Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=tveq20 Veterinary Quarterly ISSN: 0165-2176 (Print) 1875-5941 (Online) Journal homepage: https://www.tandfonline.com/loi/tveq20 Bacteriology: Review paratuberculosis: How does mycobacterium avium subsp. Paratuberculosis resist intracellular degradation? M.Z. Tessema , A.P. Koets , V.P.M.G. Rutten & E. Gruys To cite this article: M.Z. Tessema , A.P. Koets , V.P.M.G. Rutten & E. Gruys (2001) Bacteriology: Review paratuberculosis: How does mycobacterium avium subsp. Paratuberculosis resist intracellular degradation?, Veterinary Quarterly, 23:4, 153-162, DOI: 10.1080/01652176.2001.9695105 To link to this article: https://doi.org/10.1080/01652176.2001.9695105 Copyright Taylor and Francis Group, LLC Published online: 01 Nov 2011. Submit your article to this journal Article views: 988 View related articles Citing articles: 4 View citing articles
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Bacteriology: Review paratuberculosis: How does mycobacterium avium subsp. Paratuberculosis resist iFull Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=tveq20
Veterinary Quarterly
Bacteriology: Review paratuberculosis: How does mycobacterium avium subsp. Paratuberculosis resist intracellular degradation?
M.Z. Tessema , A.P. Koets , V.P.M.G. Rutten & E. Gruys
To cite this article: M.Z. Tessema , A.P. Koets , V.P.M.G. Rutten & E. Gruys (2001) Bacteriology: Review paratuberculosis: How does mycobacterium avium subsp. Paratuberculosis resist intracellular degradation?, Veterinary Quarterly, 23:4, 153-162, DOI: 10.1080/01652176.2001.9695105
To link to this article: https://doi.org/10.1080/01652176.2001.9695105
Copyright Taylor and Francis Group, LLC
Published online: 01 Nov 2011.
Submit your article to this journal
Article views: 988
View related articles
SUMMARY Paratuberculosis is a chronic, progressive disease of mainly ruminants caused by the facultative intracellular bacterium, Mycobacterium avium subsp. paratuberculo- sis. Infection usually occurs in young animals through oral uptake of food contaminated with the organisms. The ingested bacteria are transcytosed through M-cells overlying the Peyer's patches and are released in the stroma, where they are taken up by macrophages. Inside the macrophage, the mycobacteria resist enzymatic and toxic degradation and multiply until the infected macrophage ruptures. The thick, lipid-rich cell envelope is mainly responsible for micobacterial resistance. In ad- dition to its barrier effect, which provides protections, the mycobacterial cell wall also contains several biologi- cally active components that down-regulate the bacteri- cidal function of macrophages. The basic survival strat- egy of pathogenic mycobacteria can be viewed at three levels: selective use of relatively safe entry pathways that do not trigger oxidative attack, modification of the intracellular trafficking of mycobacteria-containing phagosomes, and modulation of the cooperation between the innate and specific immunity. In doing so, pathogenic mycobacteria are successful intracellular organisms that survive and multiply inside macrophages. Current un- derstanding about the survival strategies of M. a. para- tuberculosis and its implications in the epidemiology, diagnosis, and control of the disease are discussed.
INTRODUCTION Paratuberculosis, Johne's disease, is a chronic, progressive, and ultimately fatal disease of mainly ruminants. It is caused by the facultative intracellular, acid-fast bacterium, Mycobacterium avium subspecies paratuberculosis. The disease affects a wide spectrum of species, from the most commonly affected domestic ruminants (cattle, sheep, and goats) to some other members of the order artiodactyla, ro- dents, carnivores, and primates (8,38,60,63). Infected ani- mals shed a large number of bacteria in the faeces and serve as the main source of infection. Young animals in the first few months of life are the most susceptible. They become in- fected through oral uptake of the organism from contami- nated teats, feed, or water. Recovery of mycobacteria from milk, semen, uterus, and placenta of infected cattle indicates the possibility of intra-mammary and intra-uterine infections
Departmml of Pathology, Faculty of Veterinary Medicine, Utrecht University, the Netherlands.
= Department of Infections Diseases and Immunology. Faculty of Veterinary Medicine, Utrecht University, the Netherlands.
3 Department of Pathology, Faculty of Veterinary Medicine, Addis Ababa University, Ethiopia
Address correspondence to: Erik Gruys, E,[email protected],nl
Vet Quart 2001; 23: 153-62
Accepted for publication: July 9, 2001
(87,90). Generally, animals acquire tolerance to new infec- tion as they grow older, but immunity is never solid, as indi- cated by experimental infections of adult animals (56,72). In addition to reports of more paratuberculosis cases in some breeds of cattle and sheep, there is some evidence for the presence of genetic variations in the susceptibility of dairy cattle to the disease (22,52,94). Although animals are infec- ted at an early age, clinical disease does not usually develop until 2-5 years of age (22).
After ingestion, the mycobacteria are transcytosed through microfold epithelial cells (M-cells) and are taken up by mononuclear phagocytes in the intestinal mucosa and gut-as- sociated lymphoid organs (GALT) (65). Macrophages are, however, the target cells of M. a. paratuberculosis, where the pathogen survives and multiplies until the macrophage ruptures and releases the organisms into the surrounding tis- sue (9). Monocytes and lymphocytes are recruited to the site of infection and become activated by releasing a variety of cytokines. Stimulated macrophages form epithelioid cells and multinucleated giant cells. Infiltration of the infected tis- sue with a large number of inflammatory cells leads to the thickening and corrugation of the intestinal mucosa, particu- larly of the ileum, which is the typical lesion of Johne's dis- ease. Chronic lymphangitis of the intestinal lymphatics and mesenteric granulomatous lymphadenopathy are also com- mon (22,24,92).
Clinically, the disease in cattle is characterized by reduced fertility and milk production followed by chronic, progres- sive loss of body condition, and intermittent or persistent diarrhoea with faecal shedding of the organisms. Despite re- maining alert with a normal and even sometimes increased appetite, clinically ill cattle continue to waste and eventually die. Diarrhoea is not a common feature ofparatuberculosis in sheep and goats, but all species of animals in the advanced stage of the disease become cachectic and weak before death. However, such clinically detectable disease is not a common finding; instead, a large proportion (higher than 95%) of infection remains subclinical (92). The reason why so many infected animals do not manifest clinical disease is not clear, but age at the first exposure, the dose of the organ- ism and the immune status of the animal may play an impor- tant role.
Paratuberculosis is also gaining more attention as a potential threat to public health. It has clinical and pathological similari- ties to Crohn's disease, a chronic, granulomatous inflamma- tory bowel disease of humans with an unknown aetiology (21,39). In addition to the recovery of M. a. paratuberculosis in some Crohn's patients, there are reports of successful ther- apy of the disease with antibiotics targeting the mycobacte-
153 THE VETERINARY QUARTERLY, VOL 2 3 , No 4 , NOVEMBER, 2001
"sr, s-kiJV P96111WCPCO
4
I
rium (40). However, due to lack of reproducibility and specifi- city of some of the findings, the role of M a. paratuberculosis in initiating and maintaining the inflammation in Crohn's dis- ease is inconclusive.
The pathogenicity of mycobacteria in general relies on the ability of the organism to survive and multiply inside macrophages. This is believed to be the result of unique cell- wall components that promote virulence either by protecting the organisms from the destructive properties of the host macrophages and/or by modulating the immunological re- sponse of the host in favour of the pathogen (6,18). Although the modification is observed at several levels, the general strategy for mycobacterial survival inside macrophages can be approached from three different angles:
Selective entry to host cells through relatively safe entry pathways. Modification of intracellular trafficking. Intervention in the intercellular communication and inte- gration between different components of the immune sys- tem.
This paper summarizes the different survival mechanisms of mycobacteria in general and M a. paratuberculosis in parti- cular inside macrophages, which are equipped with powerful enzyme and toxic degradation systems.
BIOLOGY OF MYCOBACTERIA Mycobacteria are Gram-positive, acid-fast, non-spore for- ming, and non-motile bacteria with a straight or curved rod shape. The success of mycobacteria in entering, surviving and multipling in macrophages is partially linked to the un- usual physicochemical properties of their surface (15,31). The lipid-rich cell wall, approximately 40% of the total cell dry weight, is believed to determine many of the properties of the organism, such as resistance to intracellular degrada- tion, adjuvancity, anti-tumor activity, and virulence (62).
Structure of mycobacterial cell wall The mycobacterial envelope consists of four layers: from in- side out, the plasma membrane, electron-dense layer, elec- tron-transparent layer, and outer layer (7,68). The plasma membrane, like that of other bacteria, is composed of a lipid bilayer with embedded membrane proteins. The surrounding
electron-dense layer, which appears dense on transmission electron microscopy, consists of a peptidoglycan backbone covalently linked through a diglycosylphosphoryl bridge to a branched chain of arabinogalactan. The subsequent electron- transparent layer is mainly composed of mycolic acids that are esterified to the arabinogalactan. Components of the outer layer of mycobacteria vary from species to species. In M. avium the predominant constituents are serovar-specific glycopeptidolipids. Lipoarabinomannan is found in all the three layers of the cell wall. Other components of the cell wall include phosphatidylinositol mannosides, lipoproteins, cord factor (a-a-D- trehalose 6, 6'-dimycolate), macrophage in- hibitory factor (MIF-A3), and other glycolipids (7,15,66,78).
Biologically active cell-wall components Lipoarabinomannan is a highly immunogenic and potent in- hibitor of macrophage activation that down-regulates macro- phage effector function at several levels (Table 1). It suppresses macrophage activation and T-cell stimulation, and scavenges potentially cytotoxic substances (7,18,83). The peptidoglycan- arabinogalactan complex is responsible for the adjuvancity and induction of tumor necrosis factor (TNF) release. Cord factor, MIF-A3, and several other cell-wall glycolipids together with certain enzymes, such as catalase/peroxidase and superoxide dismutase (SOD), are involved in the detoxification of reactive oxygen intermediates (ROI) (5,15,26,42,85).
Glycopeptidolipids accumulate on the surface of M. avium during growth. When M. avium grows inside macrophages, they accumulate within the phagosomal/phagolysosomal compart- ment and appear as a peribacillary space or an electron-trans- parent zone surrounding the bacteria on transmission electron microscopy (Figure lb) (7). This electron-transparent capsule surrounding the mycobacteria may serve as a passive barrier and protect the pathogen from toxic and enzymatic attack. Secreted enzymes such as catalase/peroxidase and SOD, toxic lipids, con- tact-dependent lytic substances, and constituents that inhibit both macrophage priming and lymphoproliferation have also been found in the capsule (26).
BINDING AND UPTAKE OF MYCOBACTERIA After ingestion, M. a. paratuberculosis crosses the intestinal mucosal barrier via the M-cells that are found in the follicle-
Table 1. Some of the known biological activities of mycobacterial cell components.
Mycobacterial cell wall component Biological activity
Lipoarabinomannan (LAM) scavenges reactive oxygen intermediates (ROI), inhibits IFN-a-mediated activation of maprophages, suppresses T-cell proliferation, suppresses IL-2, IL-5, and GM-CSF production, enhances TNF- a production.
Cord factor inhibits fusion between vesicles, induces granuloma formation
.
Superoxide dismutase (SOD) scavenges ROI.
1 54 THE VETERINARY QUARTERLY, VOL 23 , No 4 , NOVEMBER, 2001
1
\-owi rrbatr-Acrio L _ _
Figure 1. Electron microscopic appearance of Mycobacteria-infected macrophages indicating:
a) Phagocytosis of a large number of M. a. paratuberculosis by bovine monocyte-derived macrophage in vitro (7800 x). b) The same pathogen detected inside a mononuclear cell in a tissue obtained from the ileum ofa cow with spontaneous paratuberculosis. Immunogold labelling technique with polyclonal antibody to paratuberculosis was used (17,800 x).
associated epithelium overlying the Peyer's patches. M-cells function to continuously sample and transport various anti- gens from the intestinal lumen to the underlying mononu- clear cells, where they are processed for the induction of ac- quired immunity and immunotolerance to food antigens (49,67). Unlike enterocytes, M-cells lack brush-border microvilli, digestive enzymes, and surface mucus, and thereby provide an easily accessible surface for attachment of microorganisms (30,49,86). In vivo experiments showed a greater transport of M. a. paratuberculosis through M-cells in the presence of anti-M. a. paratuberculosis serum than in the presence of normal bovine serum. This suggests the im- portance of maternal antibodies in the natural infection of young animals (65). Live mycobacteria that traverse the M- cells are expelled on the basolateral side without apparent metabolic change and are constantly scavenged by macro- phages (66,86). Macrophages are, however, the target cells for mycobacterial infection. The pathogens survive and mul- tiply inside macrophages until they eventually kill the infec- ted macrophage and spread to other nearby cells.
Although most cells in metazoa have some phagocytotic capa- city, professional phagocytes, which include monocytes, macrophages, dendritic cells, and neutrophils, are the main phagocytic cells. They are far more efficient at internalizing a wider range of particles at a faster rate, mainly because of the various receptors they have in their plasma membrane (Figure I a). This is illustrated by the dramatic increase in the range and rate of phagocytosis by fibroblasts and epithelial cells transfected with Fc receptors (FcRs) (45,77). Macrophage receptors involved in binding and uptake either directly recognize different molecular patterns on the surface of particles or detect and bind to the host ligands, such as anti- bodies, that cover the particle. Complex particles such as my- cobacteria usually possess different patterns and can be recognized by more than one type of receptor (Table 2). Binding to different receptors results in signals that initiate different mechanisms of internalization and intracellular pro- cessing (1). Some intracellular organisms appear to exploit this difference in their favour. The entrance of tachyzoites of Toxoplasnia gondii into macrophages through pl-integrin re- ceptors, for instance, helps the tachyzoites to protect themsel- ves from lysosomal attack while tachyzoites that are coated with antibody and use FcR succumb to the macrophage respi- ratory burst (84). Similarly, Listeria monocytogenes is de- graded by macrophages when it enters through complement receptor 3 (CR3) but when it uses an alternative receptor it sur- vives and multiplies inside the macrophage (95). Mycobacteria may also survive the interaction with host macrophages by utilizing uptake pathways that do not result in phagolysosomal fusion, or that do not signal for an appropriate respiratory burst or other cytocidal mechanisms (28).
Complement receptors (CRs) Phagocytosis of M. a. paratuberculosis by- monocytes and monocyte-derived macrophages (MDM) increases in thepre- sence of serum, indicating the importance of serum opsonins. Serum from paratuberculosis-free animals contributes to the uptake to the same extent as that of infected animals. This in- dicates the presence of sufficient serum opsonins in a healthy animal (97). Among the serum opsonins, complement pro- teins such as C3b, C4b and C3bi are known to opsonize mycobacteria for phagocytosis through the CRs found on the surface meMbrane of macrophages, namely CR1, CR3, and
1 55 THE VETERINARY QUARTERLY, VOL 2 3 , No 4 , NOVEMBER, 2001
,7111
. 1. ' A.'7.11 1 :". taif: '11- ., oN.. -I' ' "'t 0',.et 1
..:.a.t,.1 =,.... , .,-- t
,-,r., ..- ...... , , . ,2,
, 1-1,,,, . 7 g .., - ...i [... i
-'.%:.;', ,..1.1...t...--, t.:;,t 7 . ..*V''. ",
. k.
1.. , .1,:-.......
2- 4., 4.
,
Table 2 Macrophage plasma membrane components involved in the binding and uptake of mycobacteria.
Macrophage plasma membrane molecule
CD ligand Response to entry Other mycobacteria known to use the pathway
Reference
Complement receptor CD35 C3b,C4b No respiratory burst, M. tb, M.1, MAC 29,77,79 CR1 CD11a/CD18 C3bi No inflammatory- M. tb, M.1, MAC 29,77,79 CR3 CD11b/CD18 C3bi mediator release M. tb, M.1, MAC 29,77,79 CR4
Fc recepror Immunoglobulins Respiratory burst M.tb 29
Mannose receptor Mannose No respiratory burst M. tb, MAC 4,29,95
Scavenger receptor Lipopolysaccharide M.tb 29,95
Lipopolysaccharide receptor CD14 Lipopolysaccharide M.tb 70
Fibronectin receptor Fibronectin No respiratory burst MAC 12
Vitronectin receptor Vitronectin MAC 11,12
Transferrin receptor CD73 Transferrin MAC 11
Surfactant protein receptor Surfactant protein A M.tb, M.b(BCG) 29
Membrane cholesterol M.b(BCG) 35
Sialophorin ('surface mucin) CD43 M.tb, M.b(BCG) 33
M.tb = M. tuberculosis MAC = M. avium complex M.b(BCG)= M. bovis derived from BCG. M.I M. leprae
CR4, respectively (1). Mycobacteria can also generate C3b, using their C4b-like surface component to bind and enter through CR1 (1,77,81). Entry through CRs seems very critical for mycobacteria as they can also use these gates in the absence of serum opsonins. M. tuberculosis, for instance, uses its cell-wall polysaccharides to directly bind with the p-glucan binding site of CR3 (25). The significant reduction in the mycobacterial uptake of macrophages demonstrated by blockade of the CRs (11,12,79) is clear evidence for their im- portance in mycobacterial uptake. This characteristic of mycobacteria to exploit CR-mediated entry is advantageous because internalization via CR does not induce a respiratory burst and thus the mycobacteria have a better chance of escaping degradation (59,77,93).
Fc receptors (FcRs) Immunoglobulins (Igs) are the other serum constituents that opsonize particles for phagocytosis. Opsonization by anti- body and subsequent engagement with FcRs initiates the production of reactive oxygen intermediates (ROI) (45). It is, therefore, unlikely that successful intracellular pathogens such as mycobacteria would utilize FcRs as a route of entry to phagocytes. In contrast to the known role of CRs in myco- bacterial adherence and uptake, the contribution of FcR-me- diated uptake remains uncertain (29,31). In advanced clini- cal cases of bovine paratuberculosis, higher levels of antibody with little or no protective value are common (27). In the presence of such higher antibody levels, however, the mechanism(s) through which mycobacteria might avoid an- tibody opsonization, FcR-mediated entry, or initiatidn of re- spiratory burst is (are) not clear.
Mannose receptor (MR) The MR recognizes glycosylated molecules with terminal
mannose, fucose, or N-acetylglucosamine moieties and effi- ciently internalizes soluble and particulate ligands through endocytic and phagocytic pathways, respectively (71). The abundant and peripherally exposed lipoarabinomannan of the mycobacterial cell wall contains terminal mannose resi- dues that interact with the MR (80). This receptor is expres- sed on mature macrophages but not on monocytes and is in- volved in the non-opsonic phagocytosis of virulent mycobacteria (29). Therefore, MR might be responsible for the higher phagocytosis of M. a. paratuberculosis by mono- cyte derived macrophages (MDM) than by fresh monocytes, and for the phagocytosis of the same in the absence of serum (39,97). Phagocytosis through MR triggers neither ROI pro- duction nor phagosomal maturation and therefore, like up- take via CRs, it may serve as a safe portal entry for the myco- bacterial cargo (4).
Other entry pathways Lipopolysaccharide (LPS) receptor (CD14), a phosphatidyli- nositol glycan-linked membrane protein, is the other surface receptor expressed on., macrophages which is involved in mycobacterial uptake. It is best known and characterized for its high affinity for LPS of Gram-negative bacteria. It also binds lipoarabinomannan of mycobacteria and is observed to mediate binding of non-opsonized mycobacteria by human microglia (70). Macrophage scavenger receptors (SRs) (29,95), fibronectin receptors (FnRs) (12), vitronectin recep- tor (VnR) (11,12), transferrin receptor (l'fR) (11), and surfac- tant protein receptors (SpRs) (29) also mediate mycobacterial uptake. In addition to the membrane receptors, other plasma membrane molecules, such as cholesterol and sialophorin (CD43), also play an important role in the binding, uptake and intracellular survival of mycobacteria (33,35). The type of specific receptors used, the molecular mechanism of entry of
156 THE VETERINARY QUARTERLY, VOL 23 , No 4 , NOVEMBER, 2001
(--- rtICVIC int row
OM MID
a
11
a= bacteria; b = bacteria covered with serum opsonins; c = binding of opsonized or non-opsonized bacteria with macrophage surface receptors; d = phagocy- tosis; e = phagosome; f= recycling of plasma membrane components from a phagosome; g = fusion of phagosomes; h = phagolysosome formation i = secre- tion of inflammatory mediaters (cytokines); j = enzymatic and oxidative degradation of bacteria inside the phagolysosomes; k = antigen-MHC II association and presentation on the cell surface; I = binding of the antigen-MHC II-complex presented on the macrophagessurface to the T-cell receptor (TCR) of a T- cell; m = degraded products in an exocytotic vesicle; n = cytokines released from theT-cell; o = exocytosis.
M. a. paratuberculosis into macrophages, and the importance of entry pathways for intracellular survival and growth of the bacteria are not clear (22,44). However, the difference in the proportion of mycobacteria internalized and in survival after entry through different routes observed for other microorgan- isms indicate the importance of entry pathways for the suc- cess of infection.
INTRACELLULAR FATE OF MYCOBACTERIA Binding of phagocytes to particles such as microorganisms (Figure 2, c) that are covered with serum opsonin (b) or directly to the surface components of the organism (a) acti- vates the phagocyte to extend pseudopodia and engulf the particle (d). The pseudopodia fuse their tips and create a membrane-bound vesicle, the phagosome (e). The phago- some undergoes a series of fusion and fission events (g) and matures through recycling of the plasma membrane mole- cules (f), fusing with endosomes and lysosomes to finally form a phagolysosome (h). Inside the phagolysosome, the engulfed particle is degraded by lysosomal enzymes and toxic substances (j).…
Veterinary Quarterly
Bacteriology: Review paratuberculosis: How does mycobacterium avium subsp. Paratuberculosis resist intracellular degradation?
M.Z. Tessema , A.P. Koets , V.P.M.G. Rutten & E. Gruys
To cite this article: M.Z. Tessema , A.P. Koets , V.P.M.G. Rutten & E. Gruys (2001) Bacteriology: Review paratuberculosis: How does mycobacterium avium subsp. Paratuberculosis resist intracellular degradation?, Veterinary Quarterly, 23:4, 153-162, DOI: 10.1080/01652176.2001.9695105
To link to this article: https://doi.org/10.1080/01652176.2001.9695105
Copyright Taylor and Francis Group, LLC
Published online: 01 Nov 2011.
Submit your article to this journal
Article views: 988
View related articles
SUMMARY Paratuberculosis is a chronic, progressive disease of mainly ruminants caused by the facultative intracellular bacterium, Mycobacterium avium subsp. paratuberculo- sis. Infection usually occurs in young animals through oral uptake of food contaminated with the organisms. The ingested bacteria are transcytosed through M-cells overlying the Peyer's patches and are released in the stroma, where they are taken up by macrophages. Inside the macrophage, the mycobacteria resist enzymatic and toxic degradation and multiply until the infected macrophage ruptures. The thick, lipid-rich cell envelope is mainly responsible for micobacterial resistance. In ad- dition to its barrier effect, which provides protections, the mycobacterial cell wall also contains several biologi- cally active components that down-regulate the bacteri- cidal function of macrophages. The basic survival strat- egy of pathogenic mycobacteria can be viewed at three levels: selective use of relatively safe entry pathways that do not trigger oxidative attack, modification of the intracellular trafficking of mycobacteria-containing phagosomes, and modulation of the cooperation between the innate and specific immunity. In doing so, pathogenic mycobacteria are successful intracellular organisms that survive and multiply inside macrophages. Current un- derstanding about the survival strategies of M. a. para- tuberculosis and its implications in the epidemiology, diagnosis, and control of the disease are discussed.
INTRODUCTION Paratuberculosis, Johne's disease, is a chronic, progressive, and ultimately fatal disease of mainly ruminants. It is caused by the facultative intracellular, acid-fast bacterium, Mycobacterium avium subspecies paratuberculosis. The disease affects a wide spectrum of species, from the most commonly affected domestic ruminants (cattle, sheep, and goats) to some other members of the order artiodactyla, ro- dents, carnivores, and primates (8,38,60,63). Infected ani- mals shed a large number of bacteria in the faeces and serve as the main source of infection. Young animals in the first few months of life are the most susceptible. They become in- fected through oral uptake of the organism from contami- nated teats, feed, or water. Recovery of mycobacteria from milk, semen, uterus, and placenta of infected cattle indicates the possibility of intra-mammary and intra-uterine infections
Departmml of Pathology, Faculty of Veterinary Medicine, Utrecht University, the Netherlands.
= Department of Infections Diseases and Immunology. Faculty of Veterinary Medicine, Utrecht University, the Netherlands.
3 Department of Pathology, Faculty of Veterinary Medicine, Addis Ababa University, Ethiopia
Address correspondence to: Erik Gruys, E,[email protected],nl
Vet Quart 2001; 23: 153-62
Accepted for publication: July 9, 2001
(87,90). Generally, animals acquire tolerance to new infec- tion as they grow older, but immunity is never solid, as indi- cated by experimental infections of adult animals (56,72). In addition to reports of more paratuberculosis cases in some breeds of cattle and sheep, there is some evidence for the presence of genetic variations in the susceptibility of dairy cattle to the disease (22,52,94). Although animals are infec- ted at an early age, clinical disease does not usually develop until 2-5 years of age (22).
After ingestion, the mycobacteria are transcytosed through microfold epithelial cells (M-cells) and are taken up by mononuclear phagocytes in the intestinal mucosa and gut-as- sociated lymphoid organs (GALT) (65). Macrophages are, however, the target cells of M. a. paratuberculosis, where the pathogen survives and multiplies until the macrophage ruptures and releases the organisms into the surrounding tis- sue (9). Monocytes and lymphocytes are recruited to the site of infection and become activated by releasing a variety of cytokines. Stimulated macrophages form epithelioid cells and multinucleated giant cells. Infiltration of the infected tis- sue with a large number of inflammatory cells leads to the thickening and corrugation of the intestinal mucosa, particu- larly of the ileum, which is the typical lesion of Johne's dis- ease. Chronic lymphangitis of the intestinal lymphatics and mesenteric granulomatous lymphadenopathy are also com- mon (22,24,92).
Clinically, the disease in cattle is characterized by reduced fertility and milk production followed by chronic, progres- sive loss of body condition, and intermittent or persistent diarrhoea with faecal shedding of the organisms. Despite re- maining alert with a normal and even sometimes increased appetite, clinically ill cattle continue to waste and eventually die. Diarrhoea is not a common feature ofparatuberculosis in sheep and goats, but all species of animals in the advanced stage of the disease become cachectic and weak before death. However, such clinically detectable disease is not a common finding; instead, a large proportion (higher than 95%) of infection remains subclinical (92). The reason why so many infected animals do not manifest clinical disease is not clear, but age at the first exposure, the dose of the organ- ism and the immune status of the animal may play an impor- tant role.
Paratuberculosis is also gaining more attention as a potential threat to public health. It has clinical and pathological similari- ties to Crohn's disease, a chronic, granulomatous inflamma- tory bowel disease of humans with an unknown aetiology (21,39). In addition to the recovery of M. a. paratuberculosis in some Crohn's patients, there are reports of successful ther- apy of the disease with antibiotics targeting the mycobacte-
153 THE VETERINARY QUARTERLY, VOL 2 3 , No 4 , NOVEMBER, 2001
"sr, s-kiJV P96111WCPCO
4
I
rium (40). However, due to lack of reproducibility and specifi- city of some of the findings, the role of M a. paratuberculosis in initiating and maintaining the inflammation in Crohn's dis- ease is inconclusive.
The pathogenicity of mycobacteria in general relies on the ability of the organism to survive and multiply inside macrophages. This is believed to be the result of unique cell- wall components that promote virulence either by protecting the organisms from the destructive properties of the host macrophages and/or by modulating the immunological re- sponse of the host in favour of the pathogen (6,18). Although the modification is observed at several levels, the general strategy for mycobacterial survival inside macrophages can be approached from three different angles:
Selective entry to host cells through relatively safe entry pathways. Modification of intracellular trafficking. Intervention in the intercellular communication and inte- gration between different components of the immune sys- tem.
This paper summarizes the different survival mechanisms of mycobacteria in general and M a. paratuberculosis in parti- cular inside macrophages, which are equipped with powerful enzyme and toxic degradation systems.
BIOLOGY OF MYCOBACTERIA Mycobacteria are Gram-positive, acid-fast, non-spore for- ming, and non-motile bacteria with a straight or curved rod shape. The success of mycobacteria in entering, surviving and multipling in macrophages is partially linked to the un- usual physicochemical properties of their surface (15,31). The lipid-rich cell wall, approximately 40% of the total cell dry weight, is believed to determine many of the properties of the organism, such as resistance to intracellular degrada- tion, adjuvancity, anti-tumor activity, and virulence (62).
Structure of mycobacterial cell wall The mycobacterial envelope consists of four layers: from in- side out, the plasma membrane, electron-dense layer, elec- tron-transparent layer, and outer layer (7,68). The plasma membrane, like that of other bacteria, is composed of a lipid bilayer with embedded membrane proteins. The surrounding
electron-dense layer, which appears dense on transmission electron microscopy, consists of a peptidoglycan backbone covalently linked through a diglycosylphosphoryl bridge to a branched chain of arabinogalactan. The subsequent electron- transparent layer is mainly composed of mycolic acids that are esterified to the arabinogalactan. Components of the outer layer of mycobacteria vary from species to species. In M. avium the predominant constituents are serovar-specific glycopeptidolipids. Lipoarabinomannan is found in all the three layers of the cell wall. Other components of the cell wall include phosphatidylinositol mannosides, lipoproteins, cord factor (a-a-D- trehalose 6, 6'-dimycolate), macrophage in- hibitory factor (MIF-A3), and other glycolipids (7,15,66,78).
Biologically active cell-wall components Lipoarabinomannan is a highly immunogenic and potent in- hibitor of macrophage activation that down-regulates macro- phage effector function at several levels (Table 1). It suppresses macrophage activation and T-cell stimulation, and scavenges potentially cytotoxic substances (7,18,83). The peptidoglycan- arabinogalactan complex is responsible for the adjuvancity and induction of tumor necrosis factor (TNF) release. Cord factor, MIF-A3, and several other cell-wall glycolipids together with certain enzymes, such as catalase/peroxidase and superoxide dismutase (SOD), are involved in the detoxification of reactive oxygen intermediates (ROI) (5,15,26,42,85).
Glycopeptidolipids accumulate on the surface of M. avium during growth. When M. avium grows inside macrophages, they accumulate within the phagosomal/phagolysosomal compart- ment and appear as a peribacillary space or an electron-trans- parent zone surrounding the bacteria on transmission electron microscopy (Figure lb) (7). This electron-transparent capsule surrounding the mycobacteria may serve as a passive barrier and protect the pathogen from toxic and enzymatic attack. Secreted enzymes such as catalase/peroxidase and SOD, toxic lipids, con- tact-dependent lytic substances, and constituents that inhibit both macrophage priming and lymphoproliferation have also been found in the capsule (26).
BINDING AND UPTAKE OF MYCOBACTERIA After ingestion, M. a. paratuberculosis crosses the intestinal mucosal barrier via the M-cells that are found in the follicle-
Table 1. Some of the known biological activities of mycobacterial cell components.
Mycobacterial cell wall component Biological activity
Lipoarabinomannan (LAM) scavenges reactive oxygen intermediates (ROI), inhibits IFN-a-mediated activation of maprophages, suppresses T-cell proliferation, suppresses IL-2, IL-5, and GM-CSF production, enhances TNF- a production.
Cord factor inhibits fusion between vesicles, induces granuloma formation
.
Superoxide dismutase (SOD) scavenges ROI.
1 54 THE VETERINARY QUARTERLY, VOL 23 , No 4 , NOVEMBER, 2001
1
\-owi rrbatr-Acrio L _ _
Figure 1. Electron microscopic appearance of Mycobacteria-infected macrophages indicating:
a) Phagocytosis of a large number of M. a. paratuberculosis by bovine monocyte-derived macrophage in vitro (7800 x). b) The same pathogen detected inside a mononuclear cell in a tissue obtained from the ileum ofa cow with spontaneous paratuberculosis. Immunogold labelling technique with polyclonal antibody to paratuberculosis was used (17,800 x).
associated epithelium overlying the Peyer's patches. M-cells function to continuously sample and transport various anti- gens from the intestinal lumen to the underlying mononu- clear cells, where they are processed for the induction of ac- quired immunity and immunotolerance to food antigens (49,67). Unlike enterocytes, M-cells lack brush-border microvilli, digestive enzymes, and surface mucus, and thereby provide an easily accessible surface for attachment of microorganisms (30,49,86). In vivo experiments showed a greater transport of M. a. paratuberculosis through M-cells in the presence of anti-M. a. paratuberculosis serum than in the presence of normal bovine serum. This suggests the im- portance of maternal antibodies in the natural infection of young animals (65). Live mycobacteria that traverse the M- cells are expelled on the basolateral side without apparent metabolic change and are constantly scavenged by macro- phages (66,86). Macrophages are, however, the target cells for mycobacterial infection. The pathogens survive and mul- tiply inside macrophages until they eventually kill the infec- ted macrophage and spread to other nearby cells.
Although most cells in metazoa have some phagocytotic capa- city, professional phagocytes, which include monocytes, macrophages, dendritic cells, and neutrophils, are the main phagocytic cells. They are far more efficient at internalizing a wider range of particles at a faster rate, mainly because of the various receptors they have in their plasma membrane (Figure I a). This is illustrated by the dramatic increase in the range and rate of phagocytosis by fibroblasts and epithelial cells transfected with Fc receptors (FcRs) (45,77). Macrophage receptors involved in binding and uptake either directly recognize different molecular patterns on the surface of particles or detect and bind to the host ligands, such as anti- bodies, that cover the particle. Complex particles such as my- cobacteria usually possess different patterns and can be recognized by more than one type of receptor (Table 2). Binding to different receptors results in signals that initiate different mechanisms of internalization and intracellular pro- cessing (1). Some intracellular organisms appear to exploit this difference in their favour. The entrance of tachyzoites of Toxoplasnia gondii into macrophages through pl-integrin re- ceptors, for instance, helps the tachyzoites to protect themsel- ves from lysosomal attack while tachyzoites that are coated with antibody and use FcR succumb to the macrophage respi- ratory burst (84). Similarly, Listeria monocytogenes is de- graded by macrophages when it enters through complement receptor 3 (CR3) but when it uses an alternative receptor it sur- vives and multiplies inside the macrophage (95). Mycobacteria may also survive the interaction with host macrophages by utilizing uptake pathways that do not result in phagolysosomal fusion, or that do not signal for an appropriate respiratory burst or other cytocidal mechanisms (28).
Complement receptors (CRs) Phagocytosis of M. a. paratuberculosis by- monocytes and monocyte-derived macrophages (MDM) increases in thepre- sence of serum, indicating the importance of serum opsonins. Serum from paratuberculosis-free animals contributes to the uptake to the same extent as that of infected animals. This in- dicates the presence of sufficient serum opsonins in a healthy animal (97). Among the serum opsonins, complement pro- teins such as C3b, C4b and C3bi are known to opsonize mycobacteria for phagocytosis through the CRs found on the surface meMbrane of macrophages, namely CR1, CR3, and
1 55 THE VETERINARY QUARTERLY, VOL 2 3 , No 4 , NOVEMBER, 2001
,7111
. 1. ' A.'7.11 1 :". taif: '11- ., oN.. -I' ' "'t 0',.et 1
..:.a.t,.1 =,.... , .,-- t
,-,r., ..- ...... , , . ,2,
, 1-1,,,, . 7 g .., - ...i [... i
-'.%:.;', ,..1.1...t...--, t.:;,t 7 . ..*V''. ",
. k.
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,
Table 2 Macrophage plasma membrane components involved in the binding and uptake of mycobacteria.
Macrophage plasma membrane molecule
CD ligand Response to entry Other mycobacteria known to use the pathway
Reference
Complement receptor CD35 C3b,C4b No respiratory burst, M. tb, M.1, MAC 29,77,79 CR1 CD11a/CD18 C3bi No inflammatory- M. tb, M.1, MAC 29,77,79 CR3 CD11b/CD18 C3bi mediator release M. tb, M.1, MAC 29,77,79 CR4
Fc recepror Immunoglobulins Respiratory burst M.tb 29
Mannose receptor Mannose No respiratory burst M. tb, MAC 4,29,95
Scavenger receptor Lipopolysaccharide M.tb 29,95
Lipopolysaccharide receptor CD14 Lipopolysaccharide M.tb 70
Fibronectin receptor Fibronectin No respiratory burst MAC 12
Vitronectin receptor Vitronectin MAC 11,12
Transferrin receptor CD73 Transferrin MAC 11
Surfactant protein receptor Surfactant protein A M.tb, M.b(BCG) 29
Membrane cholesterol M.b(BCG) 35
Sialophorin ('surface mucin) CD43 M.tb, M.b(BCG) 33
M.tb = M. tuberculosis MAC = M. avium complex M.b(BCG)= M. bovis derived from BCG. M.I M. leprae
CR4, respectively (1). Mycobacteria can also generate C3b, using their C4b-like surface component to bind and enter through CR1 (1,77,81). Entry through CRs seems very critical for mycobacteria as they can also use these gates in the absence of serum opsonins. M. tuberculosis, for instance, uses its cell-wall polysaccharides to directly bind with the p-glucan binding site of CR3 (25). The significant reduction in the mycobacterial uptake of macrophages demonstrated by blockade of the CRs (11,12,79) is clear evidence for their im- portance in mycobacterial uptake. This characteristic of mycobacteria to exploit CR-mediated entry is advantageous because internalization via CR does not induce a respiratory burst and thus the mycobacteria have a better chance of escaping degradation (59,77,93).
Fc receptors (FcRs) Immunoglobulins (Igs) are the other serum constituents that opsonize particles for phagocytosis. Opsonization by anti- body and subsequent engagement with FcRs initiates the production of reactive oxygen intermediates (ROI) (45). It is, therefore, unlikely that successful intracellular pathogens such as mycobacteria would utilize FcRs as a route of entry to phagocytes. In contrast to the known role of CRs in myco- bacterial adherence and uptake, the contribution of FcR-me- diated uptake remains uncertain (29,31). In advanced clini- cal cases of bovine paratuberculosis, higher levels of antibody with little or no protective value are common (27). In the presence of such higher antibody levels, however, the mechanism(s) through which mycobacteria might avoid an- tibody opsonization, FcR-mediated entry, or initiatidn of re- spiratory burst is (are) not clear.
Mannose receptor (MR) The MR recognizes glycosylated molecules with terminal
mannose, fucose, or N-acetylglucosamine moieties and effi- ciently internalizes soluble and particulate ligands through endocytic and phagocytic pathways, respectively (71). The abundant and peripherally exposed lipoarabinomannan of the mycobacterial cell wall contains terminal mannose resi- dues that interact with the MR (80). This receptor is expres- sed on mature macrophages but not on monocytes and is in- volved in the non-opsonic phagocytosis of virulent mycobacteria (29). Therefore, MR might be responsible for the higher phagocytosis of M. a. paratuberculosis by mono- cyte derived macrophages (MDM) than by fresh monocytes, and for the phagocytosis of the same in the absence of serum (39,97). Phagocytosis through MR triggers neither ROI pro- duction nor phagosomal maturation and therefore, like up- take via CRs, it may serve as a safe portal entry for the myco- bacterial cargo (4).
Other entry pathways Lipopolysaccharide (LPS) receptor (CD14), a phosphatidyli- nositol glycan-linked membrane protein, is the other surface receptor expressed on., macrophages which is involved in mycobacterial uptake. It is best known and characterized for its high affinity for LPS of Gram-negative bacteria. It also binds lipoarabinomannan of mycobacteria and is observed to mediate binding of non-opsonized mycobacteria by human microglia (70). Macrophage scavenger receptors (SRs) (29,95), fibronectin receptors (FnRs) (12), vitronectin recep- tor (VnR) (11,12), transferrin receptor (l'fR) (11), and surfac- tant protein receptors (SpRs) (29) also mediate mycobacterial uptake. In addition to the membrane receptors, other plasma membrane molecules, such as cholesterol and sialophorin (CD43), also play an important role in the binding, uptake and intracellular survival of mycobacteria (33,35). The type of specific receptors used, the molecular mechanism of entry of
156 THE VETERINARY QUARTERLY, VOL 23 , No 4 , NOVEMBER, 2001
(--- rtICVIC int row
OM MID
a
11
a= bacteria; b = bacteria covered with serum opsonins; c = binding of opsonized or non-opsonized bacteria with macrophage surface receptors; d = phagocy- tosis; e = phagosome; f= recycling of plasma membrane components from a phagosome; g = fusion of phagosomes; h = phagolysosome formation i = secre- tion of inflammatory mediaters (cytokines); j = enzymatic and oxidative degradation of bacteria inside the phagolysosomes; k = antigen-MHC II association and presentation on the cell surface; I = binding of the antigen-MHC II-complex presented on the macrophagessurface to the T-cell receptor (TCR) of a T- cell; m = degraded products in an exocytotic vesicle; n = cytokines released from theT-cell; o = exocytosis.
M. a. paratuberculosis into macrophages, and the importance of entry pathways for intracellular survival and growth of the bacteria are not clear (22,44). However, the difference in the proportion of mycobacteria internalized and in survival after entry through different routes observed for other microorgan- isms indicate the importance of entry pathways for the suc- cess of infection.
INTRACELLULAR FATE OF MYCOBACTERIA Binding of phagocytes to particles such as microorganisms (Figure 2, c) that are covered with serum opsonin (b) or directly to the surface components of the organism (a) acti- vates the phagocyte to extend pseudopodia and engulf the particle (d). The pseudopodia fuse their tips and create a membrane-bound vesicle, the phagosome (e). The phago- some undergoes a series of fusion and fission events (g) and matures through recycling of the plasma membrane mole- cules (f), fusing with endosomes and lysosomes to finally form a phagolysosome (h). Inside the phagolysosome, the engulfed particle is degraded by lysosomal enzymes and toxic substances (j).…
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