13. Talaat,A.M., Reimschuessel,R., Wasserman,S.S. & Trucksis,M. Goldfish, Carassius auratus, a novel animal model for the study of Mycobacterium marinum pathogenesis. Infect. Immun. 66, 2938-2942 (1998).

14. Davis,J.M. et al. Real-time visualization of mycobacterium-macrophage interactions leading to initiation of granuloma formation in zebrafish embryos. Immunity. 17, 693-702 (2002).

15. Prouty,M.G., Correa,N.E., Barker,L.P., Jagadeeswaran,P. & Klose,K.E. Zebrafish-Mycobacterium marinum model for mycobacterial pathogenesis. FEMS Microbiol. Lett. 225, 177-182 (2003).

16. van der Sar,A.M., Appelmelk,B.J., Vandenbroucke-Grauls,C.M. & Bitter,W. A star with stripes: zebrafish as an infection model. Trends Microbiol. 12, 451-457 (2004).

17. Vos,P. et al. AFLP: a new technique for DNA fingerprinting. Nucleic Acids Res. 23, 4407-4414 (1995).

18. Anton,V., Rouge,P. & Daffe,M. Identification of the sugars involved in mycobacterial cell aggregation. FEMS Microbiol. Lett. 144, 167-170 (1996).

19. Puttinaowarat,S., K.Thompson, J.Lilley & A.Adams. Characterization of Mycobacterium spp isolated from fish by pyrolysis mass spectrometry (PyMS) analysis. AAHRI Newslett. 8, 4-8 (1999).

https://www.researchgate.net/publication/20879931_Towards_a_Phylogeny_and_Definition_of_Species_at_the_Molecular_Level_within_the_Genus_Mycobacterium?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=



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https://www.researchgate.net/publication/8334139_A_star_with_stripes_zebrafish_as_an_infection_model_Trends_Microbiol?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=

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https://www.researchgate.net/publication/12527748_Musser_JM_Amin_A_Ramaswamy_S_Negligible_genetic_diversity_of_Mycobacterium_tuberculosis_host_immune_system_protein_targets_evidence_of_limited_selective_pressure_Genetics_155_7-16?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=



https://www.researchgate.net/publication/271081594_Aquarium-borne_Mycobacterium_marinum_skin_infection_Report_of_a_case_and_review_of_the_literature?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=

https://www.researchgate.net/publication/271081594_Aquarium-borne_Mycobacterium_marinum_skin_infection_Report_of_a_case_and_review_of_the_literature?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=

https://www.researchgate.net/publication/14199999_Mycobacterium_marinum_causes_both_long-term_subclinical_infection_and_acute_disease_in_the_leopard_frog_Rana_pipiens?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=



https://www.researchgate.net/publication/278664823_Mycobacterium_marinum_infection_A_fishy_story?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=

https://www.researchgate.net/publication/278664823_Mycobacterium_marinum_infection_A_fishy_story?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=

https://www.researchgate.net/publication/285168040_Spontaneous_Tuberculosis_in_Salt_Water_Fish?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=

https://www.researchgate.net/publication/285168040_Spontaneous_Tuberculosis_in_Salt_Water_Fish?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=


20. Koeleman,J.G., Stoof,J., Biesmans,D.J., Savelkoul,P.H. & Vandenbroucke-

Grauls,C.M. Comparison of amplified ribosomal DNA restriction analysis, random amplified polymorphic DNA analysis, and amplified fragment length polymorphism fingerprinting for identification of Acinetobacter genomic species and typing of Acinetobacter baumannii. J. Clin. Microbiol. 36, 2522-2529 (1998).

21. Savelkoul,P.H. et al. Amplified-fragment length polymorphism analysis: the state of an art. J. Clin. Microbiol. 37, 3083-3091 (1999).

22. El Etr,S.H., Yan,L. & Cirillo,J.D. Fish monocytes as a model for mycobacterial host-pathogen interactions. Infect. Immun. 69, 7310-7317 (2001).

23. Miller,B.H. & Shinnick,T.M. Evaluation of Mycobacterium tuberculosis genes involved in resistance to killing by human macrophages. Infect. Immun. 68, 387-390 (2000).

24. Stinear,T.P., Jenkin,G.A., Johnson,P.D. & Davies,J.K. Comparative genetic analysis of Mycobacterium ulcerans and Mycobacterium marinum reveals evidence of recent divergence. J. Bacteriol. 182, 6322-6330 (2000).

A specific secretion system mediates PPE41 transport in pathogenic Mycobacteria

Abdallah M. Abdallah*, Theo Verboom*, Fredericke Hannes*, Mohamad Safi*, Michael Strong‡, David Eisenberg‡, René J.P. Musters§, Christina M.J.E. Vandenbroucke-Grauls*, Ben J. Appelmelk*, Joen Luirink¶ and Wilbert Bitter*

*Department of Medical Microbiology and Infection Control, VU medical centre, Amsterdam, The Netherlands. ‡UCLA-DOE Institute for Genomics and Proteomics, University of California, Los Angeles, USA. §Department of Physiology, VU medical centre, Amsterdam, The Netherlands, ¶Department of Molecular Microbiology, Vrije Universiteit, The Netherlands.

Ch

apter 4

Molecular Microbiology 62: 667-679, 2006

Chapter 4: ESX-5 secretion system identification 64

Abstract

Mycobacterial genomes contain two unique gene families, the so-called PE and PPE gene families, which are highly expanded in the pathogenic members of this genus. Here we report that one of the PPE proteins, i.e. PPE41, is secreted by pathogenic mycobacteria, both in culture and in infected macrophages. Since PPE41 lacks a signal sequence a dedicated secretion system must be involved. A single gene was identified in Mycobacterium marinum that showed strongly reduced PPE41 secretion. This gene was located in a gene cluster whose predicted proteins encode components of an ESAT-6-like secretion system. This cluster, designated ESX-5, is conserved in various pathogenic mycobacteria, but not in the saprophytic species Mycobacterium smegmatis. Therefore, different regions of this cluster were introduced in M. smegmatis. Only introduction of the complete ESX-5 locus resulted in efficient secretion of heterologously expressed PPE41. This PPE secretion system is also involved in the virulence of pathogenic mycobacteria, since the ESX-5 mutant of M. marinum was affected in spreading to uninfected macrophages.

Introduction

Mycobacterium tuberculosis is the causative agent of tuberculosis, a chronic infectious disease that is responsible for the death of over two million people each year. One of the major surprises of the M. tuberculosis genome sequence was that almost 10% of its coding capacity (167 genes) is devoted to two new gene families, the PE and PPE genes, named for the Proline and Glutamic acid (PE) and Pro–Pro–Glu (PPE) motifs near the N terminus of their gene-products1. In addition to these PE and PPE motifs, the family members share homologous N-terminal domains of approximately 110 amino acids for PE proteins and 180 amino acids for PPE proteins. Many PE and PPE proteins are composed only of these homologous domains, whereas other members have an additional C-terminal segment of variable length. These additional segments are often composed of multiple copies of polymorphic repetitive sequences, which led to the hypothesis that these proteins have a structural role1,2. PE and PPE genes have not been identified yet in any non-mycobacterial species, not even in closely related bacteria such as Nocardia farcinica3. Surprisingly, although PPE genes are widely present in pathogenic mycobacteria, such as Mycobacterium avium and Mycobacterium marinum, the non-pathogenic species Mycobacterium smegmatis shows a conspicuous lack of these genes. The M. smegmatis genome, which is more than 50% larger than that of M. tuberculosis, contains only 2 putative PPE genes (http://www.tigr.org/tigr-scripts/CMR2/gene_attribute_form .dbi). Apparently, there is a strong selection for PPE proteins in pathogenic mycobacteria. The function of these proteins is still an enigma, but the available data suggest that PPE proteins are located at the cell surface4,5 and that they are involved in virulence6. Since PPE proteins lack a distinguishable signal sequence

http://www.tigr.org/tigr-scripts/CMR2/gene_attribute_form


we reasoned that, if these proteins are indeed surface exposed, a dedicated secretion system must be present. Therefore, we analysed the expression and localization of PPE41, a small and soluble PPE protein. This hydrophilic protein, which is encoded by the M. tuberculosis Rv2430c gene, has been shown to induce a strong B-cell response in humans7. Rv2430c forms together with the PE25 encoding gene Rv2431c an operon8 and recently the structure of the heterodimeric complex formed by PPE41 and PE25 has been determined9. In this study we show that PPE41 is secreted by the pathogenic species Mycobacterium marinum and Mycobacterium bovis, but not by Mycobacterium smegmatis. Furthermore, the secretion system involved in this process has been identified and belongs to the class of ESAT-6-like secretion systems.



Three different Mycobacterial species were used in this study: M. marinum strain M10, M. smegmatis strain mc215511 and Mycobacterium bovis BCG Copenhagen. Mycobacteria were grown in shaking cultures in (i) Middlebrook 7H9 liquid medium, supplemented either with Middlebrook ADC (BD, Biosciences) and 0.2% Tween or (ii) modified Sauton's medium, enriched with 0.5% sodium pyruvate and 0.5% glucose. For secretion in M. bovis BCG and M. marinum 7H9 medium was used, supplemented with 0.2% (w/v) dextrose, 0.2% Tween and 0.1% or 0.01% of the advised amount of ADC supplement, respectively. The presence of BSA in the medium (part of the ADC supplement) is essential for secretion in these two species. For secretion in M. smegmatis we used the pre-culture method described by Converse and Cox12. As a solid medium Middlebrook 7H10 plates supplemented with OADC (BD, Biosciences) were used. Both M. smegmatis and BCG were grown at 37°C, whereas M. marinum was grown at 30°C. If M. marinum cells were cultured for electroporation experiments, 2.5 mg/ml glycine was added to the media once the culture reached an optical density (600 nm) of 0.5, to increase the electroporation efficiency (David Lakey, personal communication). For cloning experiments, Escherichia coli strain DH5α was used. Hygromycin, chloramphenicol and kanamycin were used at final concentrations of 50 µg/ml, 30 µg/ml and 25 µg/ml, respectively, both for E. coli and mycobacteria, and gentamicin was used at a final concentration of 5 µg/ml for M. smegmatis and 10 µg/ml for E. coli.

Expression of Rv2430c/Rv2431c in M. smegmatis and M. marinum

H37Rv genomic DNA was used as a template to amplify the Rv2431c/Rv2430c genes with Expand polymerase (Boehringer) using the primers Rv2430cR (GACACGAAATCCGCAGGTAT) and Rv2431cF (CTCATCTGTCACGAGCC GTA). The resulting PCR product was cloned in pUC18 digested with SmaI, which


resulted in pUC18-30c/31cL and pUC18-30c/31cR. Subsequently, the operon was cloned in pSMT3-eGFP13 and placed under the control of the Hsp60 promoter, resulting in pSMT30/31c. The PPE41 encoding gene Rv2430c was also inserted without the Rv2431c gene in pSMT3eGFP by digesting the pUC18-30c/31cL plasmid with HpaI, which overlaps the stop codon of Rv2431c and HindIII. As a second construction vector pBH10 was used, which is compatible with pAL5000 derivatives such as pSMT3. pBH10 is a derivate of pBP1014 that contains the Hsp60 promoter of pSMT3-eGFP by cloning a XbaI and BamHI fragment of pSMT3 in SpeI and BamHI-digested pBP10. Subsequently, the putative Rv2431c/30c operon was cloned in pBH10 using BamHI/PstI, resulting in plasmid pBH30c/31c. A chloramphenicol-resistant version of this plasmid, designated pBH30/31cat, was produced using pEMCat. pEMcat contains the chloramphenicol acetyl transferase encoding gene (cat) of pACYC184, cloned as a Sau3A fragment in BamHI digested pEM3715. This places the cat gene under control of the mycobacterial Ag85 promoter. pBH30/31cat was created by cloning the cat-containing Ecl136II-EcoRV fragment of pEMCat in pBH30/31c restricted with EcoRV. For reconstitution of the secretion system in M. smegmatis an integration construct expressing Rv2430c/Rv2431c was used. This construct was created by isolating the integration region of plasmid pUC-Gm-Int plasmid16 as a HindIII fragment and clone it into pBH30c/31c, thereby deleting the OriM. The resulting plasmid is designated pBH30c/31c-Int. Gentamicin-resistant colonies of M. smegmatis were analysed for correct genome insertion by PCR, using primers specific for the Rv2431c/Rv2430c operon and primers surrounding the integration site, i.e. wbINTgen (CTACCAAGCTGCGCTACA CC) and wbINTpls (TCGTTTGTCAGCATC GAAAG).

Polyclonal antiserum directed against Rv2430c

Rv2431c and Rv2430c were expressed together in E. coli with an optimised translation initiation site and a His-tag at the C-terminal end of Rv2430c9. Rv2430c was purified from disrupted E. coli cells and the Rv2431c protein co-purified. This purified material was used to raise polyclonal antibodies. Two rabbits were immunized subcutaneously (s.c.) with 0.4 ml Rv2430c/Rv2431c preparation (containing 100 µg protein) 4:5 diluted in the mineral oil-based adjuvant Stimune (Cedi Diagnostics BV, Lelystad NL) and s.c. booster-immunized after 4 weeks with 0.2 ml plain antigen. The resulting antisera were analyzed for activity against the purified proteins, which showed only a response against PPE41.

SDS-PAGE and immunoblot

Mycobacteria were grown to mid-logarithmic phase. Subsequently, secreted proteins were precipitated from cell-free supernatant with 5% TCA (w/v). The cell pellets were resuspended in PBS and disrupted with a mini-BeadBeater (BioSpec Products) using 0,1 mm zirconia/silica beads. Cell lysates and supernatants were


separated by SDS/PAGE on 12% polyacrylamide gels. For the treatment of intact cells with protease bacteria were grown to midlog phase and isolated by centrifugation. Cells were resuspended in water and left untreated or incubated with 0.1 mg ml-1 proteinase K (Qiagen). After a 30 min incubation at room temperature, sample buffer was added to all samples, samples were boiled and separated on 12% polyacrylamide gels. Proteins were visualized by immunoblotting using antibodies directed against PPE41 (see above), antibodies against PknG (kindly provided by Y. Av-Gay, Vancouver, Canada), and antibodies against GroEL and GroES. The GroEL monoclonal antibody used is CS44, obtained from J. Belisle ( Colorado State University, Fort Collins, CO, and the National Institutes of Health, Bethesda, MD, contract NO1 AI-75320). The presence of the second antibody, GARpo or GAMpo, was visualized using 4-chloronaphtol/3,3-diaminobenzidine staining or using ECL detection (PIERCE). For protein quantification nitrocellulose membranes were probed with a-PPE41 poly-clonal antibody and detected by Lumi-Light Western Blotting Substrate (Roche Applied Science). Quantification was done on a Fluor-S MultiImager (BioRad) using Biorad multi-analyst software, version 1.0.2.

Transposon mutagenesis

Transposon mutagenesis was performed using the mycobacterial specific phage phiMycoMarT7 containing the mariner-like transposon Himar117. Transductants of M. marinum strain M supplemented with the Rv2430c containing vector pSMT3-30/31c were plated on a nitrocellulose filter, which was placed on 7H10 agar plates supplemented with kanamycin (to select for the presence of the transposon) and hygromycin (pSMT3-30/31c). After visible colonies had appeared, the filter was removed and placed on a new filter, on top of another 7H10 plate. This sandwich was incubated for 5-6 hours at 30ºC and subsequently the top filter with the colonies was preserved at 4ºC, whereas the bottom filter with the secreted proteins was incubated with antibodies directed against PPE41. Colonies of interest, i.e. those that showed reduced mounts of PPE41, were first checked on a similar colony blot assay and subsequently checked in culture for PPE41 secretion. To establish the chromosomal location of the transposon insertion, ligation-mediated PCR was used essentially as described by Prod'hom and colleagues18, with the following modifications. 50 ng of chromosomal DNA, isolated from plate-grown mutants with the DNA tissue kit (Qiagen), was digested with BamHI and BglII instead of SalI. These two restriction enzymes together have approximately the same digestion frequency in mycobacterial DNA as SalI, but in addition are functional in a buffer also suited for ligation. In the same incubation reaction (overnight at room-temperature) as the digestion, an adaptor, consisting of the SalgD primer together with the Bampt primer (GATCGCTCGTGCC), is ligated to the digested DNA. This adaptor does not restore the BamHI/BglII restriction sites. Subsequently, this ligation mixture is used as a template in a PCR reaction with the pSalg primer (GCTTATTCCTCAAGGCACGA), which is identical to the single-stranded part of the adaptor, and the pMyco primer


(CCGGGGACTTATCAGCCAAC), which binds to both sides of the Himar1 transposon. PCR fragments were sequenced using an ABIprism300 (Applied biosystems) and analyzed using the sequence information of the M. marinum Sequencing Group at the Sanger Institute (http://www.sanger.ac.uk/Projects/ M_marinum/). In both mutants the MycoMar transposon inserted into the dinucleotide TA, as can be expected for this mariner-based transposon.

Infection of leukocytes

For intracellular experiments the human acute monocytic leukemia cell line THP-1 and the carp leucocyte cell line CLC were used as described previously19. THP-1 cells were cultured in RPMI-1640 with glutamax-1 media (GIBCO, BRL) supplemented with 10% fetal calf serum (FCS), and differentiated into macrophage-like cells in the presence of PMA (phorbol 12-myristate 13-acetate, 1 ng ml-1, Sigma). For phagosome isolation, cells were seeded in 75 cm diameter flasks whereas 24-well plates (Nunc) were used for survival assays and microscopy. For the infection, the mycobacteria were grown to logarithmic phase (OD600 = 0.5-0.8) in 7H9 media, washed and diluted in RPMI-1640 with 10% FCS and used at an MOI of 10 bacteria per cell. After 1 h of uptake at 33°C, infected cells were washed three times with PBS to remove extracellular bacteria and incubated in fresh medium plus amikacin (200 µg/ml; Sigma Chemicals, St. Louis, Mo.) for 2 h. The cells were then washed once with PBS and incubated in fresh medium plus amikacin (30 µg/ml) for indicated time periods. Mycobacterial survival was analyzed by lysing the infected macrophages in 0.1% Triton X-100 and plating serial dilutions on 7H10 agar (Difco) supplemented with 10% OADC. For microscopic examination, cells were differentiated with coverslips in wells and infection was done with a pretreatment of amikacin as described, but incubated in the absence of amikacin.

Mycobacterial phagosomes isolation

Isolation of mycobacterial phagosomes was performed as described previously20. Briefly, infected THP-1 cells (7-10x107 with MOI of 25 for 48 h) were homogenized in DGE buffer (10 mM triethanolamine, 10 mM acetic acid, 1 mM EDTA, 0.25 M sucrose, pH 7.4) by passaging through a 23-gauge needle. After removal of the cell nuclei by low speed centrifugation (250g, 10 min), the resulting postnuclear supernatant (PNS) was transferred to a fresh Eppendorf tube and sedimented at 35,000g for 30 min. The resulting supernatant corresponds to the macrophage cytosol whereas the pellet corresponds to the mycobacterial phagosomes. The bacteria-free phagosomes were recovered, by treating them with 1% Triton X-100 for 15 min at room temperature and sediment the intact mycobacteria at 35,000g for 30 min. The resulting supernatant corresponds to the phagosomes and the pellet to the mycobacteria.

http://www.sanger.ac.uk/Projects/


Digital imaging fluorescence microscopy

THP-1 cells (7.5x105 per well) were seeded on glass coverslips in 24-well plates in the presence of PMA and infected with different strains of mycobacteria as described in the infection procedures for 24 h. Cells were fixed with 4% paraformaldehyde in PBS for 15 min at room temperature, permeabilised with 0.1% Triton X-100 in PBS for 15 min, and blocked in 2% BSA (Sigma) in PBS for 30 min. This blocking solution was also used for dilution of antibodies. The slides were incubated with antibodies directed against PPE41, monoclonal antibody F30-521 directed against mycobacterial lipoarabinomannan (LAM), obtained from Arend Kolk (KIT, Amsterdam), or against LAMP-1 (H4A3, obtained from the Hybridoma Bank of the University of Iowa, Iowa City). After overnight incubation with primary antibodies at 4°C, coverslips were washed with PBST and incubated with Alexa fluor 488 or 546-conjugated secondary antibodies (Molecular Probes). Coverslips were mounted in Vectashield (Vector Laboratories) and analyzed using a ZEISS Axiovert 200 MarianasTM digital imaging microscopy workstation as described previously22. The microscope, camera and data viewing/processing were conducted/controlled by SlidebookTM software (Slidebook version 4.1.0.4 [Intelligent Imaging Innovations, Denver, CO]). The data acquisition protocol included confocal optical planes to obtain 3D definition.

Cloning and expression of the M. marinum ESX-5 -cluster

M. marinum genomic DNA was used as a template to amplify the ESX-5 region in two fragments with Extensor Hi-Fidelity PCR Master Mix (ABgene) using the primers AbMmfront (CAAAGTGTCCTGAGAGACGGGTA) and AbMmMiddle-H5-R (ACCTAGCTTCCCACTCAGCAAAC) for the first part and AbMmMiddle-H5-F (AAGGGAGCACCGAAAATGTTAAA) and AbMmEnd (CGAGTTGATCTCAATCCATCCAC) for the second part. The resulting PCR products were cloned in pCRII-TOPO (invitrogen). To transfer these constructs into M. smegmatis mc2155, the pAL5000 replicon and the hygromycin resistance gene was cloned from pSMT3-eGFP using the restriction enzymes DraI/EcoRV for pSMT3-eGFP and DraI for the TOPO-PCR constructs, resulting in pSMT-H5-1 and pSMT-H5-2, respectively. Subsequently, a complete ESX-5 region was constructed by digesting both plasmids with the restriction enzymes DraI/MluI and re-ligation. The resulting plasmid was designated pSMT-H5. Also the mh1798 gene alone was amplified by PCR, using chromosomal DNA of M. marinum M as a template and the primers 1798R (TGTTAACTGATGCCAATTCCGATTTC) and 1798F (CGTTAACTCGATGAGGTCTGGCTCTC). This fragment was cloned in pCRII-Topo (Invitrogen) and checked by sequence analysis. Subsequently, mh1798 was cloned downstream of the mycobacterial hsp60 promoter and the gene encoding DsRed, by ligating the HindIII/NotI fragment of pTOPO-Mh1798 with the NotI/BamHI fragment of pSMT3DsRed and the BamHI/HindIII vector fragment of pSMT3eGFP. This tripartite ligation was checked by restriction digestion and designated pMh1798.


Hydrophobicity

Cell surface hydrophobicity was assessed in three separate experiments as described previously23. Briefly, M. marinum wild type and Mx2 strains were grown on 7H9 broth for 1 week. Cells were harvested by centrifugation at 4°C and 2000g and washed twice with PBS. Bacterial suspensions were prepared in PBS to an optical density of 1.0 (OD600). Duplicate (3 ml) samples of each suspension were placed in test tubes. Xylene was added to each suspension at concentrations of 0, 0.5, 1 and 2% (v/v). The tube contents were mixed for 45 s with a vortex mixer at the maximum setting. The aqueous and organic phases were allowed to separate for 30 min, after which the aqueous phase was carefully removed with a Pasteur pipette and transferred to cuvettes. Contaminating xylene was allowed to evaporate for 60 min. After this, each cuvette was vortexed for 1 s to resuspend the remaining bacteria. The level of absorption of the cells to the xylene droplets was calculated as the loss in optical density at 600 nm of the aqueous phase compared with the initial cell suspension, expressed as a percentage. Similar results were obtained if hexadecane (0, 5, 10 and 15%) was used instead of xylene.

Results

PPE41 is secreted by M. marinum and M. bovis

PPE41 is a small hydrophilic protein, which is encoded by the M. tuberculosis Rv2430c gene. First, the expression and localization of PPE41 was examined in Mycobacterium bovis BCG, which contains an operon encoding a PPE protein with 100% identity to PPE41 of M. tuberculosis. Analysis of the culture supernatant showed that M. bovis BCG secretes a protein that is recognized by the antiserum directed against PPE41 (Fig. 1A). Introduction of the PE25/PPE41 operon placed under the control of the Hsp60 promoter into M. bovis BCG resulted in the overproduction of a protein with an identical apparent molecular weight, indicating that this protein is PPE41 (Fig. 1A). Introduction of this operon also resulted in the presence of bands at higher and lower molecular weight, which probably represent partially degraded, modified or complexed PPE41 (Fig. 1C). Because not all of PPE41 is secreted in the culture supernatant by M. bovis BCG (in Fig. 1B 63% is secreted), we also examined if the cell-associated PPE41 molecules are intracellular or located on the surface using a protease sensitivity assay (proteinase K). The cytoplasmic control protein GroEL was not affected by proteinase K treatment (Fig. 1B), although this protein was degraded completely if lysed bacteria were used (Fig. 1E). However, PPE41 present in the cell fraction could be efficiently removed by proteinase K (Fig. 1B), which indicates that the cell associated form of PPE41 is located on the surface. To determine whether PPE41 is also secreted by other mycobacterial species, the PE25/PPE41 operon placed under control of the Hsp60 promoter was introduced in M. marinum and M. smegmatis. These two species do not contain an endogenous copy of this operon, although M. marinum does contain a large number of other


PPE genes. Upon introduction of the PE25/PPE41 operon in M. marinum this bacterium efficiently secreted PPE41, 73% of the protein could be detected in the culture supernatant (Fig. 1C). Also in M. marinum leakage of the cytoplasmic protein GroEL in the culture supernatant was minimal (Fig. 1C). The non-pathogenic species M. smegmatis efficiently expressed the PPE41 protein (Fig.1C), however only minimal amounts of PPE41 (<5%) were observed in the culture supernatant. In this case, the cell associated PPE41 material was resistant to proteinase K (Fig. 1C). Apparently, M. smegmatis does not secrete PPE41 efficiently.

E

Figure 1 | Secretion of PPE41 by M. bovis BCG (A, B) and M. smegmatis (C) and M. marinum (C,D). Equivalent OD units of the cell pellet (P) and the supernatant (S) fractions were analysed for the presence of PPE41 or GroEL by immunoblot. When indicated, bacterial cell pellet fractions were treated with proteinase K (pK). (A) PPE41 secretion of M. bovis BCG and M. bovis BCG complemented with the PE25/PPE41 operon under control of the hsp60 promoter. Molecular size markers are shown in kDa on the left. (B) PPE41 secretion in M. bovis BCG using GroEL as intracellular control and analysing the protease sensitivity of cell associated PPE41. (C) Introduction of the PPE41/PE25 operon in M. smegmatis (Ms) does not result in PPE41 secretion, whereas introduction in M. marinum (Mm) results in efficient secretion. (D) In M marinum the expression and/or stability of PPE41 is dependent on PE25. (E) Immunoblot showing that GroEL proteins of M. bovis BCG (BCG), M. smegmatis (Ms) and M. marinum (Mm) are sensitive to proteinase K (pK) if lysed cells are subjected to the protease.

The PPE41 encoding gene Rv2430c forms an operon together with the PE25 encoding gene Rv2431c. These two proteins form a stable heterodimeric complex9, and therefore the secretion of PPE41 might be dependent on the presence of


PE25. Introduction in M. marinum of the Rv2430c gene, under control of the Hsp60 promoter, did not result in the presence of significant amounts of PPE41 (Fig. 1D), which indicates that PE25 is needed for secretion and or stability of PPE41.

PPE41 is secreted by M. marinum inside macrophages

Next, we determined if PPE41 was also secreted by mycobacteria inside macrophages. For these experiments we used human THP-1 cells, differentiated into macrophage-like cells in the presence of PMA, infected with M. marinum expressing PE25/PPE41. The infected macrophages were differentially disrupted (Experimental procedures) and the various fractions were analysed by immunoblot for the presence of PPE41. In addition, immunoblots containing these fractions were also incubated with antibodies directed against the cytoplasmic protein GroEL and the secreted protein PknG24 as controls. About half of the total amount of PPE41 was found associated with the bacteria in a protease-resistant form, which could represent intracellular PPE41. However, a similar amount of PPE41 was located in the Triton X-100 soluble fraction, representing the contents of macrophage-derived vesicles (Fig. 2). Since the cytoplasmic protein GroEL was exclusively present in a protease-resistant form in the bacteria-containing fraction, we concluded that bacterial lysis inside macrophages was not significant. This shows that PPE41 is indeed secreted inside macrophages. The other control protein, i.e. PknG was identified in small amounts in the f-raction representing the macrophage cytosol and mainly in the Triton X-100 soluble fraction, as expected (Fig. 2). Figure 2 | Intracellular secretion of PPE41 by M. marinum. Immunoblot analysis of THP-1 cells infected for 48 hours with M. marinum supplemented with PE25/PPE41. Infected cells were homogenized and cell debris was collected by low speed sedimentation (lane 1). Subsequently, mycobacterial phagosomes (lane 2) were separated from the cytosol (lane 3) by sedimentation. Phagosomes were isolated and lysed by Triton X-100 treatment (lane 4) and bacteria were separated by sedimentation (lane 5) and treated with trypsin (lane 6). As a control we also analysed uninfected THP-1 cells (lane 7), THP-1 cells infected with M. marinum wild-type (lane 8) and in vitro grown M. marinum expressing PE25/PPE41 (lane 9). Immunoblots were incubated with antisera directed against PPE41, GroEL (cytoplasmic mycobacterial protein) and PknG (secreted mycobacterial protein).


Intracellular secretion of PPE41 was confirmed with immunofluorescence microscopy. For this approach, macrophages were infected with either live or heat-killed M. marinum expressing PE25/PPE41. Using antibodies directed against PPE41 and against lipoarabinomannan (LAM), a surface glycolipid of M. marinum, PPE41 was found co-localized with phagocytosed bacteria (Fig. 3A). However, PPE41 was also present in vesicles devoid of bacteria (Fig. 3B), which indicates the release of PPE41 from mycobacterial phagosomes into the endocytic network of the host cell. To analyse further the intracellular localization of PPE41, infected cells were stained for LAMP-1, a late-endosome/lysosome marker. Since PPE41-stained vesicles were usually associated with LAMP-1 (Fig. 3C) extracellular PPE41 probably accumulates in late endosomal vesicles. Both heat-killed M. marinum cells complemented with the PE25/PPE41 operon (Fig. 3D) and M. marinum cells devoid of this operon (results not shown) showed no extracellular PPE41 in macrophages, which indicates that this effect is specific and requires live bacteria. Next, we also tested if PPE41 was secreted by M. bovis BCG. Although this species secretes lower amounts of PPE41, intracellular secretion of PPE41 could be observed (Fig. 4).

Identification of the PPE41 secretion system

PPE41 lacks a distinguishable signal sequence and is therefore probably not secreted via the Sec-secretion machinery. This means that a dedicated secretion system must be present in M. marinum and M. bovis. To identify this secretion system we first tested the only known Sec-independent secretion system in mycobacteria: the so-called ESAT-6 secretion system or ESX-1. ESAT-6 is a small protein, which is secreted together with CFP-10 by pathogenic and non-pathogenic mycobacteria. The secretion system involved has recently been identified and is (partially) encoded by the RD1 region (also called ESX-1)12,25-28, which is deleted in the vaccine strain Mycobacterium bovis BCG29. Although we already know that M. bovis BCG is able to secrete PPE41, we still wanted to compare the level of PPE41 secretion in wild-type and mutant strains. Therefore, two RD1 mutants of M. marinum were analysed for PPE41 secretion, i.e. M8, which is mutated in one of the components of ESX-1, and ∆CE, which contains a deletion for the cfp10/esat-6 operon27. The M8 secreted normal amounts of PPE41 in the culture supernatant (Fig. 5A), which shows that ESX-1 is indeed not required for PPE41 secretion. However, surprisingly the mutant devoid of the ESX-1 substrates (∆CE) showed significantly increased secretion of PPE41 (Fig. 5A), indicating a link between the two secretion systems. The intracellular levels of PPE41 were comparable for these strains (Fig. 5A).


Figure 3 | Intracellular secretion of PPE41 by M. marinum. THP-1 cells were seeded on glass cover-slips and infected with Mm::PE25/PPE41 (A,B,C) or heat-killed Mm::PE25/PPE41 (D) for 24 hours. Infected cells were fixed, permeabilised and incubated with antisera directed against PPE41 or against LAM and examined by 3D digital imaging fluorescence microscopy. Secreted PPE41 is shown in red and intracellular bacteria (LAM) or the lysosomal marker protein (LAMP-1) in green, the nuclei are stained with DAPI (blue). Arrows show co-localization of PPE41 and mycobacteria, whereas arrowheads show PPE41 not co-localized with bacteria. Bar represents 10 µM.


Figure 4 | Intracellular secretion of PPE41 by M. marinum mutant Mx2 supplemented with PE25/PPE41 and Mycobacterium bovis BCG. THP-1 cells were seeded on glass cover-slips and infected with M. marinum Mx2 (A) or M. bovis BCG (B) for 24 hours. Infected cells were fixed, permeabilised and incubated with antisera directed against PPE41 and LAM, and examined by 3D digital imaging fluorescence microscopy. Secreted PPE41 is shown in red and intracellular bacteria (LAM) in green, the nuclei are stained with DAPI (blue). Arrows indicate co-localization, whereas arrowheads indicate no co-localization. Bar represents 10 µM.

A genetic screen was used to identify the PPE41 secretion system in M. marinum. M. marinum strain M expressing PE25/PPE41 was subjected to transposon mutagenesis, using the mycobacterial specific phage phiMycoMarT7 containing the mariner-like transposon Himar1 (Experimental procedures). Subsequently, the mutants were grown on nitrocellulose filters and screened for PPE41 secretion using a double-filter colony blot approach (Experimental procedures, Fig. 6). Screening of 10,000 transposon mutants resulted in the isolation of two mutants with strongly reduced amounts of extracellular PPE41 (Fig. 5A). The transposon insertion sites of both mutants with reduced secretion, i.e. mutant Mx2 and Mx12,


were determined by ligation-mediated PCR and sequence analysis. Comparison of these sequences with the complete genome of M. marinum M (http://www.sanger.ac.uk/Projects/M_marinum/) showed that both mutants contained the mariner transposon at different positions in a single coding sequence (CDS). The gene product of this CDS shows high homology (93% identity) to Rv1798 of M. tuberculosis (Fig. 8) and was therefore designated Mh1798. Complementation of Mx2 and Mx12 with the corresponding gene on a mycobacterial shuttle plasmid resulted in restoration of PPE41 secretion (Fig. 5B). Mh1798 and Rv1798 belong to the AAA+ protein family of ATPases. Members of this family are chaperones that assist in the assembly and/or operation of protein complexes. Therefore, Mh1798 probably does not form the secretion channel, but is part of a multiprotein secretion machinery. Alternatively, since Mh1798 belongs to a family of chaperones, the transposon insertion could affect the folding and/or stability of the PPE41 protein. To discriminate between these two possibilities we used the non-secreting species M. smegmatis. Figure 5 | Isolation and characterization of the ESX-5 secretion mutants. (A) Equivalent OD units of cell pellets (P) and culture supernatant (S) of M. marinum wild-type (Mm wt), the ESX-1 mutants M8 and ∆CE and the isolated secretion mutants Mx2 and 12 were separated on SDS-PAGE, immunoblotted and incubated with polyclonal antiserum against PPE41 or GroEL. (B) Complementation of PPE41 secretion mutants. Equivalent amounts of culture supernatant of M. marinum wild-type, with or without PE25/PPE41, the secretion mutants Mx2 and 12, supplemented with PE25/PPE41 and with the empty vector pSMT3 or with pSMT3 containing the Mh1798 gene, were separated on SDS-PAGE and analysed for the presence of PPE41 by immunoblot .


Figure 6 | Quality of the two-filter colony blot screening procedure used in our experiments to isolate secretion mutants of M. marinum. The colony-containing filter (shown on the right) is placed for 5 hours on a second filter after 9-10 days of incubation (time needed to grow the colonies). The second filter (shown on the right) is treated with antiserum directed against PPE41 and stained for the presence of peroxidase. Non-secreting colony is indicated.

Reconstitution of the PPE41 secretion system in M. smegmatis

The genomic organization of Rv1798 and Mh1798 is highly conserved between M. tuberculosis and M. marinum (Fig. 7A). Furthermore, both regions contain CDSs homologous to the M. tuberculosis ESX-1 cluster (Table 1). Four gene clusters homologous to the ESAT-6 secretion system ESX-1 have been identified in M. tuberculosis, including the Rv1798-containing region that is called ESX-530. This could mean that ESX-5 encodes an ESAT-6-like secretion machinery involved in PPE41 transport. Interestingly, ESX-5 is conserved in M. marinum, but is not present in the non-secreting M. smegmatis30. To determine if cluster ESX-5 is directly involved in PPE41 secretion, different regions (Fig. 7A) of the ESX-5 cluster of M. marinum were isolated by long-range PCR, cloned on a mycobacterial shuttle vector and introduced in M. smegmatis. The M. smegmatis strain used already contained the Rv2430c/Rv2431c operon integrated in the genome. Introduction of the mh1798 gene alone or half of the ESX-5 cluster (PE19-mh1798) did not result in increased amounts of extracellular PPE41 (Fig. 7B). However, the presence of the entire ESX-5 cluster (mh1782-mh1798) enabled M. smegmatis to secrete PPE41 efficiently (Fig. 7B). To verify that this secretion was not due to the lysis of M. smegmatis cells, the different fractions were also analysed for GroEL. As expected, GroEL was only present in the cell pellet (Fig. 7B). Together, these data show that the ESX-5 cluster is necessary for PPE41 secretion. The secretion of PPE41 by M. smegmatis through the introduction of ESX-5 did not result in reduced intracellular amounts of PPE41 (Fig. 7B), which indicates that intracellular levels of PPE41 are tightly regulated, probably by proteolytic degradation.


A

B Figure 7 | Reconstitution of the ESX-5 secretion system in M. smegmatis. (A) ESX-5 cluster of M. marinum M compared with the corresponding region of M. tuberculosis H37Rv. Grey-blue areas indicate regions with more than 65% identity (200 bp). Genes predicted to be involved in the secretion system30 are shown in green, CFP-10/ESAT-6-like genes in red, PE in dark blue and PPE in light blue. Genes not present in other ESX systems are shown in black. Arrowheads indicate the transposon insertion sites and the regions cloned for reconstitution in M. smegmatis are indicated in red. Size is shown in basepairs. (B) Reconstitution of the PPE41 secretion system in M. smegmatis (Ms). Equivalent amounts of pellet (P) and supernatant fractions (S) of M. smegmatis supplemented with the PE25/PPE41 plasmid and the empty control plasmid (pSMT3), pSMT3-H5-2, or pSMT3-H5 (part of ESX-5 present in these plasmids is shown in A) were analysed for the presence of PPE41 and GroEL, as an intracellular control, as described in (Fig. 5).


Mh1798 MTRAQSAADDARNAMVAGLLASGISVNGLQPSHNPQVAAKMFTTATKLDPAMCDAWLARL Rv1798 MTRPQAAAEDARNAMVAGLLASGISVNGLQPSHNPQVAAQMFTTATRLDPKMCDAWLARL 120 Mh1798 LAGDQTMDVLAGAWAAVRTFGWETRRLGVTDLQFRPEVSDGLFLRLAVTSVDSLACAYAA Rv1798 LAGDQSIEVLAGAWAAVRTFGWETRRLGVTDLQFRPEVSDGLFLRLAITSVDSLACAYAA 180 Mh1798 VLAENKRYQEASDLLDTTDPKHPFDAELVSYVRGVLYFRTKRWPDVLAQFPEATPWRHPE

Mx2

Rv1798 VLAEAKRYQEAAELLDATDPRHPFDAELVSYVRGVLYFRTKRWPDVLAQFPEATQWRHPE 240 Mh1798 LKAAGAAMATTALASLGVFEEAFRRAQEAIEGDRVPGAANIALYTQGMCLRHVGREEEAV Rv1798 LKAAGAAMATTALASLGVFEEAFRRAQEAIEGDRVPGAANIALYTQGMCLRHVGREEEAV 300 Mh1798 ELLRRVYSRDAKFSPAREALDNPNYRLVLTDPETIEARKDPWDPDSAPTRAQTEAARHAE Rv1798 ELLRRVYSRDAKFTPAREALDNPNFRLILTDPETIEARTDPWDPDSAPTRAQTEAARHAE 360 Mh1798 MAAKYLAEGDAELNAMLGMEQAKKEIKLIKSTTKVNLARAKMGLPVPVTSRHTLLLGPPG Rv1798 MAAKYLAEGDAELNAMLGMEQAKKEIKLIKSTTKVNLARAKMGLPVPVTSRHTLLLGPPG 420 Mh1798 TGKTSVARAFTKQLCGLTVLRKPLVVETSRTKLLGRYMADAEKNTEEMLEGSLGGAVFFD Rv1798 TGKTSVARAFTKQLCGLTVLRKPLVVETSRTKLLGRYMADAEKNTEEMLEGALGGAVFFD 480 Mh1798 EMHTLHEKGYSQGDPYGNAIINTLLLYMENHRDELVVFGAGYAKAMEKMLEVNQGLRRRF Rv1798 EMHTLHEKGYSQGDPYGNAIINTLLLYMENHRDELVVFGAGYAKAMEKMLEVNQGLRRRF 540 Mh1798 STVIEFFSYTPEELIALTKLMGQENEDVITEEEAQVLLPSYTRFYNDQNYSEDGDLIRGI

12

Rv1798 STVIEFFSYTPQELIALTQLMGRENEDVITEEESQVLLPSYTKFYMEQSYSEDGDLIRGI 600 Mh1798 DMLGNAGFVRNVVEKARDHRSFRLDDEDLDAVLNSDLTEFSELQMRRFRELTKEDLAEGL Rv1798 DLLGNAGFVRNVVEKARDHRSFRLDDEDLDAVLASDLTEFSEDQLRRFKELTREDLAEGL 610 Mh1798 SAAVAEKKTN Rv1798 RAAVAEKKTK Figure 8 | Alignment of Mh1798 and Rv1798. Amino acids corresponding with the Tn insertion sites of Mx2 and 12 are indicated with arrows, identical residues are shown in grey and amino acid numbers are indicated.


Table I | CDSs of the ESX-5 region of M. marinum M, their homologues in the EXS-5 and EXS-1 region of M. tuberculosis H37Rv and the proposed function of their gene products, localization or protein family they belong to.

M. marinum ESX-5*

M. tuberculosis ESX-5 ESX-1

Predicted gene function/localization/gene family

Mh1782 Rv1782 Rv3869 membrane protein Mh1783/84

Rv1783Rv1784

Rv3870 Rv3871

FtsK/SpoIIIE family, membrane protein with putative ATP/GTP binding site(s)

cyp153 cyp153 - probable cytochrome P450 CDS 4 PPE25 - PPE protein PE18 PE18 PE35 PE protein CDS 6 PPE26 PPE68 PPE protein CDS 7 - - mycobacteriophage protein PE19 PE19 - PE protein EsxM EsxM EsxB ESAT-6 family, small secreted protein EsxN EsxN esxA ESAT-6 family, small secreted protein Mh1794 Rv1794 Rv3866 putative membrane protein Mh1795 Rv1795 Rv3877 membrane protein mycP5 mycP5 mycP1 secreted subtilisin-like protease, mycosin Mh1797 Rv1797 Rv3882c membrane protein Mh1798

Rv1798

Rv3868

AAA+ family, ATPase activity and putative chaperone

* CDSs with a high identity (>80%) on the protein level with their M. tuberculosis counterparts have been named as such (H37Rv nomenclature), with the prefix Mh.

Proteins secreted by ESX-5 are involved in macrophage escape

ESX-5 is specific for pathogenic mycobacteria, which could mean that the ESX-5 secreted substrates are involved in virulence. To test this possibility, we studied the interaction of the M. marinum secretion mutant Mx2 with human macrophages and fish leucocytes, as described previously19. For this experiment, Mx2 was cured for the Rv2430c/Rv2431c plasmid to study the effect of ESX-5 independent of heterologous PPE expression. Both the wild-type and the Mx2 mutant were supplemented with a DsRed encoding plasmid, which results in red fluorescent bacteria. First the surface characteristics of these two strains were examined by determining the hydrophobicity and binding to human macrophages. Although the secretion mutant showed a decreased surface hydrophobicity as compared to the wild-type (Fig. 9A), the binding and uptake of both cells by macrophages was similar (results not shown). Subsequent infection experiments showed that ESX-5 mutants survived and multiplied in both types of leukocytes. However, a clear difference was observed when the percentage of infected cells was determined (Fig. 9B). With M. marinum wild type, the percentage of infected cells increased throughout the assay, reaching a value of 53% by day 3. In contrast, the Mx2 mutant infected only 17% of the host cells by day 3. This could indicate that the secretion mutant was impaired in spreading to new cells. To examine this phenotype in more detail, also the number of bacteria per infected cell was


determined (Fig. 9C). Throughout the assay, most of M. marinum wild type infected cells contain less than 10 bacteria, although the number of M. marinum wild type infected cells increased (Fig. 9C). For the secretion mutant Mx2 the total number of infected cells remained relatively constant, whereas the percentage of cells containing more than 10 bacteria progressively increased (Fig. 9C). This result indicates that Mx2 mutant is capable of intracellular growth, but is impaired in macrophage escape. Together our data show that ESX-5 plays an important role in the infection cycle of M. marinum, possibly by facilitating host cell lysis.

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Figure 9 | (A) Cell surface hydrophobicity of M. marinum wild-type ( ) and the Mx2 mutant ( ). Effect of ESX-5 on intracellular replication and spreading of M. marinum (B and C). THP-1 macrophage-like cells were seeded on glass cover-slips and infected with the wild type (closed bars) and Mx2 mutant (open bars) expressing DsRed. At each time point the percentage of infected cells (A) and the percentage of the infected cells with more than 10 bacteria (B) was determined. The results are the average of 3 separate experiments and the standard deviation is shown. Enumeration of bacilli within THP-1 cells was visualized by microscopy, with a minimal of 50 infected cells per time point.

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Discussion

Most pathogenic bacteria are dependent on specialized secretion systems for the export of crucial virulence factors. Over the last decade a number of these secretion systems have been identified, ranging from the type I to the type V secretion pathway. Until recently, pathogenic mycobacteria were thought to be an exception to the rule. M. tuberculosis contains, apart from the omnipresent Sec-dependent transport system, no homologue of any known specialized secretion system and only relatively small amounts of extracellular proteins could be identified as compared to other bacterial species. However, three years ago this idea changed when it was shown that M. tuberculosis possesses a Sec-independent secretion system to transport two small virulence proteins, ESAT-6 and CFP-1012,25-28. This secretion pathway was called the ESAT-6 secretion pathway or ESX-1 and represented a new class of secretion systems. Genome analysis showed that ESAT-6-like secretion systems are in fact also present in other Gram-positive bacteria30,31 and a homologue of this secretion pathway was found to be involved in the secretion of two small proteins of Staphylococcus aureus and necessary for pathogenesis of this bacterium32. M. tuberculosis itself contains the genetic information for five of these ESAT-6-like secretion machineries30, which indicates that Sec-independent protein secretion is an important feature of this pathogen. These ESAT-6-like gene clusters are conserved in the genomes of other mycobacteria, although not all species contain the full extent of these gene clusters30. In this study, we show that the M. marinum orthologue of cluster ESX-5 encodes a protein secretion system, which is specific for pathogenic mycobacteria and is involved in the secretion of PPE41. Introduction of the entire ESX-5 gene cluster in M. smegmatis resulted in the secretion of heterologously expressed PPE41. Smaller genome fragments did not result in the reconstitution of this ESAT-6-like secretion system, which shows that the predictions of Gey van Pittius and colleagues30, based on genome comparisons, were very accurate. This ESX-5 cluster encodes several proteins that are likely to be directly associated with a secretion apparatus, such as a membrane protein with a putative ATP binding site (Mh1784/84) and various other putative membrane proteins (Mh1782, Mh1794, Mh1795 and Mh1797), but also proteins that are perhaps not involved, such as several PEs and PPEs, a putative cytochrome P450 and a mycobacteriophage protein (Table 1). Future experiments will have to show which of these genes are actually involved in PPE41 secretion. But, if most genes of the ESX-5 gene cluster are needed for secretion, why are only transposon insertion mutants in a single gene (Mh1798) isolated in the genetic screen in M. marinum? The results from two separate high-density transposon mutagenesis studies in M. tuberculosis provide a possible explanation for this apparent discrepancy. In both these studies transposon insertions in the Mh1798 homologue Rv1798 were isolated, but mutations in the other genes of ESX-5 encoding structural components of the secretion system, i.e. the homologues of Rv1782-Rv1784 and Rv1794-Rv1797 (CDSs shown in green in Fig. 7A) were not


detected17,33. This indicates that these transposon insertions are, in contrast to the ESX-1 secretion system, lethal. The Mh1798 mutants isolated in our screen showed only a strongly reduced secretion of PPE41, and perhaps mutations in the other ESX-5 genes could block secretion completely. Interestingly, we isolated one mutant that showed no PPE41 secretion at all on colony blot. Unfortunately, this mutant grew very slowly and could not be resuscitated after two passages and was therefore not further analysed. Future experiments will be directed to prove the essential nature of ESX-5. The saprophytic species M. smegmatis does not contain an ESX-5 cluster and because this bacterium produces a cell wall similar to that of M. tuberculosis and M. marinum, it is not likely that ESX-5-secreted substrates play an important role in cell wall biosynthesis. Therefore, the lethality of ESX-5 mutations is probably due to the accumulation of normally secreted proteins. Since ESX-5 secretes PPE41, the logical candidates for these toxic proteins are PPE proteins. M. smegmatis contains only 2 genes encoding putative PPEs (http://www.tigr.org/tigr-scripts/CMR2/gene_attribute_form.dbi) and these are located in the ESX-1 and ESX-3 region, respectively30. Apart from ESX-4, all ESX regions have one or more PPE genes. Comparison of the conserved N-terminal PPE motif of all PPE proteins of M. tuberculosis shows that the majority of PPEs cluster together with PPE41 and the PPEs of ESX-5. This clustering indicates that the expansion of PPEs is correlated with ESX-5 (Gey van Pittius, manuscript in preparation). Therefore, we hypothesise that more PPEs are secreted via the ESX-5 system. The PPEs encoded by ESX-1, -2 or -3 are more distantly related and could have a different function. Thus far, the PPE protein of the ESX-1 region (PPE68) has been most extensively studied. This protein is not involved in the secretion of ESAT-6 and CFP-1034. Overlay experiments showed that PPE68 specifically interacts with both of these secreted proteins35, which indicates that PPE68 is secreted, together with ESAT-6/CFP-10, via the ESX-1 secretion system. However, proteomic analysis of ESX-1 secretion mutants failed to show secretion of PPE68, although the secretion of the ESX-1-encoded PE35 could be determined36. Another report showed that PPE68 is in fact located in the cell wall34. Therefore the exact role and fate of PPEs in the various ESX systems needs to be examined in more detail. An interesting observation was that the ∆CE mutant strain in fact secreted significantly higher amounts of PPE41, whereas a deletion of a gene encoding a structural component of the ESX-1 system did not affect the level of PPE41 secretion. These data show that there is some level of cross-talk between the two secretion systems ESX-1 and ESX-5. Deletion of the ESAT-6 and CFP-10 encoding genes could perhaps result in up-regulation of the ESX-5 secretion system. Alternatively, the absence of the ESX-1 secreted substrates ESAT-6 and CFP-10 could result in cross-secretion of ESX-5 substrates via ESX-1. The ESX-5 secretion system seems to be specific for pathogenic mycobacteria and our data show that ESX-5 plays an important role in the macrophage infection cycle. Interestingly, the observed effects are highly similar to those described for ESX-1 mutations in M. tubercsulosis/M. bovis25-28. This probably means that ESAT-6

http://www.tigr.org/tigr-scripts/CMR2/gene_attribute_form.dbi

http://www.tigr.org/tigr-scripts/CMR2/gene_attribute_form.dbi


secretion itself is not sufficient to perturb the macrophage cell membrane and complete the macrophage infection cycle. A recent study of Li and colleagues6 identified a PPE gene of M. avium that was associated with virulence and the ability to grow in macrophages. Interestingly, this PPE gene is highly homologous to the Rv1787 gene of M. tuberculosis, which is located within the ESX-5 cluster and encodes PPE25. Therefore, this protein could very well be one of the virulence-associated ESX-5-secreted substrates needed for the macrophage infection cycle. Future experiments will be directed to unravel the role of extracellular PPE proteins within the host cell.

Acknowledgements

The authors thank Jonathan Kadouch, Widad Rifi and Corrine ten Hagen-Jongman for technical assistance, Astrid van der Sar and Marian Llamas for helpful discussions, Yossef Av-Gay, Arend Kolk, Eric Rubin, Janisha Patel, Neil Stoker, Papinavinasasundaram and Eric Brown for strains, plasmids, phages and antibodies.

Notes:

Supporting Online Material (SOM) accompanies the paper on the Nature website (http://www.blackwell-synergy.com/) and includes SOM Figures S1-S5, Z-stack shown in movie S1 and S2, and SOM Table S1

References



3. Ishikawa,J. et al. The complete genomic sequence of Nocardia farcinica IFM 10152. Proc. Natl. Acad. Sci. U. S. A 101, 14925-14930 (2004).

4. Sampson,S.L. et al. Expression, characterization and subcellular localization of the Mycobacterium tuberculosis PPE gene Rv1917c. Tuberculosis. (Edinb. ) 81, 305-317 (2001).

5. Pym,A.S., Brodin,P., Brosch,R., Huerre,M. & Cole,S.T. Loss of RD1 contributed to the attenuation of the live tuberculosis vaccines Mycobacterium bovis BCG and Mycobacterium microti. Mol. Microbiol. 46, 709-717 (2002).

http://www.blackwell-synergy.com/


6. Li,Y.J., Miltner,E., Wu,M., Petrofsky,M. & Bermudez,L.E. A Mycobacterium

avium PPE gene is associated with the ability of the bacterium to grow in macrophages and virulence in mice. Cellular Microbiology 7, 539-548 (2005).

7. Choudhary,R.K. et al. PPE antigen Rv2430c of Mycobacterium tuberculosis induces a strong B-cell response. Infect. Immun. 71, 6338-6343 (2003).

8. Tundup,S., Akhter,Y., Thiagarajan,D. & Hasnain,S.E. Clusters of PE and PPE genes of Mycobacterium tuberculosis are organized in operons: evidence that PE Rv2431c is co-transcribed with PPE Rv2430c and their gene products interact with each other. FEBS Lett. 580, 1285-1293 (2006).

9. Strong,M. et al. Toward the structural genomics of complexes: crystal structure of a PE/PPE protein complex from Mycobacterium tuberculosis. Proc. Natl. Acad. Sci. U. S. A 103, 8060-8065 (2006).

10. Ramakrishnan,L. & Falkow,S. Mycobacterium marinum persists in cultured mammalian cells in a temperature-restricted fashion. Infect. Immun. 62, 3222-3229 (1994).

11. Snapper,S.B., Melton,R.E., Mustafa,S., Kieser,T. & Jacobs,W.R., Jr. Isolation and characterization of efficient plasmid transformation mutants of Mycobacterium smegmatis. Mol. Microbiol. 4, 1911-1919 (1990).

12. Converse,S.E. & Cox,J.S. A protein secretion pathway critical for Mycobacterium tuberculosis virulence is conserved and functional in Mycobacterium smegmatis. Journal of Bacteriology 187, 1238-1245 (2005).

13. Hayward,C.M. et al. Construction and murine immunogenicity of recombinant Bacille Calmette Guerin vaccines expressing the B subunit of Escherichia coli heat labile enterotoxin. Vaccine 17, 1272-1281 (1999).

14. Bachrach,G. et al. A new single-copy mycobacterial plasmid, pMF1, from Mycobacterium fortuitum which is compatible with the pAL5000 replicon. Microbiology 146 ( Pt 2), 297-303 (2000).

15. Pashley,C.A. & Parish,T. Efficient switching of mycobacteriophage L5-based integrating plasmids in Mycobacterium tuberculosis. FEMS Microbiol. Lett. 229, 211-215 (2003).

16. Lee,M.H., Pascopella,L., Jacobs,W.R., Jr. & Hatfull,G.F. Site-specific integration of mycobacteriophage L5: integration-proficient vectors for Mycobacterium smegmatis, Mycobacterium tuberculosis, and bacille Calmette-Guerin. Proc. Natl. Acad. Sci. U. S. A 88, 3111-3115 (1991).


18. Prod'hom,G. et al. A reliable amplification technique for the characterization of genomic DNA sequences flanking insertion sequences. FEMS Microbiol. Lett. 158, 75-81 (1998).

19. van der Sar,A.M. et al. Mycobacterium marinum strains can be divided into two distinct types based on genetic diversity and virulence. Infection and Immunity 72, 6306-6312 (2004).

20. Schuller,S., Neefjes,J., Ottenhoff,T., Thole,J. & Young,D. Coronin is involved in uptake of Mycobacterium bovis BCG in human macrophages but not in phagosome maintenance. Cell Microbiol. 3, 785-793 (2001).

21. Kolk,A.H. et al. Production and characterization of monoclonal antibodies to Mycobacterium tuberculosis, M. bovis (BCG) and M. leprae. Clin. Exp. Immunol. 58, 511-521 (1984).

https://www.researchgate.net/publication/15159140_Mycobacterium_marinum_persists_in_cultured_mammalian_cells_in_a_temperature-restricted_fashion?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=



https://www.researchgate.net/publication/11619442_Coronin_is_involved_in_uptake_of_Mycobacterium_bovis_BCG_in_human_macrophages_but_not_in_phagosome_maintenance?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=



https://www.researchgate.net/publication/21253643_Site-specific_integration_of_mycobacteriophage_L5_Integration-proficient_vectors_for_Mycobacterium_smegmatis_Mycobacterium_tuberculosis_and_bacille_CALMETTE-GUERIN?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=








https://www.researchgate.net/publication/8954392_Efficient_switching_of_mycobacteriophage_L5-based_integrating_plasmids_in_Mycobacterium_tuberculosis?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=




22. van der Sar,A.M. et al. Zebrafish embryos as a model host for the real time

analysis of Salmonella typhimurium infections. Cell Microbiol. 5, 601-611 (2003). 23. Rosenberg,M., Gutnick,D. & Rosenberg,E. Adherence of bacteria to

hydrocarbons - a simple method for measuring cell-surface hydrophobicity. FEMS Microbiol Lett 9, 29-33 (1980).

24. Walburger,A. et al. Protein kinase G from pathogenic mycobacteria promotes survival within macrophages. Science 304, 1800-1804 (2004).

25. Hsu,T. et al. The primary mechanism of attenuation of bacillus Calmette-Guerin is a loss of secreted lytic function required for invasion of lung interstitial tissue. Proceedings of the National Academy of Sciences of the United States of America 100, 12420-12425 (2003).




29. Mahairas,G.G., Sabo,P.J., Hickey,M.J., Singh,D.C. & Stover,C.K. Molecular analysis of genetic differences between Mycobacterium bovis BCG and virulent M-bovis. Journal of Bacteriology 178, 1274-1282 (1996).


31. Pallen,M.J. The ESAT-6/WXG100 superfamily -- and a new Gram-positive secretion system? Trends Microbiol. 10, 209-212 (2002).

32. Burts,M.L., Williams,W.A., DeBord,K. & Missiakas,D.M. EsxA and EsxB are secreted by an ESAT-6-like system that is required for the pathogenesis of Staphylococcus aureus infections. Proc. Natl. Acad. Sci. U. S. A 102, 1169-1174 (2005).

33. Lamichhane,G. et al. A postgenomic method for predicting essential genes at subsaturation levels of mutagenesis: application to Mycobacterium tuberculosis. Proc. Natl. Acad. Sci. U. S. A 100, 7213-7218 (2003).

34. Demangel,C. et al. Cell envelope protein PPE68 contributes to Mycobacterium tuberculosis RD1 immunogenicity independently of a 10-kilodalton culture filtrate protein and ESAT-6. Infect. Immun. 72, 2170-2176 (2004).

35. Okkels,L.M. & Andersen,P. Protein-protein interactions of proteins from the ESAT-6 family of Mycobacterium tuberculosis. J. Bacteriol. 186, 2487-2491 (2004).


https://www.researchgate.net/publication/8641452_Protein-Protein_Interactions_of_Proteins_from_the_ESAT-6_Family_of_Mycobacterium_tuberculosis?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=

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https://www.researchgate.net/publication/11395613_Pallen_M_J_The_ESAT-6WXG100_superfamily_-_and_a_new_Gram-positive_secretion_system_Trends_Microbiol_10_209-212?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=

https://www.researchgate.net/publication/11395613_Pallen_M_J_The_ESAT-6WXG100_superfamily_-_and_a_new_Gram-positive_secretion_system_Trends_Microbiol_10_209-212?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=

https://www.researchgate.net/publication/228034730_Adherence_of_bacteria_to_hydrocarbons_A_simple_method_for_measuring_cell-surface_hydrophobicity_FEMS_Micro_Lett_9_29-33?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=



Mycobacterial PPE and PE_PGRS proteins are transported via a type VII secretion system

Abdallah M. Abdallah*, Theo Verboom*, Nicolaas C. Gey van Pittius‡, Phetole W. Mahasha‡, Connie Jiménez§, Marcela Parra¶, Nathalie Cadieux¶, Michael J. Brennan¶, Ben J. Appelmelk* and Wilbert Bitter*

*Department of Medical Microbiology and Infection Control, VU University Medical Centre, Amsterdam, The Netherlands. ‡Division of Molecular Biology and Human Genetics, Department of Biomedical Sciences, Stellenbosch University, Tygerberg, South Africa. §OncoProteomics Laboratory, Vumc Cancer Centre Amsterdam, VU University Medical Centre, Amsterdam, The Netherlands. ¶Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, USA.

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Chapter 5: The ESX-5 pathway secretome 88

Abstract

ESX-5 is one of the five type VII secretion systems found in mycobacteria. Here, we have determined the secretome of ESX-5 by a proteomic approach in two different strains of Mycobacterium marinum. Comparison of the secretion profile of wild-type strains and their ESX-5 mutants showed that a number of PE_PGRS and PPE proteins are dependent on ESX-5 for transport. The PE_PGRS and PPE protein families are unique to mycobacteria, are highly expanded in several pathogenic species, such as Mycobacterium tuberculosis and M. marinum, and certain family members are cell surface antigens associated with virulence. Using a monoclonal antibody directed against the PGRS domain we showed that in fact all PE_PGRS proteins are missing in the supernatant of ESX-5 mutants. In addition to PE_PGRS and PPE proteins, the ESX-5 secretion system is responsible for the secretion of a number of small ESAT-6-like proteins. Surprisingly, mutants without an active ESX-5 secretion system showed increased secretion of two ESX-1 proteins, which suggests a functional link between these two secretion systems. Together, these data show that ESX-5 is a major secretion pathway for mycobacteria and that this system is likely responsible for the secretion of all recently evolved PE_PGRS and PPE proteins.

Introduction

Many pathogenic bacteria rely on specialized protein secretion pathways to secrete effector proteins that are important for virulence1-3. An important group of pathogens that have only recently been shown to secrete virulence factors are the mycobacteria4. This genus includes the fish pathogen Mycobacterium marinum and Mycobacterium tuberculosis, which is, with an estimate of two million deaths per year, the most deadly bacterial pathogen worldwide. Most pathogenic mycobacteria are intracellular pathogens that survive and replicate within cells of the host immune system, primarily macrophages, by inhibiting the acidification and maturation of the phagosome5-7. As in other bacterial pathogens, the exported proteins of mycobacteria also play an important role in pathogenesis of the disease, by altering host resistance and by modifying the macrophage environment8-12. Proteomic studies have shown that M. tuberculosis secretes a number of different proteins13,14, although many of these proteins are produced without a classical signal sequence14,15. This fact, combined with the highly complex architecture of the mycobacterial cell wall implies that (a) specialized secretion pathway(s) must be present in these bacteria. Recently, a novel type of protein secretion system has been identified in mycobacteria16-20, the type VII secretion pathway21, which is also known as the ESAT-6 secretion pathway. The ESX-1 system is the archetype of type VII, this system is responsible for the secretion of 5 proteins, including the important T-cell antigens ESAT-6 and CFP-1020,22-24. The ESX-1 secretion system is affected by a


spontaneous deletion, called RD1, in the vaccine strain Mycobacterium bovis BCG25-27 and this deletion is (partially) responsible for the attenuated phenotype of BCG28-

30. Genome analysis has shown that there are four gene clusters homologous to the ESX-1 cluster, designated ESX-2 to ESX-531. Interestingly, whereas ESX-1 is present in most mycobacteria, the ESX-5 system is confined to the slow-growing mycobacteria, a subclass of mycobacteria that contains most pathogenic species16,32. The appearance of the ESX-5 system in slow-growing mycobacteria predates the recent expansion of two gene families, i.e. PE and PPE32. These two gene families are unique for the mycobacteria and were one of the major surprises of the M. tuberculosis genome sequence33: together they cover almost 10% of the genome (167 genes). The PE and PPE families are named after the conserved Proline and Glutamic acid (PE) and Pro–Pro–Glu (PPE) motifs near the N terminus of their gene-products33,34. However, in fact the protein family members share homologous N-terminal domains of approximately 110 amino acids for the PEs and 180 amino acids for PPE proteins. PE and PPE proteins have been shown to be secreted or located at the cell surface. Although their exact function is generally unknown, they are associated with virulence and have been hypothesized to show antigenic variation. The evolutionary link between the expansion of PE/PPEs in slow-growing mycobacteria and the appearance of ESX-5 led to he hypothesis that ESX-5 is in fact involved in the functioning of these proteins32. Previously, we have shown that an ESX-5 mutant of M. marinum is defective in the secretion of one heterologously expressed PPE, PPE4116. In this study we determined which other proteins are secreted by the ESX-5 pathway and show that ESX-5 is in fact a major secretion pathway responsible for the transport of a large number of proteins, including numerous members of the largest subfamily of PE proteins, i.e. PE_PGRS. These are glycine-rich proteins encoded by genes with a poly GC-rich sequence.



Wild-type M. marinum strains M, E11 and their mutants Mx2 and 7C1 were routinely grown in Middlebrook 7H9 liquid medium or Middlebrook 7H10 agar supplemented with 10% Middlebrook ADC or OADC respectively (BD, Biosciences) and 0.05% Tween 80. Escherichia coli strain DH5α was used for DNA manipulation experiments and propagation of plasmid DNA. Antibiotics were added at the following concentration: kanamycin, 25 µg ml-1 and hygromycin, 50 µg ml-1 for both mycobacteria and E. coli.


Molecular cloning of PPE10, 13 and PE_PGRS45

Specific primer pairs (Table 1) bearing restriction sites were designed for the PE_PGRS Rv2615c and two PPE-MPTR genes, Rv0442c and Rv0878c. A hemaglutinin (HA) tag was incorporated in the C-terminal end of the primers. Rv2615c was amplified using nested-PCR with both the outer and inner primers (Table 1) and M. tuberculosis H37Rv chromosomal DNA as template. The PCR amplicons were cloned into the pGemT-Easy T vector (Promega) and sequenced. Subsequently, the cloned products were sub-cloned into the mycobacterial expression vector p19Kpro (Koen de Smet).

SDS-PAGE and immunoblot

Mycobacteria were grown to mid-logarithmic phase in Middlebrook 7H9 liquid medium supplemented with 0.2% (w/v) dextrose, 0.05% Tween 80 and 0.1% of the advised amount of ADC supplement. The presence of BSA in the medium (part of the ADC supplement) is essential for PPE41 secretion in M. marinum16. Proteins in the cell free supernatants were precipitated with 50% TCA (w/v). Proteins were separated by SDS-PAGE on 10-15% polyacrylamide gels35 and visualized by immunoblot using mouse monoclonal antibodies HA.11 (Eurogentec) and against GroEL of M. tuberculosis (John Belisle, NIH, Bethesda, MD, contract NO1 AI-75320); rabbit antiserum reactive to EsxN (rMtb9.9A)36, Rv3881c37 and PPE4116. The presence of peroxidase-conjugated secondary antibodies was detected via 4-chloronaphtol/3,3-diaminobenzidine staining or via chemiluminescence (Pierce). For the treatment of intact cells with protease, mycobacteria were resuspended in PBS and left untreated or treated with 0.1 mg ml-1 proteinase K (Qiagen). After 30 min incubation at room temperature, cells were harvested and sample buffer was added.

Recombinant PE and PE_PGRS constructs

Recombinants of PE_PGRS and PE genes fused to a Histidine tag were constructed using the pET15b expression vector and proteins expressed in E. coli were purified as described in Brennan et al38. Monoclonal antibody directed against the PGRS domain

mAb 7C4.1F7 was produced after fusing spleen cells from BALB/c mice immunized with DNA coding for PE_PGRS 33 and boosted once with recombinant PE_PGRS protein with the P3-X63-Ag.8.653 myeloma cell line (ATCC), using the procedure of Margulies et al39. Ascites of mAb 7C4.1F7 were provided by Harlam, Bioproducts, Indianapolis, Indiana, USA.


Isolation and identification of M. marinum ESX-5 mutant

The mariner-based transposon system using the mycobacterial specific phage phiMycoMarT740 was used to generate a transposon insertion mutant library of M. marinum strain E11 supplemented with the Rv2430c as described previously16. Ligation-mediated PCR was used to localize and identify the transposon-disrupted gene, as described previously16. The resulting DNA sequence was compared with the M. marinum genome sequence (http://www.sanger.ac.uk/Projects/M _marinum/).

Two-dimensional gel analysis of culture supernatants

Short-term culture filtrates were prepared as follows. Strains were grown under agitation at 30oC in 7H9 supplemented with 0,2% Dextrose and 0,002% BSA until the culture reached an OD600 of 0,8 - 1,0. Cultures were then washed 3 times with 50 mM Tris-HCl pH 8,0 in order to remove all BSA and the pellet was resuspended in 7H9 supplemented with 0,2% Dextrose and incubated overnight at 30oC. Culture filtrate was harvested by centrifugation and filtration, first through 0,45µm filter (Whatman) and then through 0,2 µm filter. This culture filtrate was concentrated by ultrafiltration (Millipore, PLBC, nmwl: 3000) and the proteins were precipitated with TCA and dialysed overnight (10 mM Tris-HCl pH 8,0). Equal amounts of protein (150 µg) from each strain were mixed with rehydration buffer (8M urea, 2% CHAPs, 40 mM DTT and 0,5% Pharmalyte 3-10). Protein was hydrated by performing isoelectric focusing (IEF) on 24-cm Immobiline dry strips (Amersham, Biosciences) with pH interval 3-10NL using Ettan IPGphor Isoelectric Focusing system (Amersham, Biosciences). Running conditions: 20oC, 50µa/strip, 30V 12.00 hr, 500V 5.00 hr, 1000V 1.00hr, and 8000V 4.00hr. Prior to the second dimension, the strips were incubated for 15 min in equilibration buffer (6M urea, 2% SDS, 50 mM Tris-HCl pH8, 8, 30% glycerol) with 65 mM DTT first and then with 135 mM iodoacetoamide. To resolve the second dimension, the strips were embedded into 10% SDS-PAGE gel. Gel was stained using colloidal Coomassie brilliant blue.

Mass spectrometry

For MS, protein spots from Coomassie stained 2-D gels were selected and excised manually. Protein spots were destained with ammonium bicarbonate, reduced with DTT, alkylated with iodoacetamide and digested for overnight in gel with modified porcine trypsin (Promega, Madison, USA). The extracted tryptic peptides were concentrated and desalted on reversed-phase ZipTips (Millipore) and eluted with 3 ml CHCA matrix (Sigma). A 0.8 µl sample from the peptide-matrix mixture was analyzed by MALDI-TOF/TOF mass spectrometry (4800 MALDI-TOF/TOF; Applied Biosystems). The obtained mass spectra were searched against M. marinum complex database using a MASCOT search engine

http://www.sanger.ac.uk/Projects/M


(Matrix Science) with a mass tolerance set at 20 ppm for MS1 and 0.6 Da for MS/MS. The protonated trypsin autodigestion products at m/z 842.510 and 2,211.104 were used for internal calibration of the MALDI-TOF-MS spectra. All the proteins listed were identified with a confidence interval of at least 95% from the combined MS and MS/MS analysis. Table 1 | Primers list used in this study

Primer name Sequence SalgD TAGCTTATTCCTCAAGGCACGAGC Bampt GATCGCTCGTGCC PSalg GCTTATTCCTCAAGGCACGA pMyco CCGGGGACTTATCAGCCAAC Rv2615c outer f CGTGGCGGTCAGGAGGATTT Rv2615c outer r GCTCGATGAGCCCAAAGGATGT Rv2615c f GGATCCATGTCGTTTGTCAACGTGGCCCCAC Rv2615c r

AAGCTTTCAGGCGTAGTCCGGCACGTCGTACGGGTAGCCGTCGGCTCCGTTGG

Rv0442c f GGATCCGTGACAAGCCCGCATTTTGCGTGGT Rv0442c r

AAGCTTTCAGGCGTAGTCCGGCACGTCGTACGGGTACTCCGAACCGACCGGCTGCC

Rv0878c f GGATCCATGAATTTCATGGTGCTGCCGCCGG Rv0878c r

AAGCTTTCAGGCGTAGTCCGGCACGTCGTACGGGTACCCGCTGTTCCC TACTTTTT

C-terminal HA tag is shown in italics and the introduced restriction sites (BamHI and HindIII) in bold. Results

Isolation of an ESX-5 mutant in M. marinum E11

M. marinum strains can be divided into two clusters, cluster I contains strains isolated from both cold-blooded animals and humans and these strains cause an acute disease in zebrafish, whereas cluster II isolates are predominantly isolated from poikilothermic species and cause a chronic infection in zebrafish41. Previously, we identified ESX-5 as the secretion system responsible for the transport of heterologously expressed PPE41 in the M. marinum M strain, which belongs to cluster I16. To study the effect of the ESX-5 mutation in both clusters, we also isolated PPE41 secretion mutants in the cluster II strain E11 by genetic screening as described previously16. Eleven mutants with undetectable quantities of PPE41 were isolated, but analysis showed that all but one of these mutants in fact contained deletions in the PPE41 encoding plasmid. This instability was not observed in the previous screen using the M strain16 and indicates differences in recombination frequencies. The last mutant, named 7C1, contained an intact PPE41 gene and was unable to secrete PPE41 in the culture supernatant (Fig. 1A). This mutant contained a transposon insertion in a gene of the ESX-5 cluster, namely Mm2676, which is the M. marinum orthologue of M. tuberculosis Rv1794


(91% identity). Complementation of 7C1 with the corresponding gene on a mycobacterial shuttle plasmid partially restored protein secretion (Fig. 1B). These results confirm that ESX-5 is involved in PPE41 secretion.

Figure 1 | Isolation and characterization of the ESX-5 secretion mutant 7C1 of M. marinum E11. A. Immunoblot analysis of equivalent amounts of culture supernatant of M. marinum wild type E11 and the secretion mutant 7C1, with (+) or without (-) the PE25/PPE41-containing plasmid, for the presence of PPE41. B. Immunoblot analysis of the ESX-5 secretion mutant 7C1, and complemented 7C1 mutant showing cell pellets (P) and the culture supernatant (S) fraction using polyclonal antiserum directed against EsxN-like proteins (Mtb9.9) or monoclonal antibody against GroEL.

ESX-5 mediates the secretion of EsxN

To study which proteins are secreted via ESX-5 we first focused on an obvious candidate, namely EsxN, the ESAT-6 homologue that is encoded by the ESX-5 gene cluster. First we analyzed culture supernatant of M. marinum and showed that indeed a small protein is recognized by antiserum specific for EsxN and that it is partially secreted by both M. marinum strains (69% and 49% is secreted by E11 and M strains, respectively) (Fig. 2A,B). Since the intracellular control protein GroEL was not detected in the culture supernatant (Fig. 2A,B), the extracellular presence of this EsxN-like protein was not due to cell leakage or lysis. Analysis of both ESX-5 mutants showed that, although EsxN was efficiently expressed in these mutants (Fig. 2A,B), only a minimal amount of EsxN-like protein (<3% and <9% respectively) was detected in the culture supernatant, indicating ESX-5 dependence. To determine if EsxN can also be located to the cell surface, intact bacteria were subjected to proteinase K treatment (Fig 2A,B). This protease treatment efficiently removed the majority of the EsxN-like molecules of the wild-type E11 strain (Fig.


2B), whereas EsxN was only partially removed from the cell fraction of the M strain (Fig. 2A). In contrast, both ESX-5 mutants contained protease resistant EsxN molecules (Fig. 2A,B). Together, these results show that the localization of EsxN-like protein(s) in the culture supernatant and on the cell surface is dependent on ESX-5.

Figure 2 | Secretion and surface accessibility of EsxN-like proteins in M. marinum M strain and its ESX-5 mutant Mx2 (A) or the E11 strain and its mutant 7C1 (B). Immunoblot of M. marinum M and the Mx2 mutant showing cell pellet (P), cells treated with proteinase K (pk) and the culture supernatant (S) fraction using either antiserum directed against EsxN or against GroEL as a control for cell death/leakage.

PPE10, PPE13 and PE_PGRS45 are secreted via ESX-5

Since the known substrate of ESX-5 is PPE41, two PPE proteins as well as a PE_PGRS protein were tested as potential ESX-5 substrates16. The PPE10, PPE13 and PE_PGRS45 genes of M. tuberculosis were individually cloned under control of the 19kDa antigen promoter and modified to express a HA-tag at the C-terminus. Subsequently, these constructs were introduced into M. marinum M, E11 strains and their ESX-5 mutants. Upon introduction of the different constructs in M. marinum wild-type strains, 39%, <5% and 35% of PPE10, PPE13 or PE_PGRS45 could be detected in the culture supernatant, respectively by immunoblots (Fig. 3A-C, data shown for the M strain). The supernatant of PPE10-expressing wild-type strains also showed a major band with lower molecular weight, probably representing processed or partially degraded PPE10 (Fig. 3A). Bacterial lysis did not contribute significantly to the protein profiles in the culture supernatant, as GroEL was exclusively present in the cell fraction (Fig. 3A-C). Since the culture supernatants of the ESX-5 mutants did not show bands reacting with the HA antisera, these results show that PPE10 and PE_PGRS45 are actively secreted via ESX-5, whereas PPE13 was not secreted in substantial amounts.


Figure 3 | Secretion of PPE10 (A), PPE31 (B) and PE_PGRS45 (C) by M. marinum M strain and its ESX-5 mutant (Mx2). Immunoblot analysis of M. marinum strains supplemented with the various plasmids showing cell pellet (P), cells treated with proteinase K (pk) and the culture supernatant (S) fraction using either antibody directed against the HA-tag or GroEL. * Indicates processed or degraded PPE10. Similar results were obtained for strain E11 (data not shown) To study the potential surface localization of PPE13 proteinase K assay was performed. Protease treatment of intact cells showed that the majority of cell-associated PPE13 material could be removed (Fig. 3B), indicating a location at the cell surface. Since PPE13 expressed in ESX-5 mutant cells was not affected by proteinase K we conclude that surface localization of this protein is also due to ESX-5. Also substantial amounts of PPE10 and PE_PGRS45 could be removed from the cell fraction by protease treatment in wild-type cells but not in the ESX-5 mutants (Fig. 3A,C). Together, these results show that these randomly chosen PE_PGRS and PPE proteins are all secreted across the mycobacterial cell envelope via the ESX-5 pathway.

Proteomics of the ESX-5 secretome

To study ESX-5 pathway secretion in an unbiased manner, we also analyzed the short-term culture filtrates of both wild-type M and E11 strains and their ESX-5 mutants using a proteomic approach. Because the addition of BSA is essential for efficient (induction of) secretion via ESX-516, we first cultivated M. marinum in the presence of BSA, washed the cells extensively and incubated for an additional 24 hours in medium without BSA (experimental procedures). Although, the protein secretion profiles of the two wild-type strains M and E11 showed many similarities (Fig. 4AB), there were also major differences. In particular, the absence of several major spots at the 10kD range of the M strain was apparent. Mass spectrometry (MS) showed that these spots were in fact ESAT-6 and CFP-10 (Table 2). This


means that E11 secretes high amounts of these proteins whereas M does not. These results were confirmed by immunoblot using antibodies directed against M. tuberculosis ESAT-6 (Fig. 5), indicating an ESX-1 secretion defect in this strain. 2D gel analysis showed many differences between the culture filtrates of wild-types and their ESX-5 mutants (Fig. 4A,B). Some proteins are more prominent in wild-types, but surprisingly, some are also clearly more present in the ESX-5 mutants. First, we analyzed spots that were (more) present in the supernatant of the wild-type cells, and which are thereby probably secreted via ESX-5. In the supernatant of strain E11, EsxP (Rv2347c orthologue) was identified. This protein belongs to the ESAT-6 family and is highly homologous to the EsxM protein encoded by the ESX-5 locus. Furthermore, we also identified the APA protein, a putative cutinase protein (cut3, encoded by Mm1098) and the hypothetical protein MM2929 in the culture supernatant of M strain. These proteins contain a signal sequence42 and are therefore not expected to be secreted by ESX-5. Further analysis, however, showed that one of the spots specific for the ESX-5 mutants was in fact also APA (see Fig. 4A, spot 25). These two specific spots are located close together and probably represent differently processed forms of APA, as described previously43. MM2929 was also not unique for the wild-type strains, as it was also identified in 7C1 culture supernatant by MS analysis on 1D gels (results not shown).

A B

Figure 4 | 2D gels of culture filtrate proteins from M. marinum wild-type M strain and its ESX-5 mutant Mx2 (A) or E11 and its ESX-5 mutant 7C1 (B). 150 µg protein of short-term culture filtrates was hydrated into 3-10 pH strips in the first dimension. In the second dimension, proteins were separated on a 10% polyacrylamide gel and visualized with coomassie brilliant blue. Numbers indicate the proteins that were excised and identified by MS analysis (Table 2).


Figure 5 | Secretion of ESAT-6 by M. marinum wild-type M and E11 strains and their mutants Mx2 and 7C1. Immunoblot analysis of equivalent amounts of M. marinum wild-types M and E11 strains and their mutants MX2 and 7C1 showing cell pellets (P) and the culture supernatant (S) fraction using either antiserum directed against ESAT-6 proteins or against GroEL. In addition to the three proteins described above, most of the wild-type-specific spots in fact corresponded to PE_PGRS and PPE proteins (Table 2). Two of these proteins, encoded by Mm1402 (a PPE-MPTR protein) and Mm3570 (a PE_PGRS protein), are present in culture filtrates of both wild-type strains. Both proteins are present at multiple locations, indicating extensive proteolytic processing and/or degradation. One of the major spots in the highly acidic region (pH~3,5) could not be determined by trypsin cleavage, but was shown by digestion with endoGluC to be the C-terminal domain of PPE protein Mm1402. In silico analysis of the C-terminal sequence of Mm1402 indeed showed a complete absence of Lys/Arg residues needed for trypsin cleavage. In fact, difficulties in identifying PE_PGRS and PPE-MPTR proteins by a proteomic approach seems to be a general characteristic of these proteins, they all have limited numbers of charged amino acid residues and therefore only few peptides can be recovered for MS analysis with either trypsin or EndoGlu (Table 2 and 3). PPE or PE_PGRS proteins were never identified in the culture filtrates of the ESX-5 mutants. Together, these findings confirm that the ESX-5 cluster is necessary and required for extracellular presence of PPE and PE_PGRS proteins.

PE_PGRS proteins are secreted via ESX-5

The proteomics analysis described above showed that PE_PGRS proteins are secreted via ESX-5. PE_PGRS is the major subclass of PE proteins, with 69 members in M. tuberculosis H37Rv32,33,44 and 102 family members in M. marinum M (Timothy Stinear, personal communication). This family is characterized by a variable number of (imperfect) glycine-rich repeats (GGAGGX) in their C-terminal domain. To study the effect of the ESX-5 mutation on PE_PGRS proteins we used a novel monoclonal antibody raised against the PGRS domain of PE_PGRS33 (Rv1818c) of M. tuberculosis (Fig. 6A).

98

Table 2 | List of secreted proteins identified by 2-DE combined with MALDI-TOF MS from culture filtrates of M. marinum wild-types and ESX-5 mutants.

Spots #

Mm gene number

Mt ortholo

gue

Gene product

Number of

peptides

Protein coverage

Protein Score

C. I. %

Protein MW (KDa)

Protein PI

1 Mm0225 Rv0398c Possible secreted protein 4 23% 100 21.1 5.23 2 Mm1098 Rv3451 Cutinase 4 21% 100 26.4 5.79 3 Mm1098 Rv3451 Cutinase 5 24% 100 26.4 5.79 4 Mm1402 - Mm-specific PPE-MPTR 4 9% 100 61.1 3.93 5 Mm2737 Rv1860 APA 6 32% 100 33.9 4.74 7 Mm3570 - Mm-specific PE_PGRS 3 5% 100 81.9 3.78 8 Mm3570 - Mm-specific PE_PGRS 3 5% 100 81.9 3.78 9 Mm3728 - Mm- and Mu-specific PE_PGRS 1 3% 100 33.9 4.74 10 Mm1129 - Mm- and Mu-specific PPE-MPTR 3 5% 99.156 55.7 4.38 11 Mm1402 - Mm-specific PPE-MPTR 1 2% 100 61.1 3.93 12 Mm1402 - Mm-specific PPE-MPTR 4 9% 100 61.1 3.93 13 Mm2933 - M. marinum-specific PE_PGRS 1 2% 99.99 53.2 3.7 14 Mm3316 - Mm-specific PE_PGRS 1 2% 100 79.9 3.84 15 Mm3316 - Mm-specific PE_PGRS 1 2% 100 79.9 3.84 16 Mm3570 - Mm-specific PE_PGRS 3 5% 100 81.9 3.78 17 Mm3570 - Mm-specific PE_PGRS 3 5% 100 81.9 3.78 18 Mm3570 - Mm-specific PE_PGRS 2 3% 100 81.9 3.78 19 Mm3654 Rv2347c EsxP 8 57% 100 10.9 5.83 20 Mm3664 Rv2352c PPE38 2 13% 100 37.6 5.05 21 Mm5439 Rv3864 EspA-like 2 8% 99.39 43.5 4.42 22 Mm5457

Rv3881c

Ala/Gly-rich protein

9

25%

100

46.9

4.52

99

Table 2 | List of secreted proteins identified by 2-DE combined with MALDI-TOF MS from culture filtrates of M. marinum wild-types and ESX-5 mutants.

Spots #

Mm gene number

Mt ortholo

gue

Gene product

Number of

peptides

Protein coverage

Protein Score

C. I. %

Protein MW (KDa)

Protein PI

23 Mm2704 Rv1827 CFP17 6 75% 100 17.2 4.33 24 Mm2704 Rv1827 CFP17 6 75% 100 17.2 4.33 25 Mm2737 Rv1860 APA 5 29% 100 33.9 4.74 26 Mm5140 Rv3648c CspA 2 66% 100 73.4 5.17 27 Mm2737 Rv1860 APA 5 25% 100 33.9 4.74 28 Mm2737 Rv1860 APA 6 32% 100 33.9 4.74 29 Mm5439 Rv3864 EspA-like 3 15% 100 43.5 4.42 30 Mm5449 Rv3874 CFP-10 3 46% 100 10.7 4.56 31 Mm5449 Rv3874 CFP-10 5 71% 100 10.7 4.56 32 Mm5449 Rv3874 CFP-10 5 71% 100 10.7 4.56 33 Mm5449 Rv3874 CFP-10 5 71% 100 10.7 4.56 34 Mm5450 Rv3875 ESAT-6 2 38% 100 9.9 4.67 35 Mm5450 Rv3875 ESAT-6 2 38% 100 9.9 4.67 36 Mm5457 Rv3881c Ala/Gly-rich protein 9 26% 100 46.9 4.52 37 Mm5457 Rv3881c Ala/Gly-rich protein 5 13% 100 46.9 4.52

* this protein was identified by EndoGlu digestion, Mm is M. marinum; Mt, M. tuberculosis and Mu, Mycobacterium ulcerans. Wild-type strain M: spots # 1-9 Wild-type strain E11: spots # 10-22 Mx2, ESX-5 mutant of M: spot s # 23-26 7C1, ESX-5 mutant of E11: spot s # 27-37


To determine if this antibody is able to recognize also other PE_PGRS proteins three randomly chosen PE_PGRS genes were heterologously expressed in E. coli and tested. As can be seen in Fig. 6B, all three PE_PGRS proteins were recognized by this antiserum. To determine the specificity of the antibody, a number of different mycobacterial species were tested on immunoblot. M. tuberculosis, M. bovis and M. marinum all have a large amount of PE_PGRS genes32, M. avium contains ESX-5 and a number of PE genes, but no PE_PGRS genes, while fast-growing species like M. smegmatis contains only two PE genes and no PE_PGRS genes or ESX-532. As can be seen in Fig. 6C only species containing PE_PGRS genes produce proteins that react with the antiserum, indicating specificity for PE_PGRS proteins.

Fig. 6 | Secreted PE_PGRS proteins are dependent on ESX-5. (A,B,C) Characterization of the PGRS-specific monoclonal antibody and (D) analysis of culture filtrate proteins of M. marinum wild-type strains and ESX-5 mutants. (A) PGRS monoclonal antibody is directed against the PGRS domain and not against the PE domain. Purified His-tagged recombinant PE_PGRS33 (lanes 2 and 5) or the PE domain of PE_PGRS33 (lanes 3 and 6) was separated on SDS-PAGE and analyzed by coomassie stain (lanes 1-3) and immunoblot (lanes 4-6). (B) The PGRS monoclonal recognizes different, randomly chosen, and heterologously expressed PGRS proteins. Immunoblot of purified His-tagged recombinant PE_PGRS33 (lane 2), PE_PGRS1 (lane 3), PE_PGRS18 (lane 4) and PE_PGRS24 (lane 5). (C) The PGRS monoclonal is specific, it recognizes (PE_PGRS) proteins of of M. bovis BCG (lane 1), M. tuberculosis CDC 1551 (lane 2), M. marinum (lane 3), but it does not recognize any protein of M. avium 104 (lane 4) and M. smegmatis mc2155 (lane 5), which both do not contain PE_PGRS genes. (D) Immunoblot analysis of equivalent amounts of collected short-term culture filtrates of M. marinum wild-type M, E11 strains and their ESX5-mutants.


Subsequently, this monoclonal was used to study the supernatant of our two different M. marinum strains and their ESX-5 mutants. The supernatant of both the wild-type strains contained various bands cross-reacting with the antibody, whereas only a faint reaction was seen in the supernatant of the ESX-5 mutants (Fig. 6D). To confirm that different PE_PGRS proteins are visualized by this antiserum we also performed these experiments on 2D gels (results not shown) and analyzed the antibody-reacting spots by MS analysis (Table 3). All spots that could be identified by tandem MS represented PE_PGRS proteins and in total 7 different PE_PGRS proteins were identified in the culture filtrates. Together, these results indicate that all PE_PGRS proteins secreted in culture medium are dependent on an active ESX-5 system. The ESX-5 mutant secretes increased amounts of ESX-1 proteins

Analysis of proteins that are present in higher amounts in the culture supernatant of the ESX-5 mutants showed that proteins already known to be secreted into the culture supernatant, such as CFP17 and cold shock protein (CspA) (Fig. 4A-Mx2) are present. In addition, APA protein was present at a different position in the ESX-5 mutant, as already discussed above. The most surprising result, however, was the fact that two ESX-1 encoded proteins, i.e. Mm5439 and Mm5457, were present in substantially higher amounts in the culture supernatant of the ESX-5 mutant 7C1 (Fig. 4, compare spots 22 with 36 and 21 with 29). These proteins are orthologues of Rv3864 and Rv3881c, respectively. Mm5457 has recently been shown to be secreted by ESX-124, but the presence of Mm5439 in the culture supernatant is novel. These two proteins are not present in the supernatant of the M strain, which was also unable to secrete the ESX-1 substrates ESAT-6 and CFP-10. Previously, we have shown that an ESX-1 mutation results in increased secretion via ESX-516. Our present findings expand on this observation by showing that the reverse might also be true. To confirm the increased activity of the ESX-1 pathway, we studied the secretion of Mm5457 and ESAT-6 directly by immunoblot analysis. Short-term culture filtrates of wild-type strain E11 and its ESX-5 mutant showed that, whereas the secretion of ESAT-6 was comparable in both strains, Mm5457 was indeed present in increased amounts in the supernatant of the ESX-5 mutant (Fig. 7). This increased secretion of Mm5457 was correlated with a substantially decreased amount of precursor protein in the cell pellet (Fig. 7), which confirms the increased secretion of this ESX-1 substrate.

102

Table 3 | Summary of different PE_PGRS proteins visualized by PGRS antiserum from culture filtrate of M. marinum wild-type E11 strain.

Mm gene

number

Gene product

Number of

peptides

Protein

coverage

Protein Score

C. I. %

Protein

MW (KDa)

Protein

PI

Mm2933 Mm-specific PE_PGRS 1 2% 100 53.2 3.7


Mm3105 Mm- and Mu-specific PE_PGRS 2 7% 100 64.4 3.97










Fig. 7 | The M. marinum homologue of the ESX-1 substrate Rv3881c (Ala/Gly-rich protein Mm5457) is secreted in increased amounts by the ESX-5 mutant. Immunoblot analysis using monoclonal antibody against ESAT-6 or against Rv3881c after collection of short-term culture filtrates of M. marinum wild-type E11 strains and ESX-5 mutant 7C1. The cell pellet contains a band of higher molecular weight, representing the unprocessed form of Mm5457.

Discussion

In our previous study we have shown that M. marinum contains a functional ESX-5 type VII secretion system16, which is responsible for the secretion of heterologously expressed PPE4116. The ESX-5 system is recently evolved in the mycobacteria, and its appearance in evolution predates the expansion of the PE and PPE genes32. This finding led us to hypothesize that ESX-5 is a specialized protein secretion pathway that is devoted to the transport of the recently evolved subclasses of PE and PPE proteins, i.e. PE_PGRS and PPE-MPTR. By comparing the profile of proteins secreted by M. marinum wild-type and ESX-5 mutants we now show that a large number of the ESX-5-dependent proteins are indeed PE_PGRS and PPE-MPTR proteins. With 69 members in M. tuberculosis and 102 members in M. marinum, the PE_PGRS proteins form by far the largest subfamily of PE proteins34. This family is characterised by polymorphic GC-rich repeats in the C-terminal half of the protein. Despite their abundance, very little is known about the function of the proteins encoded by these multigene families. Multiple laboratories have shown that some PE_PGRS and PPE proteins are located at the cell surface28,38,44-46, although the secretion route was never established. Using a new monoclonal antibody we were able to show that all secreted PE_PGRS proteins were in fact dependent on ESX-5. This means that ESX-5 is a major secretion pathway for extracellular proteins in M. marinum and most probably also in M. tuberculosis. ESX-1, the prototypic Type VII secretion system, is responsible for the secretion of five different proteins20,22-24. In this first study on the


secretome of ESX-5 we have already identified more than 20 proteins that are secreted via this second type VII secretion system and, if indeed numerous PE_PGRS proteins are secreted via ESX-5, this would mean that in total more than 100 proteins are secreted via this pathway in M. marinum. Although we identified numerous PE_PGRS and PPE proteins among the ESX-5-dependent secreted proteins, we cannot rule out that other PE and PPE proteins may be secreted via other secretion pathways, such as via the other ESX secretion systems. For instance, other studies have shown that the M. tuberculosis PE_PGRS33 protein is surface exposed when expressed heterologously in M. smegmatis38,46. Since M. smegmatis does not have an ESX-5 secretion system this would mean that this PE_PGRS protein is secreted via another secretion pathway. Furthermore, the ancestral PE and PPE proteins, present within and duplicated from ESX clusters 1 to 3, could be secreted by their respective clusters and not through ESX-532. Probably, the PE and PPE gene families have evolved in conjunction with the ESX secretion systems and the most recently duplicated members, i.e. the PE_PGRS and PPE-MPTR subfamilies, are specifically linked to ESX-532. Proteomic analysis also showed that in many cases, the same ESX-5 substrate was identified from different 2-DE spots with different molecular mass and acidic isoelectric point (pI) regions, possibly as a consequence of posttranslational modification or proteolytic processing. Similar processing was also observed for one of the HA-tagged PPE proteins, which could mean that the N-terminal PE and PPE domains are only present during transport and/or cell surface localization. Recent experiments have shown that the PE domain of PE_PGRS proteins is responsible for the transport of these proteins, indicating that this domain could function as a sort of leader peptide47. The recently identified ESX-1 substrate Rv3881c (EspB) was also shown to be processed24, which could indicate that processing is common in ESX secretion pathways. In addition to PE_PGRS and PPE proteins, we have also identified other proteins (Table 2) as ESX-5-substrates. EsxN and EsxP are ESAT-6-like proteins and it was not surprising to detect them in the culture filtrate as EsxN is encoded by ESX-5 and EsxP is a paralogue of its partner EsxM. A problem with detecting EsxN is that both M. tuberculosis and M. marinum are coding for several (3-6) proteins that are highly homologous to EsxN (more than 90% identity)36. Thus, when antibodies are used, it cannot be concluded with confidence which of these proteins is seen on immunoblot. These proteins are therefore also collectively known as Mtb9.936. Both EsxN-like and EsxM-like proteins have been identified previously in culture filtrates of M. tuberculosis23,36,48, although the transport pathway was never identified. Furthermore, we also identified an unexpected putative ESX-5 substrate, namely cutinase 3, the M. marinum orthologue of Rv3451. However, at present it cannot be concluded that ESX-5 is involved in Cut3 secretion. For instance, the APA protein was present at different positions in the supernatant of both the wild-type and the ESX-5 mutant. Different isoforms of APA have been observed previously and it has been shown that they arise by extracellular


processing at the C-terminus43. Absence of this processing step in the ESX-5 mutant could indicate that, instead of APA, the processing enzyme is in fact an ESX-5 substrate. A similar situation could be true for Cut3. In our previous study, we have shown that a mutant devoid of the ESX-1 substrates ESAT-6/CFP-10 secretes significantly higher amounts of the ESX-5 substrate PPE4116, suggesting cross-inhibition between these two secretion systems. Consistent with the above finding, we now show that the ESX-5 mutant 7C1 in fact secretes significantly higher amounts of two proteins encoded by the ESX-1 cluster, i.e. the M. marinum homologues of Rv3864 and Rv3881c. Previous studies have already shown that Rv3881c (MTB48) is secreted by M. tuberculosis37, and this secretion is dependent on the ESX-1 pathway24. The Rv3864 homologue has not been shown previously to be secreted, but because this protein is homologous to the ESX-1 secreted protein EspA22, it is probably also a substrate of the ESX-1 secretion pathway. Taken together, these results reveal that there is cross-inhibition between these two systems. Blockage of the ESX-5 pathway may result in the upregulation of ESX-1 or in enhanced ESX-1 activity. Paradoxically, the secretion of ESAT-6, the most well-known substrate of ESX-1, was not increased in the ESX-5 mutant. Further studies are required to substantiate the cross-talk between the ESX-1 and ESX-5 system. An unexpected finding of these experiments was that, although the E11 strain secretes high amounts of ESAT-6 and CFP-10, the M strain does not. However, from the literature it is known that the M strain does secrete ESAT-6 efficiently18. Analysis of a different M isolate obtained form the Brown lab also confirmed these results (results not shown), which means that our first M. marinum M isolate, now designated Mvu, somehow acquired a mutation affecting ESAT-6 expression and/or secretion. Because the extended RD1 region is, together with the espA locus (Rv3614c-Rv3616c), the only known region that is required for ESAT-6 secretion18,22,23, it seems likely that the Mvu strain was disturbed in ESAT-6 secretion by a spontaneous mutation in this region. However, our efforts to determine the precise component responsible for this defect have thus far been unsuccessful (results not shown). In summary, we have provided experimental data that shows that ESX-5 is responsible for the transport of many PE_PGRS proteins secreted in culture and various PPE proteins (specifically of the MPTR subfamily). The ESX-5 substrates are secreted in the culture supernatant or present on the bacterial cell surface. Because specialized secretion systems are key virulence determinants of many bacterial pathogens2, studies are underway to determine the role of the ESX-5 cluster in virulence. Acknowledgements

We acknowledge Mark Alderson (Corixa Corporation, Seattle, USA), and Bryan McLaughlin (UCSF, San Francisco, USA) for the Mtb9.9a and Rv3881c antibodies, respectively. We also thank Jaco Knol for help with MS.


References

1. Thanassi,D.G. & Hultgren,S.J. Multiple pathways allow protein secretion across the bacterial outer membrane. Current Opinion in Cell Biology 12, 420-430 (2000).

2. Lee,V.T. & Schneewind,O. Protein secretion and the pathogenesis of bacterial infections. Genes & Development 15, 1725-1752 (2001).

3. Finlay,B.B. & Falkow,S. Common themes in microbial pathogenicity revisited. Microbiology and Molecular Biology Reviews 61, 136-& (1997).

4. DiGiuseppe Champion,P.A. & Cox,J.S. Protein secretion systems in Mycobacteria. Cell Microbiol. 9, 1376-1384 (2007).

5. Fratti,R.A., Chua,J., Vergne,I. & Deretic,V. Mycobacterium tuberculosis glycosylated phosphatidylinositol causes phagosome maturation arrest. Proceedings of the National Academy of Sciences of the United States of America 100, 5437-5442 (2003).

6. Stewart,G.R., Patel,J., Robertson,B.D., Rae,A. & Young,D.B. Mycobacterial mutants with defective control of phagosomal acidification. Plos Pathogens 1, 269-278 (2005).

7. Malik,Z.A. et al. Mycobacterium tuberculosis blocks Ca2+ signaling and phagosome maturation in human macrophages via specific inhibition of sphingosine kinase. Journal of Immunology 170, 2811-2815 (2003).

8. Beatty,W.L. & Russell,D.G. Identification of mycobacterial surface proteins released into subcellular compartments of infected macrophages. Infection and Immunity 68, 6997-7002 (2000).

9. Andersen,P. Effective Vaccination of Mice Against Mycobacterium-Tuberculosis Infection with A Soluble Mixture of Secreted Mycobacterial Proteins. Infection and Immunity 62, 2536-2544 (1994).

10. Roberts,A.D. et al. Characteristics of Protective Immunity Engendered by Vaccination of Mice with Purified Culture Filtrate Protein Antigens of Mycobacterium-Tuberculosis. Immunology 85, 502-508 (1995).

11. Horwitz,M.A., Lee,B.W.E., Dillon,B.J. & Harth,G. Protective Immunity Against Tuberculosis Induced by Vaccination with Major Extracellular Proteins of Mycobacterium-Tuberculosis. Proceedings of the National Academy of Sciences of the United States of America 92, 1530-1534 (1995).

12. Wiker,H.G. & Harboe,M. The Antigen-85 Complex - A Major Secretion Product of Mycobacterium-Tuberculosis. Microbiological Reviews 56, 648-661 (1992).

13. Jungblut,P.R. et al. Comparative proteome analysis of Mycobacterium tuberculosis and Mycobacterium bovis BCG strains: towards functional genomics of microbial pathogens. Molecular Microbiology 33, 1103-1117 (1999).

14. Rosenkrands,I. et al. Mapping and identification of Mycobacterium tuberculosis proteins by two-dimensional gel electrophoresis, microsequencing and immunodetection. Electrophoresis 21, 935-948 (2000).

15. Gomez,L., Johnson,S. & Gennaro,M.L. Identification of secreted proteins of Mycobacterium tuberculosis by a bioinformatic approach. Infection and Immunity 68, 2323-2327 (2000).


17. Converse,S.E. & Cox,J.S. A protein secretion pathway critical for Mycobacterium tuberculosis virulence is conserved and functional in Mycobacterium smegmatis. Journal of Bacteriology 187, 1238-1245 (2005).

https://www.researchgate.net/publication/12444786_Multiple_pathways_allow_protein_secretion_across_the_bacterial_outer_membrane?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=





https://www.researchgate.net/publication/12592565_Identification_of_Secreted_Proteins_of_Mycobacterium_tuberculosis_by_a_Bioinformatic_Approach?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=



https://www.researchgate.net/publication/21675833_The_Antigen_85_Complex_a_Major_Secretion_Product_of_Mycobacterium_tuberculosis?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=

https://www.researchgate.net/publication/21675833_The_Antigen_85_Complex_a_Major_Secretion_Product_of_Mycobacterium_tuberculosis?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=

https://www.researchgate.net/publication/15287828_Effective_vaccination_of_mice_against_M_tuberculosis_infection_with_a_soluble_mixture_of_secreted_mycobacterial_proteins?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=



https://www.researchgate.net/publication/12243617_Identification_of_Mycobacterial_Surface_Proteins_Released_into_Subcellular_Compartments_of_Infected_Macrophages?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=



https://www.researchgate.net/publication/10797716_Mycobacterium_tuberculosis_glycosylated_phosphatidylinositol_causes_phagosome_maturation_arrest?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=




https://www.researchgate.net/publication/15321242_Protective_immunity_against_tuberculosis_induced_by_vaccination_with_major_extracellular_protein_of_M_tuberculosis?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=







https://www.researchgate.net/publication/11881842_Protein_secretion_and_the_pathogenesis_of_bacterial_infections?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=

https://www.researchgate.net/publication/11881842_Protein_secretion_and_the_pathogenesis_of_bacterial_infections?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=


18. Gao,L.Y. et al. A mycobacterial virulence gene cluster extending RD1 is required

for cytolysis, bacterial spreading and ESAT-6 secretion. Molecular Microbiology 53, 1677-1693 (2004).



21. Abdallah,A.M. et al. Type VII secretion--mycobacteria show the way. Nat. Rev. Microbiol. 5, 883-891 (2007).


23. MacGurn,J.A., Raghavan,S., Stanley,S.A. & Cox,J.S. A non-RD1 gene cluster is required for Snm secretion in Mycobacterium tuberculosis. Molecular Microbiology 57, 1653-1663 (2005).

24. McLaughlin,B. et al. A Mycobacterium ESX-1-Secreted Virulence Factor with Unique Requirements for Export. PLoS Pathog. 3, e105 (2007).

25. Mahairas,G.G., Sabo,P.J., Hickey,M.J., Singh,D.C. & Stover,C.K. Molecular analysis of genetic differences between Mycobacterium bovis BCG and virulent M-bovis. Journal of Bacteriology 178, 1274-1282 (1996).

26. Gordon,S.V. et al. Identification of variable regions in the genomes of tubercle bacilli using bacterial artificial chromosome arrays. Molecular Microbiology 32, 643-655 (1999).

27. Behr,M.A. et al. Comparative genomics of BCG vaccines by whole-genome DNA microarray. Science 284, 1520-1523 (1999).

28. Pym,A.S., Brodin,P., Brosch,R., Huerre,M. & Cole,S.T. Loss of RD1 contributed to the attenuation of the live tuberculosis vaccines Mycobacterium bovis BCG and Mycobacterium microti. Molecular Microbiology 46, 709-717 (2002).

29. Lewis,K.N. et al. Deletion of RD1 from Mycobacterium tuberculosis mimics bacille Calmette-Guerin attenuation. Journal of Infectious Diseases 187, 117-123 (2003).

30. Majlessi,L. et al. Influence of ESAT-6 secretion system 1 (RD1) of Mycobacterium tuberculosis on the interaction between mycobacteria and the host immune system. J. Immunol. 174, 3570-3579 (2005).


32. Gey van Pittius,N.C. et al. Evolution and expansion of the Mycobacterium tuberculosis PE and PPE multigene families and their association with the duplication of the ESAT-6 (esx) gene cluster regions. Bmc Evolutionary Biology 6, (2006).

33. Cole,S.T. et al. Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393, 537-+ (1998).

34. Brennan,M.J., Espitia,C. & Gey von Pittius,N. S.Cole, D.N.McMurray, K.Eisenach, B.Gicquel & W.R.Jacobs (eds.), pp. 513-525 (ASM Press, Washington, DC.,2004).

https://www.researchgate.net/publication/277532544_Comparative_Genomics_of_BCG_Vaccines_by_Whole-Genome_DNA_Microarray?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=

https://www.researchgate.net/publication/277532544_Comparative_Genomics_of_BCG_Vaccines_by_Whole-Genome_DNA_Microarray?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=


35. Towbin,H., Staehelin,T. & Gordon,J. Electrophoretic Transfer of Proteins from

Polyacrylamide Gels to Nitrocellulose Sheets - Procedure and Some Applications. Proceedings of the National Academy of Sciences of the United States of America 76, 4350-4354 (1979).

36. Alderson,M.R. et al. Expression cloning of an immunodominant family of Mycobacterium tuberculosis antigens using human CD4(+) T cells. Journal of Experimental Medicine 191, 551-559 (2000).

37. Lodes,M.J. et al. Serological expression cloning and immunological evaluation of MTB48, a novel Mycobacterium tuberculosis antigen. Journal of Clinical Microbiology 39, 2485-2493 (2001).

38. Brennan,M.J. et al. Evidence that mycobacterial PE_PGRS proteins are cell surface constituents that influence interactions with other cells. Infection and Immunity 69, 7326-7333 (2001).

39. Margulies,D.H. et al. Regulation of Immunoglobulin Expression in Mouse Myeloma Cells. Cold Spring Harbor Symposia on Quantitative Biology 41, 781-791 (1976).


41. van der Sar,A.M. et al. Mycobacterium marinum strains can be divided into two distinct types based on genetic diversity and virulence. Infection and Immunity 72, 6306-6312 (2004).

42. Laqueyrerie,A. et al. Cloning, Sequencing, and Expression of the Apa Gene Coding for the Mycobacterium-Tuberculosis 45/47-Kilodalton Secreted Antigen Complex. Infection and Immunity 63, 4003-4010 (1995).

43. Horn,C. et al. Decreased capacity of recombinant 45/47-kDa molecules (Apa) of Mycobacterium tuberculosis to stimulate T lymphocyte responses related to changes in their mannosylation pattern. Journal of Biological Chemistry 274, 32023-32030 (1999).

44. Banu,S. et al. Are the PE-PGRS proteins of Mycobacterium tuberculosis variable surface antigens? Molecular Microbiology 44, 9-19 (2002).

45. Sampson,S.L. et al. Expression, characterization and subcellular localization of the Mycobacterium tuberculosis PPE gene Rv1917c. Tuberculosis 81, 305-317 (2001).

46. Delogu,G. et al. Rv1818c-encoded PE_PGRS protein of Mycobacterium tuberculosis is surface exposed and influences bacterial cell structure. Molecular Microbiology 52, 725-733 (2004).

47. Cascioferro,A. et al. PE is a functional domain responsible for protein translocation and localization on mycobacterial cell wall. Mol. Microbiol. 66, 1536-1547 (2007).

48. Mattow,J. et al. Comparative proteome analysis of culture supernatant proteins from virulent Mycobacterium tuberculosis H37Rv and attenuated M-bovis BCG Copenhagen. Electrophoresis 24, 3405-3420 (2003).

https://www.researchgate.net/publication/22754145_Electrophoretic_Transfer_of_Proteins_From_Polyacrylamide_Gels_to_Nitrocellulose_Sheets_Procedure_and_Some_Applications?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=




The ESX-5 secretion system of Mycobacterium marinum modulates the macrophage response

Abdallah M. Abdallah*, Nigel DL. Savage‡, Fredericke Hannes*, Maaike van Zon§, Christina M.J.E. Vandenbroucke-Grauls*, Nicole van der Wel*,§, Tom H. M. Ottenhoff‡ and Wilbert Bitter*

*Department of Medical Microbiology and Infection Control, VU University medical centre, Amsterdam, The Netherlands. ‡Department of Immunohematology & Blood Transfusion, and Department of Infectious Diseases, Leiden University Medical Centre, Leiden, The Netherlands. §The Netherlands Cancer institute, Antoni van Leeuwenhoek Hospital, Amsterdam, The Netherlands.

Ch

apter 6

Manuscript in preparation

Chapter 6: The ESX-5 secretion system modulates the macrophage response 110

Abstract

The ESX-5 secretion system of pathogenic mycobacteria is responsible for the secretion of various PPE and PE_PGRS proteins. To better understand the role of ESX-5 effector proteins in virulence, we analysed interactions of ESX-5 Mycobacterium marinum mutants and human peripheral blood monocyte-derived macrophages. Both wild type and the ESX-5 mutant were internalised, resulting in activation of macrophages as reflected by enhanced expression of HLA-DR, CD80 and CD86 surface antigens. Furthermore, the ESX-5 mutation did not affect the escape of mycobacteria from phagolysosomes into cytosol, as was shown by electron microscopy. However, whereas wild-type M. marinum failed to induce appreciable levels of IL-12p40, TNF-α and IL-6, the ESX-5 mutants strongly induced the production of these pro-inflammatory cytokines. By contrast, infection with M. marinum wild-type strain resulted in a significant induction of IL-1β production as compared to the ESX-5 mutants. These results show that ESX-5 plays an essential role in the secretion of immune cytokines. Subsequently, we have shown that an intact ESX-5 secretion system is needed for the active suppression of various TLR receptor-signalling events in macrophages. Finally, the ESX-5 mutant was significantly impaired in the induction of macrophage cell death, as was shown by different techniques. Together, our results show that ESX-5 substrates, directly or indirectly, manipulate the immune response at various different levels.

Introduction

Most pathogenic mycobacteria are intracellular pathogens that reside and multiply within the macrophages (Mϕ) of their host. These intracellular pathogens have evolved sophisticated mechanisms to manipulate host cell organelles and membrane trafficking1. They modify natural biological processes of host cells in order to create an environment that is favourable to their survival. During early stages of infection, the extent of mycobacterial survival and proliferation is mainly determined by the efficacy of the innate immune response2. For instance, Mϕ express a variety of antimicrobial responses to control intracellular bacilli, such as bactericidal peptides and reactive oxygen- and nitrogen-intermediates. In addition, infected Mϕ initiate adaptive T-cell immunity by antigen presentation and the induction of cellular immune responses3,4. Among these cellular immune responses, production of pro-inflammatory cytokines plays a crucial role5-9. These cytokines include TNF-α, which plays a key role in granuloma formation10,11, IL-612 and IL-12, a pivotal cytokine in the host defence against mycobacteria8,13,14. One of the mechanisms used by mycobacteria to modify Mϕ functional properties is the ability to alter Mϕ signalling required for the secretion of pro-inflammatory


cytokines15,16. However, how pathogenic mycobacteria are able to subvert cytokine responses is still unclear. Subversion of eukaryotic host responses by bacterial pathogens often requires specialized secretion systems that deliver effector proteins near or directly into host cells. Transport of proteins across bacterial membranes is a complex process requiring multi-component machineries spanning the bacterial cell wall. Recently, a novel secretion pathway has been identified in mycobacteria, which has been classified as type VII secretion system17. The best-characterized type VII secretion system is encoded by the ESX-1 locus. This locus includes the RD1 region, which is deleted in the BCG vaccine strain. ESX-1 is involved in the secretion of the potent T cell antigens ESAT-6 and CFP-1018,19 and this secretion system plays a major role in the virulence of different pathogenic mycobacteria, such as M. tuberculosis and M. marinum17,20. An active ESX-1 system is crucial for the escape of M. tuberculosis from the phagolysosome into the cytosol of infected Mϕ21. This cytosolic location predates and is important for induced cell death of infected Mϕ21. ESX-1 is however not the only type VII secretion system in mycobacteria, pathogenic mycobacteria may contain as many as five different type VII secretion systems. Recently, we have shown that one of these systems, i.e. ESX-5, is responsible for the transport of PE_PGRS and PPE-MPTR proteins22,23. The PE and PPE protein families are unique to mycobacteria and both are highly expanded in several pathogenic species, such as M. tuberculosis and M. marinum24. Although their exact function is unknown a role in virulence25,26, antigenic variation or immune evasion has been predicted27-30. In this study we characterize the effects of M. marinum ESX-5 system on Mϕ function and on bacterial growth in vivo. Our results show that ESX-5 effector molecules seem to manipulate the induction of different Mϕ cytokines and TLRs. Furthermore; we present evidence that ESX-5 system is involved in Mϕ cell death.



Wild-type M. marinum E11 strain, mutant 7C1 and complemented 7C1 were routinely grown in Middlebrook 7H9 liquid medium or Middlebrook 7H10 agar supplemented with 10% Middlebrook ADC or OADC respectively (BD, Biosciences) and 0.05% Tween 80. For infection experiments, M. marinum cultures were grown to logarithmic phase (OD600 = 0.5-0.8) in 7H9 media, washed and diluted in RPMI-1640 with 10% foetal calf serum (FCS). To eliminate clumps of bacteria, the M. marinum suspensions were subjected to low speed centrifugation (300g, 10 min) after which the supernatant was passed through a 5 µm filter and used at an MOI of 10 bacteria per cell, unless indicated otherwise. Antibiotics were added at the following concentrations: kanamycin, 25 µg ml-1, hygromycin, 50 µg ml-1.


Cells lines and culture conditions

For apoptosis and phagosome isolation experiments the human acute pro-monocytic leukaemia THP-1 cell line and the mouse monocyte Mϕ RAW cell line were used. Cells were cultured in RPMI-1640 with glutamax-1 media (GIBCO, BRL) supplemented with 10% FCS, streptomycin and penicillin. Monocytic differentiation into Mϕ-like cells was induced with PMA (phorbol 12-myristate 13-acetate, 1 ng ml-1, Sigma) at 10ng/ml final concentration (overnight incubation).

Isolation and Culture of Human Macrophages

Monocytes were isolated to high purity from peripheral blood of healthy donors by magnetic cell sorting using anti-CD14-coated beads; isolated cells were polarized with granulocyte–Mϕ colony-stimulating factor to obtain type 1 Mϕ (Mϕ-1) or with Mϕ colony-stimulating factor to generate type 2 Mϕ (Mϕ-2) as described previously33.

Infection of macrophages

THP-1 and RAW cells were used between passages 13 and 20. For phagosome isolation, cells were seeded in 7.5 cm diameter flasks. For apoptosis assays, cells were seeded at 2 x 105 and 5 x 104 cells per well in 96-well flat-bottomed tissue culture plates or 5 x 105 and 2 x 105 cells per well in 24-well plates containing round glass coverslips, respectively. Human Mϕ-1 and Mϕ-2 were cultured in duplicate at density of 3 x 105 per well in 24-well culture plates or 9 x 105 per well in 6-well culture plates. Mononuclear cells were infected with bacteria and incubated for 1 h at 33°C and 5% CO2. The supernatant was removed and the infected cells were washed three times with medium to remove extracellular bacteria. Subsequently, the cells were incubated in fresh medium with FCS at 33°C for indicated time periods. For apoptosis assays, at each time point, 96-well tissue culture plates were centrifuged for 10 min at 200g, and the supernatants from the cells were used for measuring the release of histone-DNA complexes. The remaining cells were then lysed and immediately tested for sequestering of histone-DNA complexes in affected cells by enzyme-linked immunosorbant assay (ELISA). All infections were conducted in triplicate, and experiments were performed in duplicate. `

Cell death assays

The Cell Death Detection ELISAPLUS kit (Roche Molecular Biochemicals) was used to quantify M. marinum-induced DNA fragmentation as recommended by the manufacturers. To determine the presence of apoptosis in infected Mϕ, the TUNEL assay (Roche Molecular Biochemicals) was used. Cells were considered apoptotic if they were TUNEL positive (green fluorescence nuclear staining). The


percentage of apoptotic cells was also determined using Annexin V staining of membrane alterations and propidium iodide (PI) staining of dead cells. For annexin V-PE binding, cells were collected, washed twice with cold PBS and resuspended in annexin V-PE binding buffer (10 mM HEPES, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2). An aliquot of 100 µl (~1 x 105 cells) was removed and mixed with 5 µl of annexin V-PE and 2 µl of PI. The mixture was vortexed and incubated for 15 min at room temperature in the dark. The cells were washed once with binding buffer and resuspended in 500 µl binding buffer for analysis by flow cytometry. 10,000 events were collected for each condition, and the percentages of Annexin-V-positive and PI-negative cells were determined.

Cytokine measurements

Cultured human Mϕ (Mϕ-1 and Mϕ-2) supernatants were assayed for cytokine levels using specific ELISAs for IL-10 and IL-12p40 (purchased from BioSource International, Camarillo, CA; sensitivity: 20 pg/ml). The concentration of IL-1β, IL-6 and TNF-α was measured by Fluorescent Bead Immunoassay (Bio-Plex human cytokine assay, Bio-Rad).

Mycobacterial phagosomes isolation and analysis

Isolation of mycobacterial phagosomes was performed as described previously22. Briefly, infected THP-1 cells (7-10x107 with MOI of 25 for 48 h) were homogenized in DGE buffer (10 mM triethanolamine, 10 mM acetic acid, 1 mM EDTA, 0.25 M sucrose, pH 7.4) by passaging through a 23-gauge needle. After removal of the cell nuclei by low speed centrifugation (250g, 10 min), the resulting post-nuclear supernatant (PNS) was transferred to a fresh Eppendorf tube and sedimented at 35,000g for 30 min. The resulting supernatant corresponds to the Mϕ cytosol, whereas the pellet corresponds to the phagosomes containing the mycobacteria. The bacteria-free phagosomes were recovered, by treating them with 1% Triton X-100 for 15 min at room temperature and sedimenting the intact mycobacteria at 35,000g for 30 min. The resulting supernatant corresponds to the phagosomes and the pellet to the mycobacteria. All protein mixtures were separated by SDS/PAGE using 10-12% polyacrylamide gels. Proteins were visualized by immunoblotting using antibodies directed against PE_PGRS23; GroEL (Colorado State University, Fort Collins, CO, and the National Institutes of Health, Bethesda, MD, contract NO1 AI-75320); and LAMP-1 (H4A3, obtained from the Hybridoma Bank of the University of Iowa, Iowa City).

Infection of zebrafish embryos

Zebrafish embryos were infected with 100 colony-forming units (CFU) at 32 h post-fertilization by microinjection in the caudal vein. To assess the virulence of the ESX-5 mutant, cohorts of 50 embryos for each strain, hatched at the same


time, were injected with PBS as a control or infected with either the M. marinum E11 wild type, the ESX-5 mutant or the complemented strain. These infected embryos were monitored at 1 h post-infection, to verify that the inocula were similar, and day 1, 3 and 5 post-infection (p.i.) for mycobacterial growth by whole embryo plating. Ten embryos were plated for each time point.

Results

ESX-5 is not required for escape of M. marinum into the cytosol

The intracellular mycobacterial pathogens M. marinum and M. tuberculosis have recently been shown to escape from the phagolysosome into the cytosol of host cells21,31, and effector molecules of the ESX-1 pathway are required for this translocation21. To gain further insight into this process, we examined the effect of the ESX-5 pathway on the routing of mycobacteria in Mϕ. First, we sought to determine the role of ESX-5 pathway on translocation of M. marinum into the cytosol. To address this, we compared the intracellular localization of the M. marinum wild-type versus ESX-5 mutant by cryo-immunogold electron microscopy as described previously32. THP-1 cells infected with M. marinum wild type or ESX-5 mutant were fixed and processed for cryo-immunogold labelling with anti-LAMP-1 antibodies. After 48 hr of infection, a substantial proportion of both M. marinum wild type and ESX-5 mutant were not detected within LAMP1 positive vesicles. Significantly, these bacteria were not present in membrane-enclosed compartment and were localized to the cytosol of the infected Mϕ (Fig. 1). This means that ESX-5 effector molecules have no effect on the routing of these bacteria in the infected Mϕ.

Figure 1 | M. marinum wild-type E11 strain and ESX-5 mutant translocate from the phagolysosome to the cytosol of the host cell. Representative electron micrograph (EM) image of THP-1 cell infected with M. marinum wild-type E11 strain (A), or ESX-5 mutant (B) for 48 h, immunolabeled for LAMP-1 with gold particles. Asterisks indicates mycobacteria, G indicates Golgi, M indicates mitochondrium, and N indicates nucleus.


Next, we tested whether the ESX-5 effector molecules are secreted in human Mϕ. To do this, THP-1 cells differentiated into Mϕ-like cells in the presence of PMA were infected with M. marinum E11 and differentially disrupted. The various subcellular fractions were analysed by immunoblot for the presence of PE_PGRS proteins, the major class of ESX-5 substrates23. In addition, immunoblots containing these fractions were incubated with antibodies directed against the cytoplasmic protein GroEL and the late-endosome/lysosome marker LAMP-1 as controls. Approximately half of the PE_PGRS proteins were found associated with the bacteria, which could represent intrabacillary or surface located PE_PGRS molecules. However, cytosol fractions of cells infected for 48 hr also showed a significant amount of PE_PGRS proteins (Fig. 2, lane 2). As the cytoplasmic protein GroEL was exclusively present in the bacteria-containing fraction (Fig. 2), bacterial lysis inside Mϕ was not significant. The other control protein, LAMP-1, was identified mainly in the Triton X-100-soluble fraction, representing the contents of Mϕ-derived vesicles as expected (Fig. 2). In conclusion, this experiment shows that PE_PGRS proteins are indeed secreted inside Mϕ and in particular after 48 hours of infection and in the fraction representing the Mϕ cytosol. Figure 2 | Intracellular secretion of PE_PGRS by M. marinum. Immunoblot analysis of THP-1 cells infected for 24or 48 h with M. marinum wild-type E11 strain. Infected cells were homogenized and cell debris was removed by low-speed centrifugation. Subsequently, mycobacteria-containing phagsosomes and other vesicles (lane 1) were separated from the cell cytosol (lane 2) by sedimentation. Isolated phagosomes were lysed by Triton X-100 treatment (lane 3) and bacteria were collected by sedimentation (lane 4). As a control we also analysed uninfected THP-1 cells (lane 5). Immunoblot was analysed for the presence of PE_PGRS proteins, using monoclonal antibodies directed against the PGRS domain, for the presence of the cytoplasmic mycobacteria protein GroEL and for the presence of the late-endosome/lysosome marker protein (LAMP-1).


Dose response of M. marinum-induced macrophage cell death

Further, we assessed the relationship between intracellular bacillary load and macrophage viability, in order to determine the multiplicity of infection (MOI) at which infected cells showed minimal signs of cell death after 24 hours p.i. To this purpose, we used freshly isolated human peripheral blood monocytes differentiated into type 1 and type 2 Mϕ subsets as described previously33,34. Both Mϕ subsets were challenged with M. marinum wild-type E11 strain or ESX-5 mutants over an MOI ranging from 5 to 80. Mϕ viability was assessed by flow cytometry following propidium iodide (PI) and annexin V staining. Both strains caused low levels of Mϕ cell damage at low MOI, but a steep increase in PI-positive cells by 24 h was observed with MOIs above 10 (Fig. 3, data shown for Mϕ-1). In addition, M. marinum wild-type cells induced considerable stronger cytolytic activity as compared to the ESX-5 mutant (Fig. 3). Therefore, in the next experiments an MOI of 10 was used to analyse the cell surface phenotypes and to measure the cytokine response of infected Mϕ-1 and Mϕ-2 cells.

Figure 3 | High multiplicity of infection (MOI) challenge with M. marinum rapidly induces cell death in Mϕ-1 cells, whereas the ESX-5 mutant showed a more reduced effect. Mϕ-2 showed the same results (data not shown). Mϕ subsets were infected with M. marinum wild-type E11 strain or its isogenic ESX-5 mutant over a range of MOI for 24 h and stained with PI and Annexin V for analysis by flow cytometry to assess cell viability. A total of 10,000 cells were analysed per sample. Results are expressed as the mean percentage of PI-positive cells ± SE for two donors. Similar results were obtained in two individual experiments.


ESX-5 effector molecules modify phenotypic maturation of macrophages

Next, we wanted to analyse whether ESX-5 effector molecules play a role in altering cell surface expression of markers involved in antigen presentation and T-cell activation. Mφ-1 and Mφ-2 were infected with M. marinum wild type and the ESX-5 mutant and subsequently surface expression of MHC class II DR (HLA-DR), CD80 (B7.1), and CD86 (B7.2) was examined (Fig. 4). Mφ-1 cells infected with either M. marinum wild type or ESX-5-mutant showed no changes in the surface expression of the co-stimulatory molecules CD80, CD86 or HLA-DR, whereas the addition of LPS as a control resulted in the induction of CD80 (Fig. 4A). However, the Mφ-2 cells infected with the ESX-5 mutant showed marked differences when compared to cells infected with wild-type bacteria. Whereas M. marinum wild type suppressed the expression of CD80, CD86 and HLA-DR in Mφ-2 cells, cells infected with ESX-5 mutant showed some induction of these molecules (Fig. 4B). Down-regulation of HLA-DR, CD80 and CD86 in Mφ-2 by M. marinum is in line with previous results describing an attenuated capacity for MHC class II-restricted cells in antigen presentation and co-stimulation of Th cells following mycobacterial infection35-37. Therefore, we conclude that ESX-5 effector molecules may diminish the capacity of Mφ-2 for processing and presentation of mycobacterial antigens. Figure 4 | Expression of HLA and co-stimulatory molecules CD80 and CD86 on Mϕ-1 (A) and Mϕ-2 cells (B) infected with M. marinum wild-type E11 strain or ESX-5 mutant. Cells were infected with MOI of 10 for 24 h, and the cell surface phenotypes were analysed by FACS. A total of 10,000 cells were analysed per sample. As a control the cells were also stimulated with LPS. These are representative FACS profiles of one experiment, which was repeated three times, using Mϕ from different blood donors.


ESX-5 alters the innate immune responses of infected macrophages

Mϕ secrete a number of pro-inflammatory cytokines that are essential to elicit a protective immune response against intracellular pathogens such as mycobacteria. To determine whether ESX-5 secreted proteins are involved in the manipulation of these cytokine responses, we investigated the response of human Mϕ to infection with wild-type M. marinum strain E11 or with its isogenic ESX-5-mutant. Mϕ-1 cells infected with wild-type M. marinum produced low concentrations of IL-12p40 (Fig. 5A), as has been shown previously also for infection with M. tuberculosis13,14. On the other hand, Mϕ-1 infected with the ESX-5 mutant readily secreted high amounts of IL-12p40, in levels comparable with cells induced by LPS (Fig. 5A). IL-12p40 can form either IL-12 or IL-23, depending on which molecule it pairs with. It has been shown previously that Mϕ-1 cells mainly produce IL-23 upon infection with M. tuberculosis, whereas the production of IL-12 is dependent on the presence of exogenous IFN-γ33. Therefore, we also tested the expression of IL-12p40 in the presence of IFN-γ. For cells infected with M. marinum wild type, the presence of exogenous IFN-γ hardly affected the level of IL-12p40 production (Fig. 5A), whereas the ESX-5-mutant induced even higher levels of IL-12p40 in the presence of exogenous IFN-γ (Fig. 5A). A similar effect was observed for the induction of IL-6 and TNF-α, i.e. both these pro-inflammatory cytokines were strongly enhanced in Mϕ-1 infected with the ESX-5-mutant strain as compared to the parent strain (Fig. 5B, C). Together, these results show that the ESX-5 mutant seems to be unable to suppress the production of various pro-inflammatory cytokines by Mϕ-1. Mϕ-2 cells did not secrete significant amounts of IL-12p40, IL-6, or TNF-α upon mycobacterial infection (data not shown), as was also shown previously for M. tuberculosis33. Because IL-10 is a potent down-regulator of the immune response and known as an attenuator of IL-12p40 production in Mϕ13,38, we also measured the amount of IL-10 elicited by wild-type bacteria and the ESX-5 mutant from both Mϕ subsets. However, no IL-10 was detected in any of the conditions (data not shown).

Recognition of M. tuberculosis by human monocytes leads to IL-1β production13. Surprisingly, whereas IL-1β production was indeed enhanced in Mϕ-1 infected with wild-type M. marinum, no significant induction was detected in Mϕ-1 infected with ESX-5-mutant strain (Fig. 5D). Similar result was obtained for Mϕ-2 infected cells (data not shown). Since processing and secretion of IL-1β is dependent on the activation of caspase-139,40, our data may indicate that ESX-5 effector molecules somehow interfere with caspase-1 activation.


Figure 5 | ESX-5 effector proteins manipulate the Mϕ inflammatory response. Human monocyte-derived Mϕ-1 were infected with M. marinum wild type and the ESX-5 mutant, or treated with LPS as a control. Culture supernatants were collected after 24 h of infection and analysed for the presence of IL-12p40 by ELISA (A), or for IL-6 (B), TNF-α (C) and IL-1β (D) by Fluorescent Bead Immunoassay. Each sample was assayed in triplicate; error bars represent the means ± SE from at least three experiments. Similar results cytokine profiles were obtained with cells from at least 5 independent donors. To determine if an active process was responsible for the suppression of IL-12p40 production in cells infected with wild-type M. marinum, we mixed wild-type bacilli with either the ESX-5 mutant or with LPS. We found that the presence of wild-type bacilli suppressed the release of IL-12p40, normally induced by the ESX-5 mutant and LPS (Fig. 6A). In addition, wild-type M. marinum cells also suppressed the release of IL-6 and TNF-α, although for these cytokines the effect was less dramatic (Fig. 6B, C). Finally, the presence of wild-type E11 always resulted in the induction of significant amounts of IL-1β, irrespective of the presence of the ESX-5 mutant (Fig. 6D). From these experiments we conclude that ESX-5 effector proteins actively manipulate the cytokine responses of Mϕ.


addition of IFN-γ had no effect on mycobacterial suppression of TLR signalling.

Figure 6 | ESX-5 inhibits the heterologous induction of type 1 cytokines. M. marinum wild-type cells were mixed with the ESX-5 mutant or with LPS to measure active suppression of cytokine induction. Mϕ-1 was infected and culture supernatants were collected and the concentration of IL-12p40 (A), IL-6 (B), TNF-α (C) and IL-1β (D) was measured as mentioned previously. The results represent the means ± SE of 2 separate experiments.

ESX-5 pathway inhibits TLR signalling

Toll-like receptors (TLRs) play an important role in the detection of pathogen-associated molecules. However, because of their importance, they also may be targeted by mycobacteria as a mode of immune evasion. Mycobacterial suppression of TLR-signalling would be in accordance with the observed suppression of the LPS-mediated induction of IL-12p40 by wild-type M. marinum (Fig. 6). Therefore, we tested the possibility that ESX-5 effector proteins exert an attenuating effect on TLR signalling. To address this, we stimulated Mϕ-1 cells with various prototypical TLR ligands in the presence or absence of M. marinum E11 wild type or ESX-5 mutant bacilli. The TLR ligands used, i.e. LTA, LPS, Zymosan A, and CL075 stimulate TLR2, TLR4, TLR2/6 and TLR8/7 respectively. All these TLRs signal through the adapter molecule MyD88. Release of IL-12p40 was used as a readout of TLR activation. Infection with wild-type bacteria resulted in the suppression of IL-12p40 production triggered by all of the different TLR ligands (Fig. 7A), whereas the ESX-5 mutant was unable to suppress IL-12p40 production. The


igure 7 | ESX-5 inhibits cytokine production elicited by TLR2, TLR4, TLR2/6 and

We also analysed whether ESX-5 effector proteins could affect the secretion of IL-1β, IL-6 and TNF-α. Akin to the IL-12p40 result, only the wild-type bacteria were able to suppress the release of IL-6 secretion triggered by the different TLR ligands (Fig. 7B). The results for TNFα were more variable, and wild-type cells only seemed to affect induction by LPS and CL075 (Fig. 7C). Conversely, whereas wild-type cells always showed induction of IL-1β, no considerable release of IL-1β was observed if the cells were stimulated through TLR2, TLR4 or TLR8/7 ligands in the presence of the ESX-5 mutant. Only the addition of the TLR2/6 ligand Zymosan triggered significant IL-1β secretion (Fig. 7D). From these results we conclude that ESX-5 effector molecules attenuate MyD88-dependent TLR signalling.

FTLR8/7. Mϕ-1 were infected and stimulated with various TLR ligands. After 24 hours, culture supernatants were collected and the concentration of IL-12p40 (A), IL-6 (B), TNF-α (C) and IL-1β (D) was measured as previously mentioned. The results represent the means ± SE of 2 separate experiments.


ESX-5 pathway mediates macrophage cell death

Previous reports have shown that infection of human Mϕ with M. tuberculosis and M. marinum induces apoptosis or cell death41,42. Our experiments used to determine the optimal MOI for Mϕ infections (Fig. 3) indicated that ESX-5 secretion has an effect on cell death. To prove this, PMA-differentiated THP-1 cells and RAW cells were challenged with M. marinum wild type or ESX-5 mutant and monitored for 1, 3 and 6 days. Cell death was assessed by determining the amount of DNA fragmentation in infected and uninfected cells by nucleosome ELISA. As shown in Figure 8 (data shown for the THP-1 cells), wild-type M. marinum induced significantly more apoptosis than the ESX-5 mutant, which seems to confirm our hypothesis that ESX-5 is involved in Mϕ cell death. Similar results were obtained with TUNEL assay (data not shown).

Figure 8 | M. marinum ESX-5 effector molecules induce cell death. PMA-differentiated THP-1 cells were infected either with M. marinum wild-type E11 strain or ESX-5 mutant for different periods. Cell death was analysed by measuring the DNA fragmentation of the infected cells by ELISA. The y-axis show the relative enrichment of DNA fragmentation as compared to the uninfected control cells. The results shown are the means ± SE of at least two independent experiments, each performed in duplicate.

ESX-5 secretory pathway is required for virulence

To determine whether the ESX-5 pathway is required for virulence in vivo, we used the zebrafish embryo infection model43. We infected 32 hour-old zebrafish embryos by microinjection with 100 CFU of either the M. marinum E11 wild type, the ESX-5 mutant or the complemented strain and examined mycobacterial growth in vivo by whole embryo plating. Control embryos were inoculated with PBS. Ten embryos were injected per strain and were plated 1 hr p.i., to verify that the inocula were similar, and then again at day 1, 3, 5 p.i. As shown in Figure 9, the overall CFU count of bacteria was similar for all strains at 1 hr and 1 day p.i.


However, at 3 and especially at 5 days p.i., the embryos infected with wild-type bacteria harboured on average 5 times more colony-forming bacteria per embryo than those infected with ESX-5 mutant (Fig. 9). Unfortunately, at day 5 these experiments were terminated for animal ethical reasons, but these results indicated that, in the absence of an adaptive immune response, the ESX-5 mutant is impaired in virulence.

Figure 9: ESX-5 mutant exhibits reduced virulence and growth in zebrafish embryos. Embryos were infected with 100 CFU of M. marinum E11 wild-type, the ESX-5 mutant or the complemented strain for different periods and mycobacterial growth was examined by whole embryo plating for CFU counts.

Discussion

Pathogenic mycobacteria such as M. tuberculosis and M. marinum manipulate innate immune responses in order to achieve a balance of inflammation, which allows long-term persistence. Although several studies have demonstrated that mycobacteria are able to dampen or subvert the innate immune response13,14,16, the molecular mechanism underlying this phenomenon is still largely unknown. Our previous results have indicated that the ESX-5 secretion system might be responsible for a defect in the Mϕ infection cycle22. However, a problem of these experiments is that they were performed in a strain that was later shown to be impaired in ESAT-6 secretion23. Therefore, we analysed here the effect of a virulent wild-type strain and its isogenic ESX-5 mutant on cell infection, cytokine induction and immune regulation. ESX-5 is responsible for the secretion of a large number of proteins, including the PE_PGRS and PPE-MPTR proteins23. The PE and PPE protein family are both specific for mycobacteria, but their function is as yet unknown. However, their number is dramatically increased in several pathogenic mycobacteria, such as M. marinum and M. tuberculosis, suggesting a role in virulence.


First, the expression of Mϕ cell surface markers was analysed. Mϕ-2 showed a significant down-modulation of HLA-DR, CD80 and CD86 following M. marinum wild-type infection as previously reported35,44. Conversely, ESX-5 mutant infected Mϕ-2 showed a somewhat induced expression of the surface markers. On the other hand, the enhanced binding, uptake and endocytosis of mycobacteria by Mϕ-2 compared with Mϕ-133, may account for the difference in the expression of co-stimulatory and molecules. Thus, our results suggest that the ESX-5 pathway impairs the ability of nonclassical Mϕ, i.e. Mϕ-2, to process and/or present soluble antigen and in turn, to serve as accessory cell in T-cell activation. Next, the production of cytokines by Mϕ infected with the ESX-5 mutant was studied. It is well established that the production of pro-inflammatory cytokines, such as IL-12 and TNFα, is crucial for optimal host defence against mycobacterial infection6,8. Furthermore, the data available on the interaction of pathogenic versus non-pathogenic mycobacteria within host Mϕ show that mycobacterial virulence is inversely correlated with the secretion of these cytokines by infected Mϕ, and hence on the modulation of this secretion by the infecting bacteria45,46. In the present study, we demonstrate that only the wild-type bacteria are able to suppress the production of these Mϕ cytokines. A possible explanation for this effect is that ESX-5 effector proteins are directly involved in suppressing the cytokine response. Alternatively, an active ESX-5 secretion system might, just like the ESX-1 system21, be essential for the proper routing of the mycobacteria within the Mϕ in order to manipulate the host immune system. This latter possibility was ruled out by showing that the ESX-5 mutant is, just like wild-type bacteria, able to escape from the phagolysosome into the cytosol. Interestingly, although the cellular localisation is different, the observed effects of ESX-5 on Mϕ function are comparable to those described for the ESX-1 pathway18. An explanation for this observation is that the ESX-1 system is needed for the cytosolic localisation of M. marinum, which allows ESX-5 effector molecules to be secreted in the cytosol and manipulate the immune response from within the infected cells. Verreck et al., (2004) demonstrated that mycobacterial stimulation of Mϕ-1 initially results in the secretion of IL-23, but that the secretion of IL-12 is dependent on IFN-γ as an essential second signal. Therefore, the high IL-12p40 levels, observed in response to ESX-5 mutant infection after addition of exogenous IFN-γ suggest that the ESX-5 effector molecules suppress the capacity of Mϕ-1 to produce both IL-12 and IL-23. Future experiments are necessary to prove this supposition. Targeting TLR signalling is a strategy of immune evasion employed by different intracellular bacteria and viruses. There are several negative regulators of IL-1 receptor-TLR signalling, most of which seem to target the MyD88-dependent signalling cascade, including MyD88s (the short form of MyD88), IRAK-M, SOCS1, and Toll-interacting protein (Tollip), depending on the cell type and the nature of the stimuli47. In our present study, we examined the effect of ESX-5 on TLRs signalling and show that many different TLRs are repressed by an ESX-5


dependent mechanism. Although the data presented here are preliminary, ESX-5 could exert its inhibitory effect by attenuating MyD88 dependent TLR signalling. However, further studies are required to rigorously assess the detailed mechanisms underlying the inhibitory effect of ESX-5. Although the ESX-5 mutant enhanced induction of IL-12p40, IL-6 and TNF-α in infected Mϕ, the cells infected with the ESX-5 mutant triggered no IL-1β secretion. In contrast, M. marinum wild-type cells infected Mϕ induced IL-1β secretion, suggesting that a different mechanism is involved in this process. A possible explanation for this observation is that ESX-5 effectors are, either directly or indirectly, involved in the induction of IL-1β activation, possibly through activation of caspase-148,49. Activation of caspase-1 via microbial components in the host cytosol through NLR proteins has been described previously. For instance, Salmonella typhimurium uses a type III secretion system to secrete SipB into the cytosol of Mϕ, which binds and activates caspase-1 in infected cells50,51. Similarly, Helicobacter pylori uses a type IV secretion system for the delivery in the cytosol of infected host cells of peptidoglycan-derived molecules, which activates Nod1, another NLR family member52. Thus, ESX-5 effector molecules may interact; either directly or through another host factor in the cytosol, with inflammasome adaptors to promote the activation of caspase-1. This activation will lead to the concomitant release of pro-inflammatory cytokine IL-1β and IL-18 and the induction of osmotic death of the infected cells in vitro. Additional studies are required to unravel the mechanism that is responsible of the absence of IL-1β production from ESX-5 mutant infected Mϕ. Wild-type mycobacteria were able to induce cell death, whereas the ESX-5 mutant was significantly impaired in this process. At present it is unclear, however, whether host or bacterial cell factors are driving M. marinum-specific apoptosis. Although M. marinum wild-type infected Mϕ-1 did not secrete significant amounts of TNF-α, they did secrete IL-1β, which may contribute to cytotoxicity and cell death. Additional studies to explore the precise mechanisms leading to apoptosis and cell death are required. In conclusion, we have demonstrated that a functional ESX-5 pathway is important for the manipulation of the host Mϕ by mycobacteria. Future experiments are necessary to determine which effector molecule or, more likely, molecules are involved in this process and what the cellular targets are of these secreted effector molecules.

Acknowledgements

We thank Ben Appelmelk for valuable discussion and Peter Peters for collaborative work. The expert technical assistance of Dennis Langenberg, Louis Wilson, Tjitske de Boer and Kimberley Walburg is acknowledged. We are grateful to Michael Brennan (Centre for Biologics Evaluation and Research, USA) for providing PE_PGRS antibody.


References

1. Finlay,B.B. & Cossart,P. Exploitation of mammalian host cell functions by bacterial pathogens. Science 276, 718-725 (1997).

2. Armstrong,J.A. & Hart,P.D. Phagosome-Lysosome Interactions in Cultured Macrophages Infected with Virulent Tubercle-Bacilli - Reversal of Usual Nonfusion Pattern and Observations on Bacterial Survival. Journal of Experimental Medicine 142, 1-16 (1975).

3. Wallis,R.S. & Ellner,J.J. Cytokines and Tuberculosis. Journal of Leukocyte Biology 55, 676-681 (1994).

4. Fenton,M.J. & Vermeulen,M.W. Immunopathology of tuberculosis: roles of macrophages and monocytes. Infect. Immun. 64, 683-690 (1996).

5. Orme,I.M. & Cooper,A.M. Cytokine chemokine cascades in immunity to tuberculosis. Immunology Today 20, 307-312 (1999).

6. Flynn,J.L. & Chan,J. Immunology of tuberculosis. Annual Review of Immunology 19, 93-129 (2001).

7. van Crevel,R., Ottenhoff,T.H.M. & van der Meer,J.W.M. Innate immunity to Mycobacterium tuberculosis. Clinical Microbiology Reviews 15, 294-+ (2002).

8. Ottenhoff,T.H.M. et al. Genetics, cytokines and human infectious disease: lessons from weakly pathogenic mycobacteria and salmonellae. Nature Genetics 32, 97-105 (2002).

9. Raja,A. Immunology of tuberculosis. Indian Journal of Medical Research 120, 213-232 (2004).

10. Kindler,V., Sappino,A.P., Grau,G.E., Piguet,P.F. & Vassalli,P. The inducing role of tumor necrosis factor in the development of bactericidal granulomas during BCG infection. Cell 56, 731-740 (1989).

11. Senaldi,G. et al. Corynebacterium parvum- and Mycobacterium bovis bacillus Calmette-Guerin-induced granuloma formation is inhibited in TNF receptor I (TNF-RI) knockout mice and by treatment with soluble TNF-RI. J. Immunol. 157, 5022-5026 (1996).

12. Ladel,C.H. et al. Lethal tuberculosis in interleukin-6-deficient mutant mice. Infection and Immunity 65, 4843-4849 (1997).

13. Giacomini,E. et al. Infection of human macrophages and dendritic cells with mycobacterium tuberculosis induces a differential cytokine gene expression that modulates T cell response. Journal of Immunology 166, 7033-7041 (2001).

14. Nau,G.J. et al. Human macrophage activation programs induced by bacterial pathogens. Proceedings of the National Academy of Sciences of the United States of America 99, 1503-1508 (2002).

15. Falcone,V., Bassey,E.B., Toniolo,A., Conaldi,P.G. & Collins,F.M. Differential release of tumor necrosis factor-alpha from murine peritoneal macrophages stimulated with virulent and avirulent species of mycobacteria. FEMS Immunol. Med. Microbiol. 8, 225-232 (1994).

16. Beltan,E., Horgen,L. & Rastogi,N. Secretion of cytokines by human macrophages upon infection by pathogenic and non-pathogenic mycobacteria. Microb. Pathog. 28, 313-318 (2000).

17. Abdallah,A.M. et al. Type VII secretion--mycobacteria show the way. Nat. Rev. Microbiol. 5, 883-891 (2007).

https://www.researchgate.net/publication/12088287_Immunology_of_tuberculsis?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=

https://www.researchgate.net/publication/12088287_Immunology_of_tuberculsis?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=

https://www.researchgate.net/publication/15195302_Differential_release_of_tumor_necrosis_factor-I_from_murine_peritoneal_macrophages_stimulated_with_virulent_and_avirulent_species_of_mycobacteria?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=




https://www.researchgate.net/publication/10614760_Innate_Immunity_to_Mycobacterium_Tuberculosis?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=

https://www.researchgate.net/publication/10614760_Innate_Immunity_to_Mycobacterium_Tuberculosis?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=

https://www.researchgate.net/publication/12518682_Secretion_of_cytokines_by_human_macrophages_upon_infection_by_pathogenic_and_non-pathogenic_mycobacteria?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=



https://www.researchgate.net/publication/22345569_Armstrong_J_A_Hart_P_D_Phagosome-lysosome_interactions_in_cultured_macrophages_infected_with_virulent_tubercle_bacilli_Reversal_of_the_usual_nonfusion_pattern_and_observations_on_bacterial_survival_J_?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=




https://www.researchgate.net/publication/12920174_Cytokinechemokine_cascades_in_immunity_to_TB?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=

https://www.researchgate.net/publication/12920174_Cytokinechemokine_cascades_in_immunity_to_TB?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=

https://www.researchgate.net/publication/14558331_Immunopathology_of_tuberculosis_Roles_of_macrophages_and_monocytes?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=

https://www.researchgate.net/publication/14558331_Immunopathology_of_tuberculosis_Roles_of_macrophages_and_monocytes?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=

https://www.researchgate.net/publication/15017609_Cytokines_and_tuberculosis?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=

https://www.researchgate.net/publication/15017609_Cytokines_and_tuberculosis?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=

https://www.researchgate.net/publication/20509520_The_inducing_role_of_TNF-a_in_the_development_of_bactericidal_granulomas_during_BCG_infection?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=




18. Stanley,S.A., Raghavan,S., Hwang,W.W. & Cox,J.S. Acute infection and

macrophage subversion by Mycobacterium tuberculosis require a specialized secretion system. Proceedings of the National Academy of Sciences of the United States of America 100, 13001-13006 (2003).


20. Brodin,P., Rosenkrands,I., Andersen,P., Cole,S.T. & Brosch,R. ESAT-6 proteins: protective antigens and virulence factors? Trends Microbiol. 12, 500-508 (2004).

21. van der Wel,N. et al. M. tuberculosis and M. leprae translocate from the phagolysosome to the cytosol in myeloid cells. Cell 129, 1287-1298 (2007).


23. Abdallah,A.M. et al. Mycobacterial PPE and PE_PGRS proteins are transported via a type VII secretion system. Molecular Microbiology Submitted, (2008).

24. Brennan,M.J., Espitia,C. & Gey von Pittius,N. S.Cole, D.N.McMurray, K.Eisenach, B.Gicquel & W.R.Jacobs (eds.), pp. 513-525 (ASM Press, Washington, DC.,2004).




28. Banu,S. et al. Are the PE-PGRS proteins of Mycobacterium tuberculosis variable surface antigens? Molecular Microbiology 44, 9-19 (2002).

29. Brennan,M.J. & Delogu,G. The PE multigene family: a 'molecular mantra' for mycobacteria. Trends in Microbiology 10, 246-249 (2002).

30. Delogu,G. & Brennan,M.J. Comparative immune response to PE and PE_PGRS antigens of Mycobacterium tuberculosis. Infection and Immunity 69, 5606-5611 (2001).

31. Stamm,L.M. et al. Mycobacterium marinum escapes from phagosomes and is propelled by actin-based motility. Journal of Experimental Medicine 198, 1361-1368 (2003).

32. Peters,P.J.B.E.a.G.A. Cryo-immunogold electron microscopy. In Current Protocols Cell Biology (Hoboken, NJ: John Wiley & Sons) 4.7.1-4.7.19 (2006).

33. Verreck,F.A. et al. Human IL-23-producing type 1 macrophages promote but IL-10-producing type 2 macrophages subvert immunity to (myco)bacteria. Proc. Natl. Acad. Sci. U. S. A 101, 4560-4565 (2004).

34. Verreck,F.A., de Boer,T., Langenberg,D.M., van der,Z.L. & Ottenhoff,T.H. Phenotypic and functional profiling of human proinflammatory type-1 and anti-inflammatory type-2 macrophages in response to microbial antigens and IFN-gamma- and CD40L-mediated costimulation. J. Leukoc. Biol. 79, 285-293 (2006).

35. Gercken,J., Pryjma,J., Ernst,M. & Flad,H.D. Defective antigen presentation by Mycobacterium tuberculosis-infected monocytes. Infect. Immun. 62, 3472-3478 (1994).

https://www.researchgate.net/publication/11395620_The_PE_multigene_family_A_'molecular_mantra'_for_mycobacteria?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=

https://www.researchgate.net/publication/11395620_The_PE_multigene_family_A_'molecular_mantra'_for_mycobacteria?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=



https://www.researchgate.net/publication/8229194_ESAT-6_proteins_Protective_antigens_and_virulence_factors?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=

https://www.researchgate.net/publication/8229194_ESAT-6_proteins_Protective_antigens_and_virulence_factors?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=

https://www.researchgate.net/publication/15159166_Defective_antigen_presentation_by_Mycobacterium_tuberculosis-infected_monocytes?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=



https://www.researchgate.net/publication/11845322_Comparative_Immune_Response_to_PE_and_PE_PGRS_Antigens_of_Mycobacterium_tuberculosis?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=



https://www.researchgate.net/publication/229680711_Cryo-Immunogold_Electron_Microscopy?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=

https://www.researchgate.net/publication/229680711_Cryo-Immunogold_Electron_Microscopy?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=


36. Noss,E.H., Harding,C.V. & Boom,W.H. Mycobacterium tuberculosis inhibits

MHC class II antigen processing in murine bone marrow macrophages. Cell Immunol. 201, 63-74 (2000).

37. Sendide,K., Deghmane,A.E., Reyrat,J.M., Talal,A. & Hmama,Z. Mycobacterium bovis BCG urease attenuates major histocompatibility complex class II trafficking to the macrophage cell surface. Infect. Immun. 72, 4200-4209 (2004).

38. Oswald,I.P., Wynn,T.A., Sher,A. & James,S.L. Interleukin-10 Inhibits Macrophage Microbicidal Activity by Blocking the Endogenous Production of Tumor-Necrosis-Factor-Alpha Required As A Costimulatory Factor for Interferon-Gamma-Induced Activation. Proceedings of the National Academy of Sciences of the United States of America 89, 8676-8680 (1992).

39. Delbridge,L.M. & O'Riordan,M.X.D. Innate recognition of intracellular bacteria. Current Opinion in Immunology 19, 10-16 (2007).

40. Henry,T. & Monack,D.M. Activation of the inflammasome upon Francisella tularensis infection: interplay of innate immune pathways and virulence factors. Cell Microbiol. (2007).

41. Keane,J. et al. Infection by Mycobacterium tuberculosis promotes human alveolar macrophage apoptosis. Infect. Immun. 65, 298-304 (1997).

42. Ramakrishnan,L. & Falkow,S. Mycobacterium-Marinum Persists in Cultured-Mammalian-Cells in A Temperature-Restricted Fashion. Infection and Immunity 62, 3222-3229 (1994).

43. Davis,J.M. et al. Real-time visualization of Mycobacterium-macrophage interactions leading to initiation of granuloma formation in zebrafish embryos. Immunity 17, 693-702 (2002).

44. Hmama,Z., Gabathuler,R., Jefferies,W.A., de Jong,G. & Reiner,N.E. Attenuation of HLA-DR expression by mononuclear phagocytes infected with Mycobacterium tuberculosis is related to intracellular sequestration of immature class II heterodimers. J. Immunol. 161, 4882-4893 (1998).

45. Fattorini,L. et al. Induction of IL-1 beta, IL-6, TNF-alpha, GM-CSF and G-CSF in human macrophages by smooth transparent and smooth opaque colonial variants of Mycobacterium avium. J. Med. Microbiol. 40, 129-133 (1994).

46. Stauffer,F., Petrow,E.P., Burgmann,H., Graninger,W. & Georgopoulos,A. Release of TNF alpha and IL6 from human monocytes infected with Mycobacterium kansasii: a comparison to Mycobacterium avium. Infection 22, 326-329 (1994).

47. Liew,F.Y., Xu,D., Brint,E.K. & O'Neill,L.A. Negative regulation of toll-like receptor-mediated immune responses. Nat. Rev. Immunol. 5, 446-458 (2005).

48. Martinon,F., Burns,K. & Tschopp,J. The inflammasome: a molecular platform triggering activation of inflammatory caspases and processing of proIL-beta. Mol. Cell 10, 417-426 (2002).

49. Ogura,Y., Sutterwala,F.S. & Flavell,R.A. The inflammasome: first line of the immune response to cell stress. Cell 126, 659-662 (2006).

50. Hersh,D. et al. The Salmonella invasin SipB induces macrophage apoptosis by binding to caspase-1. Proc. Natl. Acad. Sci. U. S. A 96, 2396-2401 (1999).

51. Mariathasan,S. et al. Differential activation of the inflammasome by caspase-1 adaptors ASC and Ipaf. Nature 430, 213-218 (2004).

52. Viala,J. et al. Nod1 responds to peptidoglycan delivered by the Helicobacter pylori cag pathogenicity island. Nat. Immunol. 5, 1166-1174 (2004).

https://www.researchgate.net/publication/12511789_Mycobacterium_tuberculosis_Inhibits_MHC_Class_II_Antigen_Processing_in_Murine_Bone_Marrow_Macrophages?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=



https://www.researchgate.net/publication/15355035_Release_of_TNFa_and_IL6_from_human_monocytes_infected_with_Mycobacterium_kansasii_A_comparison_to_Mycobacterium_avium?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=



https://www.researchgate.net/publication/13492845_Attenuation_of_HLA-DR_Expression_by_Mononuclear_Phagocytes_Infected_with_Mycobacterium_tuberculosis_Is_Related_to_Intracellular_Sequestration_of_Immature_Class_II_Heterodimers?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=








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https://www.researchgate.net/publication/11195678_Martinon_F_Burns_K_Tschopp_J_The_inflammasome_a_molecular_platform_triggering_activation_of_inflammatory_caspases_and_processing_of_proIL-_Mol_Cell_10_417-426?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=



https://www.researchgate.net/publication/21626604_Oswald_IP_Wynn_TA_Sher_A_James_SL_Interleukin_10_inhibits_macrophage_microbicidal_activity_by_blocking_the_endogenous_production_of_tumor_necrosis_factor_alpha_required_as_a_costimulatory_factor_for_i?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=





Ch

apter 7

GENERAL DISCUSSION

Chapter 7: General Discussion 130

In this study, we have used Mycobacterium marinum to learn more about mycobacterial pathogens in general and about how they secrete virulence factors in particular. M. marinum has a number of practical advantages as compared to M. tuberculosis, such as increased growth rate, and lower virulence (for humans); in addition it provides a natural infection model. Furthermore, M. marinum is, together with Mycobacterium ulcerans, the closest relative of the different species belonging to the M. tuberculosis complex1. Using M. marinum as a model organism we have made a number of interesting and important observations; these are described in this thesis and discussed below. The highlight of my work has been the identification and the partial characterization of a novel secretion system, designated ESX-5, that is responsible for the secretion of PE and PPE proteins. Because both the ESX-5 secretion system and its substrates are conserved in M. tuberculosis, I predict that my results can be translated to this human pathogen. However, because my time was limited, it was not possible to test this hypothesis myself and future experiments will have to prove this point. Strain variation

Species variation and strain variation in bacteria has long been recognized as an important feature in infectious diseases and has been an important focus of study in the genomics era. This is also true for the mycobacteria and especially M. tuberculosis2. M. tuberculosis belongs to the so-called M. tuberculosis complex, which traditionally consisted of four members: M. tuberculosis, the causative agent of human tuberculosis; Mycobacterium bovis, which mainly infects cattle, but can also cause disease in a broad range of other mammals, including man; Mycobacterium africanum, a tuberculosis causing agent endemic in western Africa; and Mycobacterium microti, the causative agent of tuberculosis in voles. In addition, also the set of attenuated M. bovis strains, generally known as Bacille Calmette–Guérin (BCG), that are used for vaccination belongs to this cluster. Although all these strains share the same 16S rRNA gene sequence3 and are defined as a single species by DNA-DNA hybridization4, they can be distinguished by a limited number of phenotypic and genotypic characteristics5,6. Moreover, because they differ remarkably with respect to their host range and pathogenicity, they have been regarded as separate species7. Recently, even three novel species belonging to the M. tuberculosis complex have been described: Mycobacterium canettii, a novel smooth variant of M. tuberculosis that seems to be restricted to the horn of Africa8,9; Mycobacterium caprae, a strain that is isolated primarily from Spanish goats10-12, and Mycobacterium pinnipedii, which can be found primarily in seals13. Between and within these different species of the tuberculosis complex there is relatively little variation, mainly limited to point mutations, deletions and variation in the number and location of IS elements. This lack of variation is mainly due to the fact that, with the possible exception of M. canettii14, these species show a conspicuous lack of horizontal gene transfer. Therefore, all these species were generated by clonal


descent and their evolution can be faithfully reconstructed14,15. However, despite this relative lack of genetic variation it has recently become clear that strain variation in M. tuberculosis is an important factor for the outcome of the disease, which is exemplified by the recent success of the Beijing lineage of tuberculosis strains16,17. Although there is a wealth of knowledge on genetic variation of M. tuberculosis complex, relatively little was known about the M. marinum species. The 16S rDNA of M. marinum and M. ulcerans are nearly identical, suggesting that these species recently originated from a common ancestor18,19. However, phenotypically these bacteria show an enormous variation: they have completely different growth rates; M. marinum is an intracellular pathogen, just like M. tuberculosis, whereas M. ulcerans is mainly growing extracellulary; and, most importantly, they cause different diseases in humans. M. marinum gives rise to the mild skin lesions known as swimming pool granulomas or aquarium granulomas, whereas M. ulcerans is the causative agent of the debilitating and disfiguring disease Buruli ulcer18,20. Like M. bovis, M. marinum appears to have a broad host range, causing a systemic tuberculosis-like granulomatous infection in a variety of poikilothermic animals, including fish (both salt- and fresh-water), amphibians and reptiles21. In the past few years investigators have suggested that M. marinum isolates from humans and freshwater are genetically different as compared to isolates from marine environments18,22. Our infection experiments showed that strains from the “human” cluster caused significantly increased morbidity and mortality in zebrafish as compared to strains form the other cluster (Chapter 3). Since all the human isolates were obtained from hospitalised patients, this means that they represent the most serious and persistent human pathogens. Together, these data indicate that M. marinum species can be divided in two groups, both genetically and phenotypically. Furthermore, this finding also suggests that only some M. marinum strains have zoonotic potential. The absence of horizontal gene transfer in members of the tuberculosis complex also means that the population structure is dominated by population bottlenecks and that new host-adapted groups developed sequentially14,15. This means that the phylogeny of the animal-adapted strains is in fact a series of nested clades. Now, it is not known to what extent M. marinum allows horizontal gene transfer, but the possibility exists that each clade of M. marinum strains is in fact specialized for a specific host or group of hosts. Although this is an interesting hypothesis, there is at present only very limited data on the natural variation of M. marinum species in relation to their natural hosts. On the other hand M. marinum might also be adapted to different hosts, considering the fact that this bacterium can be isolated from a large range of different animals. In addition, M. marinum is also able to infect and grow in the slime mold Dictyostelium discoideum23. In fact, interaction with lower eukaryotes might be a way to survive for longer periods outside the vertebrate host. Previous studies have shown that passage of Mycobacterium avium through amoebae resulted in increased virulence in mice24. This could also be true for M. marinum, which seems to be, unlike M. tuberculosis, a


facultative pathogen that is able to survive and multiply in the absence of a mammalian host. As discussed above, genetic variation in M. tuberculosis is limited. However, this limited variation may cause large phenotypic changes. It has been shown that single nucleotide or amino acid changes can considerably modify virulence of in M. tuberculosis complex for guinea pigs25. In addition, also insertion sequences play an important role in mycobacterial genetic variation26,27. In M. tuberculosis the presence and activity of IS6110 is most likely responsible for the rapid evolution of new distinct clones28,29. Similarly, M. avium and M. ulcerans also acquired at least two IS elements that act as defining characteristics for M. avium subspecies paratuberculosis30 and for the ongoing genome rearrangements in M. ulcerans. Furthermore, in silico analysis of the various genome sequences of members of the M. tuberculosis complex has shown that the PE and PPE gene families contribute another major source of genetic variation. In fact, extensive polymorphisms in the PE family were already recognized for a longer period by molecular epidemiologist, who used DNA probes based on repetitive motifs within this family for high-resolution molecular typing methods31. Interestingly, the M. marinum genome contains in fact more PE and PPE gene family members than M. tuberculosis and the majority of the family members are unique. This raises the possibility that the variation of PE and PPE proteins has a role in the phenotypic variation of M. marinum isolates described in chapter 3. PE and PPE gene families

The PE and PPE gene families, which together constitute approximately 10% of the M. tuberculosis genome32, are both mycobacterium specific and are highly expanded in certain pathogenic species. The most striking feature of these gene families is that some of the most recently evolved members are encoding extremely long (up to 3,500 amino acids) and unusually glycine-rich (more than 50%) proteins of repetitive structure. On the other hand, the most ancestral PE and PPE genes are frequently associated with ESAT-6 gene clusters33, suggesting that these families are (originally) functionally linked. Although PE and PPE gene families are highly expanded in several pathogenic species33,34, only 13 intact PE and PPE genes were found in the Mycobacterium leprae genome33. The genome sequence of M. leprae is known to have undergone extensive gene loss during evolution, in which more than half of the genes present in M. tuberculosis and the majority of the PE and PPE genes have been (partially) deleted35. Furthermore, no intact members of the PE_PGRS or PPE-MPTR subfamilies were found in this M. lepare. Apparently, the lifestyle of M. leprae, although an obligate human pathogen like M. tuberculosis, allowed for the almost complete removal of all PE and PPE genes. In chapter 4 and 5, I have shown that the ESX-5 secretion system is involved in the extracellular secretion of the recently evolved PE_PGRS and PPE-MPTR proteins. M. leprae does contain a copy of the


ESX-5 gene cluster in its genome, but several of the genes from this cluster are deleted or have become pseudogenes as a result of extensive point mutation and frameshifts during genome downsizing35. This could suggest that this cluster is not functionally active in M. leprae. However, a closer scrutiny of the remaining intact genes shows that this set of genes is coding for proteins that probably form the core of the ESX-5 secretion system. Future experiments will have to show if this system is indeed still functioning. However, what M. lepare does show is that mycobacterial pathogens can be successful with a low number of PE and PPE genes. This fact only adds to the mystery of PE and PPE functioning in M. tuberculosis and M. marinum. M. ulcerans is another mycobacterial pathogen that has evolved extensive chromosomal rearrangements and genome downsizing, which resulted in the accumulation of pseudogenes36,37. Foremost among the pseudogenes in M. ulcerans are again members of the PE and PPE families. M. ulcerans is closely related to M. marinum and therefore has had an enormous specific expansion of the PE and PPE in the recent past, but a large number of these genes have become pseudogenes. Whereas M. marinum has 170 PE and 105 PPE genes, respectively, M. ulcerans has retained only 70 intact PE and 57 PPE, with the remaining orthologs showing widespread sequence variation33,36. Because the degeneration process of M. ulcerans seems to be in an intermediate stage of reductive evolution, this species could be heading towards a similar contracted genome state as that of M. leprae, with a similar reduction in PE and PPE genes. Another commonality of M. leprae and M. ulcerans is that they both have specialised in the production of a specific toxin: M. leprae produces high amounts of phenolic glycolipids (PGL), whereas M. ulcerans virulence largely depends on the production of a macrolide toxin. Perhaps the production of these specialised toxins allows for the downsizing of the number of PE and PPE genes. Linkage of glycine-rich sequences encoded by Polymorphic GC-rich Repetitive Sequences (PGRS) to the C-terminal ends of the PE domains is characteristic of the PE_PGRS, the largest subfamily of the PE family proteins. To date, evidence suggests that the occurrence of abundant PE_PGRS genes is restricted to members of the M. tuberculosis complex and a few other closely related mycobacterial species, such as M. marinum33,38,39. Because these species all infect vertebrates with an adaptive immune system, these genes could function as a source of antigenic variation in pathogenic mycobacteria in order to evade the host immune response32,40. Interestingly, the PGRS domain shows similarity with the Epstein-Bar virus (EBV) nuclear antigen 1 (EBNA-1)41, which might suggest that the PE_PGRS proteins have a similar role. Previous studies postulated that the EBNA-1 protein inhibits antigen processing and presentation through the major histocompatibility complex class I (MHC-I) pathway by interfering with proteosome-dependent antigen processing42,43. This raises the possibility that the Gly-Ala-rich PGRS domain, by a similar mechanism of immune interference, inhibits antigen processing and presentation through the MHC-I pathway. Akin to this conjecture, the few published data showed that at least some PE_PGRS genes are expressed by pathogenic mycobacteria during infection of the host41.


In conclusion, the presence of the numerous PE_PGRS genes in a number of pathogenic mycobacteria implies that these genes have been maintained under evolutionary pressure by these organisms. The reason for their existence remains an intriguing subject for scientific investigation. Type VII secretion systems in mycobacteria

The initial clues that a specialized secretion systems exist in mycobacteria came from studies that identified secreted proteins that lack obvious Sec signal sequences44. These small proteins, i.e. ESAT-6 and CFP-10, were originally identified as immunodominant antigens of M. tuberculosis. Recently, the first specialized secretion system has been identified in mycobacteria, termed ESAT-6 secretion system or ESX-1. Rapidly, this secretion pathway has gained broad interest in the M. tuberculosis field, especially because this secretion system is an important virulence factor that is absent in the vaccine strain BCG. The ESX-1 system, the archetype of type VII secretion system (Chapter 2), was shown to be responsible for the secretion of these two proteins. Most genes encoding the ESX-1 secretion system are located in a single locus on the genome. This locus, which also contains the genes encoding ESAT-6 and CFP-10, is partially deleted in M. bovis BCG. ESX-1 is conserved in most mycobacterial species, both pathogenic and non-pathogenic. This means that either ESX-1 fulfils a role in pathogenesis that is conserved in non-pathogenic mycobacteria, or the ESX-1 secretion system has been extended in pathogenic mycobacteria with novel or modified effector molecules. Using M. marinum as a host organism, I have been able to identify a second type VII secretion system in mycobacteria. This system, ESX-5, is involved in the secretion of a number of PE and PPE proteins and works to subvert normal macrophage responses (Chapters 4, 5, 6). These proteins all belong to the most recently expanded subfamilies of PE_PGRS and PPE-MPTR proteins. Because of the recent discovery of the ESX-5 system, research on this system and the effect of ESX-5 on virulence is mainly limited to M. marinum. However, there is some interesting data available for the species of the M. avium complex, which indicates that ESX-5 is indeed also important for other pathogenic mycobacteria. M. avium affects mainly birds, but is also an important opportunistic pathogen for humans, where it mainly affects immunocompromised individuals such as AIDS patients. M. avium also contains an ESX-5 locus and it was recently shown that one of the PPE genes within this locus, namely PPE25, is crucial for the macrophage infection cycle of this pathogen45. Even more intriguing is a set of experiments performed by Gerry Cangelosi and co-workers. They were able to show that M. avium undergoes a reversible switch between red and white colony morphotypes on agar plates containing Congo red46,47. The white morphotypes probably have an altered cell wall that blocks the binding of this stain, and which also makes them more resistant to multiple antibiotics in vitro. Furthermore, the white morphotypes


are the predominant form isolated from infected humans and are also more virulent in mouse infection models as compared to isogenic red morphotypes46,48. Further study on this phenomenon showed that this reversible red-white morphotypic switching is associated with the expression of the components of the ESX-5 secretion system in M. avium and various PE and PPE genes (Cangelosi et al., Keystone Symposium; Tuberculosis: From Lab Research to Field Trials; 2007). Together, these data strongly suggest that ESX-5 plays an important role in cell wall permeability and in M. avium virulence. Perhaps the ESX-5 system of M. avium delivers a number of PE and PPE proteins to the outer layer of the cell wall where these proteins are either a structural component of this layer or modify this outer layer. In this respect it is interesting to note that different colony morphologies have been observed in M. canetti8. Normally, M. canetti strain So93 grows with smooth colonies, but it is able to switch to variants with a rough colony morphology in vitro and especially in vivo. The tentative hypothesis that ESX-5 is involved in this process will be studied in the near future by my successor.

Conclusion

The studies presented in this thesis focused on several aspects of protein secretion mechanisms in mycobacteria. Increased knowledge of proteins secreted by pathogenic mycobacteria proteins and the respective systems responsible may benefit to the development of intervention strategies to reduce the burden of tuberculosis. The presented results have provided more insight in the value of M. marinum model for the tuberculosis research. In these studies, we have identified a major protein secretion pathway in pathogenic mycobacteria, i.e. the ESX-5 pathway. Subsequently, we have shown that the ESX-5 pathway is required for the extracellular accumulation of a large number of proteins, including all PE_PGRS and PPE proteins. These ESX-5-secreted proteins are probably used by this bacterium to assert a profound inhibitory influence on its cellular host cell, since cells infected with the ESX-5 mutant were able to induce a number of pro-inflammatory cytokines that were normally repressed. Because the ESX-5 mutant is also profoundly attenuated in an embryo model of infection, this secretion system seems to be a major determinant of M. marinum virulence. We believe that these results also apply to M. tuberculosis, but only future experiments can prove this hypothesis. If proven to be true, research should be focused on this major virulence secretion pathway and its secreted effector proteins to see if ESX-5 can be used to our advantage.


References

1. Tonjum,T., Welty,D.B., Jantzen,E. & Small,P.L. Differentiation of Mycobacterium ulcerans, M. marinum, and M. haemophilum: mapping of their relationships to M. tuberculosis by fatty acid profile analysis, DNA-DNA hybridization, and 16S rRNA gene sequence analysis. J. Clin. Microbiol. 36, 918-925 (1998).

2. Cole,S.T. Comparative and functional genomics of the Mycobacterium tuberculosis complex. Microbiology 148, 2919-2928 (2002).

3. Rogall,T., Wolters,J., Flohr,T. & Bottger,E.C. Towards a phylogeny and definition of species at the molecular level within the genus Mycobacterium. Int J. Syst. Bacteriol. 40, 323-330 (1990).

4. Imaeda,T. Deoxyribonucleic acid relatedness among theselected strains of M. tuberculosis, M. bovis, M. bovis BCG, M. microti and M. africanum. Int J. Syst. Bacteriol. 35, 147-150 (1985).

5. Wayne,L.G.a.G.P.K. Bergey's manual of systematic bacteriology. In P.H.A.Sneath and J.G.Holt (ed.) (ed.), pp. 1435-1457 (The Williams & Wilkins Co., Baltimore, Md.,1986).

6. Richter,E., Weizenegger,M., Rusch-Gerdes,S. & Niemann,S. Evaluation of genotype MTBC assay for differentiation of clinical Mycobacterium tuberculosis complex isolates. J. Clin. Microbiol. 41, 2672-2675 (2003).

7. Collins,C.H., Yates,M.D. & Grange,J.M. Subdivision of Mycobacterium tuberculosis into five variants for epidemiological purposes: methods and nomenclature. J. Hyg. (Lond) 89, 235-242 (1982).

8. van Soolingen,D. et al. A novel pathogenic taxon of the Mycobacterium tuberculosis complex, Canetti: characterization of an exceptional isolate from Africa. Int J. Syst. Bacteriol. 47, 1236-1245 (1997).

9. Pfyffer,G.E., Auckenthaler,R., van Embden,J.D. & van Soolingen,D. Mycobacterium canettii, the smooth variant of M. tuberculosis, isolated from a Swiss patient exposed in Africa. Emerg. Infect. Dis. 4, 631-634 (1998).

10. Aranaz,A. et al. Mycobacterium tuberculosis subsp. caprae subsp. nov.: a taxonomic study of a new member of the Mycobacterium tuberculosis complex isolated from goats in Spain. Int J. Syst. Bacteriol. 49 Pt 3, 1263-1273 (1999).

11. Aranaz,A., Cousins,D., Mateos,A. & Dominguez,L. Elevation of Mycobacterium tuberculosis subsp. caprae Aranaz et al. 1999 to species rank as Mycobacterium caprae comb. nov., sp. nov. Int J. Syst. Evol. Microbiol. 53, 1785-1789 (2003).

12. Niemann,S., Richter,E. & Rusch-Gerdes,S. Biochemical and genetic evidence for the transfer of Mycobacterium tuberculosis subsp. caprae Aranaz et al. 1999 to the species Mycobacterium bovis Karlson and Lessel 1970 (approved lists 1980) as Mycobacterium bovis subsp. caprae comb. nov. Int J. Syst. Evol. Microbiol. 52, 433-436 (2002).

13. Ahmed,N. et al. Genome sequence based, comparative analysis of the fluorescent amplified fragment length polymorphisms (FAFLP) of tubercle bacilli from seals provides molecular evidence for a new species within the Mycobacterium tuberculosis complex. Infect. Genet. Evol. 2, 193-199 (2003).

14. Brosch,R. et al. A new evolutionary scenario for the Mycobacterium tuberculosis complex. Proc. Natl. Acad. Sci. U. S. A 99, 3684-3689 (2002).

https://www.researchgate.net/publication/51333791_Differentiation_of_Mycobacterium_ulcerans_M_marinum_and_M_haemophilum_Mapping_of_Their_Relationships_to_M_tuberculosis_by_Fatty_Acid_Profile_Analysis_DNA-DNA_Hybridization_and_16S_rRNA_Gene_Sequence_A?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=






https://www.researchgate.net/publication/11091878_Comparative_and_functional_genomics_of_the_Mycobacterium_tuberculosis_complex?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=

https://www.researchgate.net/publication/11091878_Comparative_and_functional_genomics_of_the_Mycobacterium_tuberculosis_complex?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=

https://www.researchgate.net/publication/10720840_Evaluation_of_Genotype_MTBC_Assay_for_Differentiation_of_Clinical_Mycobacterium_tuberculosis_Complex_Isolates?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=



https://www.researchgate.net/publication/16386392_Subdivision_of_Mycobacterium_tuberculosis_into_five_variants_for_epidemiological_purposes_Methods_and_nomenclature?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=



https://www.researchgate.net/publication/13420850_Mycobacterium_canettii_the_Smooth_Variant_of_M_tuberculosis_Isolated_from_a_Swiss_Patient_Exposed_in_Africa?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=



https://www.researchgate.net/publication/8975108_Elevation_of_Mycobacterium_tuberculosis_subsp_caprae_Aranaz_et_al_1999_to_species_rank_as_Mycobacterium_caprae_comb_nov_sp_nov?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=



https://www.researchgate.net/publication/11434681_Biochemical_and_genetic_evidence_for_the_transfer_of_Mycobacterium_tuberculosis_subsp_caprae_Aranaz_et_al_1999_to_the_species_Mycobacterium_bovis_Karlson_and_Lessel_1970_Approved_Lists_1980_as_Mycobac?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=





https://www.researchgate.net/publication/242194289_Deoxyribonucleic_Acid_Relatedness_Among_Selected_Strains_of_Mycobacterium_tuberculosis_Mycobacterium_bovis_Mycobacterium_bovis_BCG_Mycobacterium_microti_and_Mycobacterium_africanum?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=




15. Smith,N.H., Gordon,S.V., Rua-Domenech,R., Clifton-Hadley,R.S. &

Hewinson,R.G. Bottlenecks and broomsticks: the molecular evolution of Mycobacterium bovis. Nat. Rev. Microbiol. 4, 670-681 (2006).

16. van Soolingen,D. et al. Predominance of a single genotype of Mycobacterium tuberculosis in countries of east Asia. J. Clin. Microbiol. 33, 3234-3238 (1995).

17. Malik,A.N. & Godfrey-Faussett,P. Effects of genetic variability of Mycobacterium tuberculosis strains on the presentation of disease. Lancet Infect. Dis. 5, 174-183 (2005).

18. Stinear,T.P., Jenkin,G.A., Johnson,P.D. & Davies,J.K. Comparative genetic analysis of Mycobacterium ulcerans and Mycobacterium marinum reveals evidence of recent divergence. J. Bacteriol. 182, 6322-6330 (2000).

19. Yip,M.J. et al. Evolution of Mycobacterium ulcerans and other mycolactone-producing mycobacteria from a common Mycobacterium marinum progenitor. J. Bacteriol. 189, 2021-2029 (2007).

20. Brosch,R., Pym,A.S., Gordon,S.V. & Cole,S.T. The evolution of mycobacterial pathogenicity: clues from comparative genomics. Trends Microbiol. 9, 452-458 (2001).


22. Ucko,M. et al. Strain variation in Mycobacterium marinum fish isolates. Appl. Environ. Microbiol. 68, 5281-5287 (2002).

23. Solomon,J.M., Leung,G.S. & Isberg,R.R. Intracellular replication of Mycobacterium marinum within Dictyostelium discoideum: efficient replication in the absence of host coronin. Infect. Immun. 71, 3578-3586 (2003).

24. Cirillo,J.D., Falkow,S., Tompkins,L.S. & Bermudez,L.E. Interaction of Mycobacterium avium with environmental amoebae enhances virulence. Infect. Immun. 65, 3759-3767 (1997).

25. Collins,D.M. et al. Mutation of the principal sigma factor causes loss of virulence in a strain of the Mycobacterium tuberculosis complex. Proc. Natl. Acad. Sci. U. S. A 92, 8036-8040 (1995).

26. Dale,J.W. Mobile genetic elements in mycobacteria. Eur. Respir. J. Suppl 20, 633s-648s (1995).

27. Poulet,S. & Cole,S.T. Repeated DNA sequences in mycobacteria. Arch. Microbiol. 163, 79-86 (1995).

28. Bifani,P.J. et al. Origin and interstate spread of a New York City multidrug-resistant Mycobacterium tuberculosis clone family. JAMA 275, 452-457 (1996).

29. Sreevatsan,S. et al. Restricted structural gene polymorphism in the Mycobacterium tuberculosis complex indicates evolutionarily recent global dissemination. Proc. Natl. Acad. Sci. U. S. A 94, 9869-9874 (1997).

30. Green,E.P. et al. Sequence and characteristics of IS900, an insertion element identified in a human Crohn's disease isolate of Mycobacterium paratuberculosis. Nucleic Acids Res. 17, 9063-9073 (1989).

31. Kremer,K. et al. Comparison of methods based on different molecular epidemiological markers for typing of Mycobacterium tuberculosis complex strains: interlaboratory study of discriminatory power and reproducibility. J. Clin. Microbiol. 37, 2607-2618 (1999).





https://www.researchgate.net/publication/11795112_The_Evolution_of_Mycobacterial_Pathogenicity_Clues_From_Comparative_Genomics?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=



https://www.researchgate.net/publication/9486015_Effect_of_Environmental_Temperatures_on_Infection_with_Mycobacterium_Marinum_Balnei_of_Mice_and_a_Number_of_Poikilothermic_Species?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=



https://www.researchgate.net/publication/10748570_Intracellular_Replication_of_Mycobacterium_marinum_within_Dictyostelium_discoideum_Efficient_Replication_in_the_Absence_of_Host_Coronin?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=



https://www.researchgate.net/publication/13937083_Interaction_of_Mycobacterium_avium_with_environmental_amoebae_enhances_virulence?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=



https://www.researchgate.net/publication/14609115_Mobile_genetic_elements_in_mycobacteria?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=

https://www.researchgate.net/publication/14609115_Mobile_genetic_elements_in_mycobacteria?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=

https://www.researchgate.net/publication/15489514_Repeated_DNA_sequences_in_mycobacteria?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=

https://www.researchgate.net/publication/15489514_Repeated_DNA_sequences_in_mycobacteria?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=








https://www.researchgate.net/publication/291837288_Effects_of_genetic_variability_of_Mycobacterium_tuberculosis_strains_on_the_presentation_of_disease?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=




33. Gey van Pittius,N.C. et al. Evolution and expansion of the Mycobacterium

tuberculosis PE and PPE multigene families and their association with the duplication of the ESAT-6 (esx) gene cluster regions. BMC Evol. Biol. 6, 95 (2006).

34. Brennan,M.J.E.C.a.G.v.P.N. The PE and PPE multigene families of Mycobacterium tuberculosis. Tuberculosis, 2nd Edition 513-525 (2004).

35. Cole,S.T. et al. Massive gene decay in the leprosy bacillus. Nature 409, 1007-1011 (2001).

36. Stinear,T.P. et al. Reductive evolution and niche adaptation inferred from the genome of Mycobacterium ulcerans, the causative agent of Buruli ulcer. Genome Res. 17, 192-200 (2007).

37. Bentley,S. & Sebaihia,M. Bacterial therapeutics. Nat. Rev. Microbiol. 5, 170-171 (2007).

38. Poulet,S. & Cole,S.T. Characterization of the highly abundant polymorphic GC-rich-repetitive sequence (PGRS) present in Mycobacterium tuberculosis. Arch. Microbiol. 163, 87-95 (1995).

39. Espitia,C. et al. The PE-PGRS glycine-rich proteins of Mycobacterium tuberculosis: a new family of fibronectin-binding proteins? Microbiology 145 ( Pt 12), 3487-3495 (1999).

40. Tekaia,F. et al. Analysis of the proteome of Mycobacterium tuberculosis in silico. Tuber. Lung Dis. 79, 329-342 (1999).


42. Levitskaya,J. et al. Inhibition of antigen processing by the internal repeat region of the Epstein-Barr virus nuclear antigen-1. Nature 375, 685-688 (1995).

43. Levitskaya,J., Sharipo,A., Leonchiks,A., Ciechanover,A. & Masucci,M.G. Inhibition of ubiquitin/proteasome-dependent protein degradation by the Gly-Ala repeat domain of the Epstein-Barr virus nuclear antigen 1. Proc. Natl. Acad. Sci. U. S. A 94, 12616-12621 (1997).

44. Sorensen,A.L., Nagai,S., Houen,G., Andersen,P. & Andersen,A.B. Purification and characterization of a low-molecular-mass T-cell antigen secreted by Mycobacterium tuberculosis. Infect. Immun. 63, 1710-1717 (1995).


46. Cangelosi,G.A., Palermo,C.O., Laurent,J.P., Hamlin,A.M. & Brabant,W.H. Colony morphotypes on Congo red agar segregate along species and drug susceptibility lines in the Mycobacterium avium-intracellulare complex. Microbiology 145 ( Pt 6), 1317-1324 (1999).

47. Cangelosi,G.A., Palermo,C.O. & Bermudez,L.E. Phenotypic consequences of red-white colony type variation in Mycobacterium avium. Microbiology 147, 527-533 (2001).

48. Mukherjee,S., Petrofsky,M., Yaraei,K., Bermudez,L.E. & Cangelosi,G.A. The white morphotype of Mycobacterium avium-intracellulare is common in infected humans and virulent in infection models. J. Infect. Dis. 184, 1480-1484 (2001).

https://www.researchgate.net/publication/12097875_Massive_Gene_Decay_in_the_Leprosy_Bacillus?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=

https://www.researchgate.net/publication/12097875_Massive_Gene_Decay_in_the_Leprosy_Bacillus?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=

https://www.researchgate.net/publication/15489515_Characterization_of_the_polymorphic_GC-rich_repetitive_sequence_PGRS_present_in_M_tuberculosis?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=



https://www.researchgate.net/publication/15657841_Inhibition_of_antigen_processing_by_the_internal_repeat_region_of_the_Epstein-Barr_Virus_nuclear_Antigen-1?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=

https://www.researchgate.net/publication/15657841_Inhibition_of_antigen_processing_by_the_internal_repeat_region_of_the_Epstein-Barr_Virus_nuclear_Antigen-1?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=

https://www.researchgate.net/publication/15469064_Sorensen_AL_Nagai_S_Houen_G_Andersen_P_Andersen_AB_Purification_and_characterization_of_a_low-molecular-mass_T-cell_antigen_secreted_by_Mycobacterium_tuberculosis_Infect_Immun_63_1710-1717?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=



https://www.researchgate.net/publication/12888711_Colony_morphotypes_on_Congo_red_agar_segregate_along_species_and_drug_susceptibility_lines_in_the_Mycobacterium_avium-intracellulare_complex?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=




https://www.researchgate.net/publication/12092990_Phenotypic_consequences_of_red-white_colony_type_variation_in_Mycobacterium_avium?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=



https://www.researchgate.net/publication/11644914_The_White_Morphotype_of_Mycobacterium_avium-intracellulare_Is_Common_in_Infected_Humans_and_Virulent_in_Infection_Models?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=



https://www.researchgate.net/publication/247023972_Inhibition_of_ubiquitin-proteasome_dependent_protein_degradation_by_the_Gly-Ala_repeat_domain_of_the_Epstein-Barr_virus_EBV_nuclear_antigen_EBNA-1?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=




https://www.researchgate.net/publication/232752644_Genome_watch_Bacterial_therapeutics?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=

https://www.researchgate.net/publication/232752644_Genome_watch_Bacterial_therapeutics?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=

Ch

apter 8

SUMMARY AND

SAMENVATTING

Summary - Samenvatting 140

Summary

Mycobacterium tuberculosis, the causative agent of tuberculosis, remains one of the most devastating pathogens. Globally, over one-third of the world's population has been exposed to the tuberculosis bacterium, and new infections occur at a rate of one person per second. In total more than 8 million people become sick with tuberculosis every year, and nearly two million die of the disease. This is the highest rate claimed by a single infectious bacterial agent. In this thesis I investigated in detail protein secretion by Mycobacterium species, with specific emphasis on the secretion of the mysterious PE and PPE proteins, using Mycobacterium marinum as a model. The PE and PPE gene families were one of the major surprises of the M. tuberculosis genome-sequencing project, because these gene families are unique for the mycobacteria and together they make up almost 10% of the coding region of the genome. However, thus far the exact function of these proteins is still largely unknown. The studies presented in this thesis are focused on several aspects of protein secretion mechanisms in mycobacteria. Increased knowledge of proteins secreted by pathogenic mycobacteria and the respective systems responsible for this may benefit the development of intervention strategies. In addition, the presented results have provided more insight in the value of M. marinum as a model for tuberculosis research. In Chapter 1, a brief general introduction of tuberculosis is presented, including the basic principles of the mycobacterial cell wall and protein secretion. Chapter 2 describes the current knowledge of a novel secretion system identified in mycobacteria, type VII secretion. The striking similarities between the type VII secretion systems in mycobacteria and related secretion systems in other Gram-positive bacteria are also discussed. Finally, this chapter contains a description of the role of these secretion systems in virulence. Chapter 3 focuses on the genetic variation between different isolates of M. marinum and the effect of this strain variation on pathogenicity in adult zebrafish. This study shows that M. marinum isolates can be grouped, based on genetic analysis, into two distinct clusters, designated cluster I and cluster II. Strains belonging to cluster II gave rise to chronic infections in adult zebrafish, whereas infection with cluster I strains resulted in acute disease. This study also correlates specific M. marinum genotypes to virulence in humans. Finally, the results of Chapter 3 were used to further develop the M. marinum zebrafish infection model in our laboratory. Once the tools to investigate M. marinum were established, my research focussed on the secretion of (potential) virulence factors. Chapter 4 describes the first experiments on this topic, where we decided to study the secretion of the small and soluble PPE41 protein. This study resulted in the identification of a novel protein secretion pathway in pathogenic mycobacteria, i.e. the ESX-5 pathway. This secretion system is present in all slow-growing pathogenic mycobacteria, but can not be identified in fast-growing species, such as Mycobacterium smegmatis. By introducing the complete ESX-5 locus we were able to successfully reconstitute the


PPE41 secretion pathway in M. smegmatis. Subsequently, the secretome of the ESX-5 system of M. marinum was determined, as described in Chapter 5. Here it is shown that ESX-5 is a major secretion pathway for mycobacteria, which is responsible for the extracellular accumulation of a large number of proteins, including probably all recently evolved PE and PPE proteins. Because of their high homology, PE/PPE proteins might have overlapping functions. By studying ESX-5 mutants one can now evaluate the effect of all recently evolved PE/PPE proteins. This approach was followed in Chapter 6, where different aspects of the virulence and pathogenesis of the M. marinum ESX-5 mutant as compared to the wild-type strain are described. This study shows that ESX-5 effector molecules subvert the normal innate immune response of the macrophage and therefore, these ESX-5-secreted proteins are probably used by this bacterium to assert a profound inhibitory influence on its cellular host cell. Furthermore, this study shows that the ESX-5 pathway is probably involved in macrophage cell death, suggesting that this secretion pathway is a major determinant of mycobacterial virulence. Finally, in Chapter 7, the results of the previous chapters are discussed, with emphasis on strain variation, PE and PPE proteins and Type VII secretion system in mycobacteria. All the experiments described in this thesis are performed on M. marinum, and although we hypothesize that these results also apply to M. tuberculosis, future experiments will have to prove this. If true, ESX-5 could be a major focus of mycobacterial research in the near future and hopefully we can use the generated knowledge of this pathway and its effector proteins to our advantage in the ongoing battle against this bacterial pathogen.


Samenvatting

Mycobacterium tuberculosis, de verwekker van tuberculose, is nog steeds een belangrijk probleem voor de internationale gezondheidszorg. Meer dan een derde van de wereldbevolking is geïnfecteerd met deze bacterie en elke seconde wordt er een nieuw persoon besmet. Jaarlijks krijgen 8 miljoen mensen tuberculose en sterven er ongeveer 2 miljoen mensen aan deze ziekte. Hiermee is tuberculose de belangrijkste infectieziekte veroorzaakt door een bacterie. In dit proefschrift is het onderzoek beschreven dat ik gedaan heb naar eiwitsecretie in Mycobacterium marinum, een nauwe verwant van M. tuberculosis, met in het bijzonder aandacht voor de mysterieuze PE en PPE eiwitten. De PE en PPE genen waren een van de grote verassingen van de ontcijfering van het M. tuberculosis genoom; deze genen zijn uniek voor de mycobacteria en vormen samen maar liefst bijna 10% van het coderende gedeelte van het genoom. Desondanks is de functie van deze eiwitten helaas nog grotendeels onbekend. Bij dit promotieonderzoek zijn verschillende aspecten van eiwitsecretie door mycobacteriën onderzocht. Kennis van deze processen zal kunnen bijdragen aan de ontwikkeling van (nieuwe) interventiestrategieën. Daarnaast hebben we met dit onderzoek meer inzicht gekregen in het gebruik van M. marinum als alternatief voor het tuberculose onderzoek. In hoofdstuk 1 is een korte inleiding gegeven over tuberculose, met nadruk op de celwand van de tuberculose bacterie en de eiwitsecretie door deze bacterie. Dit is nader beschreven in hoofdstuk 2, dat ingaat op de huidige kennis omtrent een nieuw type secretie systeem dat als eerste ontdekt is in Mycobacterium, namelijk het type VII secretie systeem. Dit secretie systeem is niet specifiek voor dit genus, het blijkt ook aanwezig te zijn in verschillende andere Gram-positieve bacteriën. Ook wordt in dit hoofdstuk het belang van type VII secretie voor de virulentie beschreven. Hoofdstuk 3 is het eerste hoofdstuk dat het experimentele onderzoek beschrijft en dit hoofdstuk behandelt de genetische variatie tussen verschillende M. marinum stammen en het effect van deze variatie op de virulentie van deze stammen in volwassen zebravissen. Deze studie laat zien dat de verschillende M. marinum stammen op grond van hun genetische verwantschap gegroepeerd kunnen worden in twee verschillende clusters, genaamd cluster I en cluster II. Deze twee clusters vertonen ook duidelijk verschillen in virulentie, waarbij stammen van cluster II zorgen voor chronische infecties in zebravissen, terwijl de andere stammen juist een acute infectie veroorzaken. Ook blijkt uit deze resultaten dat met name cluster I stammen problematische infecties veroorzaken bij mensen. De resultaten van hoofdstuk 3 zijn daarnaast ook gebruikt om het zebravissen infectie model verder te ontwikkelen aan de VUmc. Nu de gereedschappen aanwezig waren om M. marinum goed te kunnen bestuderen, richtte mijn onderzoek zich op de secretie van (potentiële) virulentiefactoren. Hoofdtsuk 4 beschrijft de eerste experimenten in deze richting, waar besloten is om de secretie te bestuderen van het kleine en goed oplosbare PPE41 eiwit. Het onderzoek in dit hoofdstuk laat zien dat PPE41 uitgescheiden


wordt via een nieuw type VII secretie systeem, dat ESX-5 is genoemd. Dit secretie systeem is aanwezig in langzaam-groeiende mycobacteriën, waaronder de belangrijkste pathogene Mycobacterium soorten vallen, terwijl het afwezig is in de snel-groeiende soorten zoals Mycobacterium smegmatis. Echter, wanneer het hele ESX-5 gencluster in M. smegmatis tot expressie wordt gebracht is deze bacterie wel in staat PPE41 te secreteren. Vervolgens is in hoofdstuk 5 beschreven welke eiwitten nog meer via ESX-5 uitgescheiden worden. Hier laat ik zien dat ESX-5 een zeer belangrijk secretiesysteem is, dat verantwoordelijk is voor de uitscheiding van alle recent geëvolueerde PE en PPE eiwitten. Vanwege hun hoge homologie hadden we al voorspeld dat deze eiwitten via dezelfde route gesecreteerd zouden worden en ze hebben mogelijk ook (deels) dezelfde of overlappende functies. Door nu de virulentie van de ESX-5 mutant te bestuderen is het mogelijk om het effect van al deze PE/PPE eiwitten in een keer te bestuderen. Deze benadering werd gevolgd in hoofdstuk 6, waar verschillende aspecten van de interactie van de ESX-5 mutant met het immuunsysteem van de gastheer zijn beschreven. Deze studie laat zien dat de eiwitten die gewoonlijk door ESX-5 gesecreteerd worden de aangeboren (innate) immuunreactie van de macrofaag in belangrijke mate manipuleren. Zo wordt de productie van een aantal belangrijke cytokines onderdrukt terwijl de productie van andere cytokines juist gestimuleerd worden. Daarnaast is de ESX-5 mutant verstoord in het induceren van celdood van de macrofaag. Deze resultaten impliceren dat ESX-5 een belangrijke rol speelt in de interactie van pathogene mycobacteriën met het immuunsysteem. Ten slotte worden in hoofdstuk 7 de resultaten van de voorgaande hoofdstukken als geheel bediscussieerd met nadruk op het nieuw ontdekt type VII secretie systeem ESX-5. Alle experimenten beschreven in dit proefschrift zijn gedaan aan M. marinum, en hoewel we verwachten dat deze resultaten ook toepasbaar zijn voor M. tuberculosis, zullen toekomstige experimenten dat moeten uitwijzen. Als onze hypothese klopt, dan zal het onderzoek aan ESX-5 waarschijnlijk een belangrijke rol krijgen in het tuberculose onderzoek en het is te hopen dat onze kennis over dit secretie systeem zal bijdragen tot een oplossing in onze strijd tegen deze belangrijke pathogeen.

Acknowledgements - Curriculum Vitae – List of publications 145

Acknowledgements

Curriculum Vitae

List of Publications


Acknowledgements

I would like to take this opportunity to express my sincerest gratitude to all that have in one way or another contributed to the realization of this thesis, to begin with all the people of the Department of Medical Microbiology, and in particular those of the Research Group. First it was a privilege to complete this work under the guidance of my promoter Prof. Christina Vandenbroucke-Grauls. I would like to thank her for giving me the possibility to perform my PhD study in her Department and for her valuable support and care about the project. Wilbert Bitter, my co-promoter, and as such the main person working behind the scene to realize my thesis. I can’t thank him enough or really express my feelings of gratitude for what he has done for me during my period of study. I am especially grateful for his advice, patience, unlimited personal help, support, and encouragement and more important for providing me with an ideal environment to carry out my work. I am greatly indebted to Ben Appelmelk for his advice, support, constructive critics and stimulating discussions. Also I am thankful to Astrid van der Sar and Marian llamas for their support and valuable discussions. The work presented in this thesis has also greatly benefited from several collaborations. I am grateful to Prof. Tom Ottenhoff and Nigel Savage, from Leiden University Medical Centre, Leiden for helpful discussions and for providing the opportunity to perform immunological assays. I would also like to thank Prof. Peter Peters, Nicole van der Wel and Maaike van Zon from the Netherlands Cancer Institute in Amsterdam for their support, performing the EM experiments and good discussion about the results. Also a number of international contacts have helped to make this work a success, I sincerely thank Bryant McLaughlin (UCSF, San Francisco, USA), Michael Strong (UCLA-DOE, Los Angeles, USA), Michael Brennan (FDA, Bethesda, USA) and Nico Gey van Pittius (Stellenbosch University, Tygerberg, South Africa) for their reagents, their support and their helpful suggestions. I wish to express my thanks to a number of people who made my stay in the Medical Microbiology Department both pleasant and fruitful. Thanks to my roommates Gert Blaauw, Marlies Mooij and Nicole Driessen; we always had nice discussions. My regards are also extended to Wim Schouten, Theo Verboom, Fredericke Hannes, Janneke Maaskant, Roy Ummels, Marion Sparrius, and Sandra Thonhauser for their kind and invaluable help for which I am greatly indebted, especially Theo’s and Fredericke’s expert assistance helped to make the ESX-5 work successful. During my PhD, I had a pleasure to meet very wonderful colleagues of Oral Microbiology group, who made the research life more attractive. I would also like to thank my colleagues Edith Houben and Jeroen Geurtsen for sharing fate and my students Tim Blaak, Widad Rifi, Desiree Steenbeek and Birnaz Adsiz for their brave efforts and great help.


I am most grateful to my best friends Mustafa Ghorashi and Murtada Muaaz for their unlimited moral support and continuous motivation. I would also like to thank Khalil Boutaga for his help in realizing the layout of the manuscript of this thesis. My gratitude is also extended to my Sudanese friends in the Netherlands for their encouragement and wishing me all the best. Certainly, at this point, I would like to give my heartily thanks to my father, my dearest brothers and sisters, my very dear wife Sarra and beloved kid Mohammed, for their wisdom, care and never-lasting patience. Lastly, and above all, most thanks goes to God (Allah) who has been my support, not only during the period of this PhD study, but also during my whole life.


Curriculum Vitae

The author of this thesis was born on the 21st of December 1973 in Elobeid (Sudan). After finishing higher secondary education in Sudan, he started to study Medical Laboratory Sciences at the Lyceum Northwestern University, Dagupan city, Philippines. In May 1994, he obtained his Bachelor Degree (BSc). After a short break, he moved to the Netherlands and he obtained his second degree in Medical Microbiology in 1998. From July 1998 to September 2001 he worked on a EU-funded research project on measles ‘‘Contribution to the elimination of measles from east Africa’’ at the Department of Virology, Erasmus University Medical Centre, Rotterdam, The Netherlands, in close collaboration with the Institute of Endemic Disease of the University of Khartoum, Sudan, under supervision of Prof. Dr. A.D.M.E. Osterhaus and Dr. R.L. de Swart. In September 2001, the author started to study Biomedical Sciences at the University of Leiden, The Netherlands. In July 2003, he defended his master thesis after he spent one year at the Department of Parasitology, Leiden University Medical centre, there he performed his graduation research on the subject: ‘‘Pfs48/45 Gene as A Potential Candidate Molecule for a transmission-blocking vaccine against Malaria’’ under supervision of Prof. Dr. A.P. Waters and Dr. M.R. van Dijk. In July 2003, he continued in science as a Ph.D. student at the Department of Medical Microbiology and Infection Control of the Vrije Universiteit Medical Centre, Amsterdam, The Netherlands. In this Department he studied molecular biology and protein secretion of pathogenic mycobacteria using Mycobacterium marinum as a model. This study was conducted under the supervision of Prof. Dr. C.M.J.E. Vandenbroucke-Grauls and Dr. W. Bitter, and resulted in the present thesis.


List of Publications

De Swart R.L., Y. Nur, Abdallah, A.M., H, Kruining, H.S. El Mubarak, S.A. Ibrahim, B. van den Hoogen, J. Groen, and A.D.M.E. Osterhaus. (2001). Combination of RT-PCR analysis and IgM detection on filter paper blood samples allows diagnostic and epidemiological studies of measles. J. Clin. Microbiol., 39:270-273. De Swart R.L., H.S. El Mubarak, H.W. Vos, O.A. Mustafa, Abdallah, A.M., J. Groen, M.M. Mukhtar, E.E. Zijlstra, A.M. El Hassan, T.F. Wild, S.A. Ibrahim, and A.D.M.E. Osterhaus. (2001). Prevention of measles in Sudan: a prospective study on vaccination, diagnosis and epidemiology. Vaccine., 19:2254-2257. S.A. Ibrahim, O.M. Mustafa, M.M. Mukhtar, I.A. Salih, H.S. El Mubarak, Abdallah, A.M., A.M. El Hassan, A.D.M.E. Osterhaus, J. Groen, R.L. de Swart, and E.E. Zijlstra. (2002). Measles in suburban Khartoum: an epidemiological and clinical study. Trop. Med. & Int. Health., 7(5): 442-449. Y.Nur, Abdallah, A.M., J. Groen, H. Kruining, R.L. de Swart, and A.D.M.E. Osterhaus. (2002). Retrospective identification of three undiagnosed cases of measles encephalitis. Eur J Clin Microbiol Infect Dis., 21(12): 900-1. S. A. Ibrahim, Abdallah, A.M., E. A. Saleh, A. D. M. E. Osterhaus, and R. L. DE Swart. (2006). Measles virus-specific antibody levels in Sudanese infants: A prospective study using filter paper blood samples. Epidemiol. Infect., 134, 79–85. van der Sar A.M., Abdallah, A.M., Sparrius M, Reinders E, Vandenbroucke-Grauls CM, Bitter W. (2004) Mycobacterium marinum strains can be divided into two distinct types based on genetic diversity and virulence. Infect Immun., 72(11): 6306-12. Abdallah, A.M., Theo Verboom, Fredericke Hannes, Mohamad Safi, Michael Strong, David Eisenberg, Rene Musters, Christina M.J.E. Vandenbroucke-Grauls, Ben J. Appelmelk, Joen Luirink and Wilbert Bitter (2006). A specific secretion system mediates PPE41 transport in pathogenic Mycobacteria. Mol. Mictobiol., 662(3):667-79. Abdallah, A.M., Patricia A DiGiuseppe Champion, Jeffery Cox, Nicolaas C Gey van Pittius, Joen Luirink, Christina MJE Vandenbroucke-Grauls, Ben J Appelmelk and Wilbert Bitter (2007). Type VII--mycobacteria show the way. Nat. Rev. Microbiol. 5 (11): 883-891.




https://www.researchgate.net/publication/10952265_Retrospective_identification_of_three_undiagnosed_cases_of_measles_encephalitis?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=







https://www.researchgate.net/publication/8214909_Mycobacterium_marinum_Strains_Can_Be_Divided_into_Two_Distinct_Types_Based_on_Genetic_Diversity_and_Virulence?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=




https://www.researchgate.net/publication/11369606_Measles_in_suburban_Khartoum_an_epidemiological_and_clinical_study?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=





Abdallah, A.M., Theo Verboom, Connie Jiménez, Christina M.J.E. Vandenbroucke-Grauls and Wilbert Bitter (2007). The ESX-5 secretion system is involved in PE_PGRS & PPE proteins secretion. Submitted for publication.. Abdallah, A.M., Nigel DL. Savage, Fredericke Hannes, Maaike van Zon, Christina M.J.E. Vandenbroucke-Grauls, Nicole van der Wel, Tom H. M. Ottenhoff and Wilbert Bitter. The ESX-5 secretion system of Mycobacterium marinum modulates the macrophage response. Manuscript in preparation.

https://www.researchgate.net/publication/23448510_The_ESX-5_Secretion_System_of_Mycobacterium_marinum_Modulates_the_Macrophage_Response?el=1_x_8&enrichId=rgreq-d973c6ad-d3d7-40b5-b0c4-17fe57d62ff6&enrichSource=Y292ZXJQYWdlOzU5MjMwODc7QVM6MTA0NDQ4NTUwNTA2NTE1QDE0MDE5MTM4ODU5NjQ=




Type VII secretion — mycobacteria show the way

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