su.diva-portal.orgsu.diva-portal.org/smash/get/diva2:1153263/FULLTEXT01.pdf · antibiotics that we use today to treat bacterial infections (1, 2). Based on Based on their antimicrobial
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
I. YfgM is an Ancillary Subunit of the SecYEG Translocon in Escherichia coli Götzke H, Palombo I, Muheim C, Perrody E, Genevaux P, Kudva R, Müller M, Daley DO. J Biol Chem (2014) 289(27), 19089-19097
II. Identification of Putative Substrates for the Periplasmic
Chaperone YfgM in Escherichia coli Using Quantitative Proteomics Götzke H, Muheim C, Maarten AF, Heck AJ, Maddalo G, Daley DO. Mol Cell Proteomics (2015) 14(1), 216–226
III. Increasing the Permeability of Escherichia coli using
IV. Identification of a Fragment-Based Scaffold that Inhibits the
Glycosyltransferase WaaG from Escherichia coli. Muheim C, Bakali A, Engström O, Wieslander Å, Daley DO, Widmalm G.
Antibiotics (2016) 5(1), 10
Author’s contribution to the publications
I. I performed the antibiotic disc diffusion assays and was involved in the interpretation of the results.
II. I cloned the plasmid constructs and performed the acid-stress
assays.
III. I was involved in the design of all experiments. I performed all of the experiments apart from the high-throughput screen. I contributed significantly in writing the final manuscript.
IV. I designed and performed all experiments apart from the in vitro lipid binding assay and the O-Deacylation of LPS. I contributed significantly in writing the final manuscript.
Abstract
The increasing emergence and spread of antibiotic-resistant bacteria is a
serious threat to public health. Of particular concern are Gram-negative
bacteria such as Escherichia coli, Acinetobacter baumannii, Klebsiella
pneumoniae or Pseudomonas aeruginosa. Some of these strains are resistant
to a large number of antibiotics and thus our treatment options are rapidly
declining. In addition to the increasing number of antibiotic-resistant
bacteria, a major problem is that many of the antibiotics at our disposal are
ineffective against Gram-negative bacteria. This is partly due to the
properties of the outer membrane (OM) which prevents efficient uptake. The
overarching goal of this thesis was to investigate how the OM of the Gram-
negative bacterium E. coli could be weakened to improve the activity of
antibiotics.
In the first two papers of my thesis (paper I + II), I investigated the
periplasmic chaperone network which consists of the two parallel pathways
SurA and Skp/DegP. This network is essential for the integrity of the OM
and strains lacking either SurA or Skp are defective in the assembly of the
OM, which results in an increased sensitivity towards vancomycin and other
antimicrobials. We identified a novel component of the periplasmic
chaperone network, namely YfgM, and showed that it operates in the same
network as Skp and SurA/DegP. In particular, we demonstrated that deletion
of YfgM in strains with either a surA or skp background further
compromised the integrity of the OM, as evidenced by an increased
sensitivity towards vancomycin.
In the remaining two papers of my thesis (paper III + IV), the goal was to
characterize small molecules that permeabilize the OM and thus could be
used to improve the activity of antibiotics. Towards this goal, we performed
a high-throughput screen and identified an inhibitor of the periplasmic
chaperone LolA, namely MAC-13243, and showed that it can be used to
permeabilize the OM of E. coli (paper III). We further demonstrated that
MAC-13243 can be used to potentiate the activity of antibiotics which are
normally ineffective against E. coli. In the last paper of my thesis (paper IV),
we undertook a more specific approach and wanted to identify an inhibitor
against the glycosyltransferase WaaG. This enzyme is involved in the
synthesis of LPS and genetic inactivation of WaaG results in a defect in the
OM, which leads to an increased sensitivity to various antibiotics. In this
paper, we identified a small molecular fragment (compound L1) and showed
that it can be used to inhibit the activity of WaaG in vitro.
To summarize, this thesis provides novel insights into how the OM of the
Gram-negative bacterium E. coli can be weakened by using small molecules.
We believe that the two identified small molecules represent important first
steps towards the design of more potent inhibitors that could be used in
clinics to enhance the activity of antibiotics.
Contents
List of publications .................................................................................... viii
Author’s contribution to the publications ................................................. ix
Abstract .......................................................................................................... x
Contents ...................................................................................................... xii
Abbreviations ............................................................................................. xiv
phosphatidylglycerol; PVCL2, 1,10-palmitoyl-2,20-vacenoyl cardiolipin. Figure taken from
(141). Reprinted with permission.
54
When I started to develop the assay for WaaG, I initially had problems with
its low activity. Since WaaG is a peripherally attached IM protein, I explored
if its activity could be improved by the addition of various lipids. After
several rounds of optimization, I discovered that I could improve the activity
of WaaG by including two E. coli membrane lipids, namely PG and CL, and
the non-ionic detergent CHAPS to the reaction mixture. Using these
optimized in vitro conditions, I showed that one of these small molecular
scaffolds, namely L1, is a weak inhibitor of WaaG with an IC50 of ~ 1 mM
(Figure 15).
Figure 15. L1 inhibits WaaG in vitro. Left panel: Mixed micelles consisting of 20 mM
CHAPS, 10 mM PG and 1 mM CL were added to LPS-TRUNC and UDP-Glc*. The reaction
was initiated by adding WaaG and incubated either with 2.5% DMSO or 25 mM of ligand
L1, L2 or L3. Samples were collected after various time points and inactivated by using
Laemmli buffer. Then, samples were separated by SDS-PAGE and detected by
autoradiography. Right panel: Relative activity of WaaG in the presence of either DMSO
or 25 mM L1, L2 or L3. Figure taken from (142). Reprinted with permission.
The observation that L1 could inhibit WaaG in vitro was an interesting
finding. Such small molecular fragments with low affinity for their target
(KD’s from high M to low mM) often represent a good starting point for the
55
design of a high-affinity inhibitor (143, 144). In this process, also known as
fragment-based drug design, small molecular fragments with low affinity for
their target are chemically elaborated or linked to produce a high affinity
inhibitor. Since L1 could be used to inhibit WaaG in vitro, we expanded
chemical space around L1 (and also L2 - L3), and created a fragment-based
library that included an additional 17 small molecular fragments (Figure 16,
unpublished data). At this point, the aim was to identify additional small
molecular fragments that either compete with the natural substrate UDP-Glc
for binding or inhibit WaaG in the in vitro activity assay.
Figure 16. Small molecular scaffold library L1-20.
56
Hence, I tested all small molecular fragments in the in vitro activity assay to
evaluate their inhibitory activity. Surprisingly, the results indicated that none
of the additional fragments had inhibitory activity (data not shown). These
findings were surprising since some of these fragments are structurally
closely related to L1 (Figure 16). The Widmalm group is currently
investigating by NMR spectroscopy if any of the additional small molecular
fragments compete with the natural substrate UDP-Glc for binding. Our
preliminary results indicate that one of these fragments, namely L8, also
binds to WaaG (data not shown). Despite the fact that the expanded library
did not contain a more potent inhibitor than L1, we could identify at least
one additional small molecular fragment that binds to WaaG. This provides
further insight for the design of a potent inhibitor against WaaG.
57
Conclusions and future perspectives
Gram-negative bacteria have developed sophisticated mechanisms to protect
themselves against noxious molecules such as antibiotics. Two of the major
mechanisms that limit the activity of many antibiotics include active efflux
by efflux pumps and reduced uptake across the OM barrier. The presented
doctoral thesis had the objective to investigate how the reduced uptake
across the OM barrier could be improved by destabilizing the OM. To
address this problem, I initially started investigating the periplasmic
chaperone network and how it contributes to the permeability of the OM
(paper I). In this paper, we identified a novel component of the SecYEG
translocon, namely YfgM, and showed that it operates in the same pathway
as the periplasmic chaperone network SurA/Skp. However, YfgM plays only
a minor role in this network since strains lacking YfgM did not have any
obvious defects in the OM. The molecular function of YfgM remains to be
determined but we speculate that it might act as a docking platform for the
periplasmic chaperones SurA/Skp. Interestingly, the periplasmic domain of
YfgM contains tetratricopeptide repeat (TPR) domains whose function has
not been determined yet. These domains are often involved in protein-
protein interactions and thus it might be interesting to further investigate the
function of the TPR domains in YfgM (145). There is also evidence that Skp
interacts with OMPs during their early translocation through the SecYEG
translocon (146). Hence, it is possible that Skp might dock to the SecYEG
translocon or a protein in close vicinity to it.
To better understand the function of YfgM, we performed a comparative
proteomic approach to identify potential substrates of YfgM (paper II). We
hypothesized that strains lacking YfgM might have a changed OMP profile
58
since YfgM operates in the same network as SurA/Skp. Although we did not
observe any significant changes in the levels of OMPs and lipoproteins in
strains lacking yfgM, the proteomic data revealed an unexpected insight into
the physiological role of YfgM. We identified a number of proteins that are
involved in acid-stress response, which were lower in abundance in strains
lacking YfgM. These findings were unexpected since YfgM had not been
shown to be induced during acid-stress previously (147–150). In an
additional experiment, we could confirm that strains devoid of YfgM had a
decreased survival rate at low pH. At this stage, it remains unclear if the
decreased survival rate at low pH in strains lacking YfgM is caused by a
defect in the cell envelope or by another secondary effect.
To address the reduced uptake of antibiotics across the OM, we performed a
high-throughput screen to look for small molecules that destabilize the OM
(paper III). In this study, we identified an inhibitor of the periplasmic
chaperone LolA, named MAC-13243. This small molecule had been
previously identified as a novel antimicrobial lead (136). However, I could
show that MAC-13243 can also be repurposed to an antibiotic adjuvant. I
observed that MAC-13243 synergized with the large-scaffold antibiotics
novobiocin and erythromycin, but not with vancomycin and rifampicin. At
this point, it remains unclear to us why MAC-13243 works synergistically
with some large-scaffold antibiotics but not with others. It is worth noting
that Krishnamoorthy et al. reported that the activity of these 4 antibiotics is
significantly limited by the OM and/or by active efflux (72). Surprisingly,
they found that the OM presented no major obstacle for novobiocin whereas
active efflux drastically reduced its activity. In the case of rifampicin and
vancomycin, they found that the OM barrier significantly limited their
activity whereas inactivation of active efflux only minor improved their
activity. In the case of erythromycin, a combination of both reduced uptake
and active efflux limited its activity.
59
Despite the preliminary findings, I believe that MAC-13243 has certain
limitations in its current form that prevent it from being used as an antibiotic
adjuvant. First of all, the synergistic interactions between MAC-13243 and
either novobiocin or erythromycin were moderate and only observed under
specific conditions. One of the major limitations of MAC-13243 might be its
limited stability in aqueous solution (151). MAC-13243 degrades in aqueous
solution into one molecule of 3,4-dimethoxyphenethylamine, two molecules
of formaldehyde and one molecule of S-(4-chlorobenzyl)isothiourea (Figure
17A). Thus, an attempt to improve the chemical stability of MAC-13243
could improve its activity as an antibiotic adjuvant. This could be achieved
by stabilizing the central triazine ring of MAC-13243, which is prone to
hydrolysis (151).
Figure 17. Degradation of MAC-13243 in aqueous solution. (A) MAC-13243 is
degraded into one molecule 3,4-dimethoxyphenethylamine, two molecules of
formaldehyde and one molecule S-(4-chlorobenzyl)isothiourea (151). Both MAC-13243
and the degradation product S-(4-chlorobenzyl)isothiourea bind to LolA (151) (B) An
analogue of the degradation product, named A22 or S-(4-dichlorobenzyl)isothiourea, has
been shown to bind to LolA. Interestingly this compound is an inhibitor of the
cytoskeletal protein MreB (138). Figure adjusted from (151). Reprinted with permission.
60
Interestingly, one of the degradation products of MAC-13243, namely S-(4-
chlorobenzyl)isothiourea, is a structural analogue of compound A22, which
has been identified as an inhibitor of the cytoskeletal protein MreB (Figure
17B) (138). Taylor et al. showed that A22 works synergistically with both
novobiocin and rifampicin in E. coli (115). Hence, the design of a molecule
which inhibits both LolA and MreB could be an interesting concept to
further enhance the synergistic effects of MAC-13243.
I personally think that it would be most interesting to expand on the findings
of paper IV. In this paper, I showed that the small molecular fragment,
namely compound L1, can be used to inhibit the glycosyltransferase WaaG
in vitro. However, due to its weak inhibitory activity, L1 is not a molecule
that can be used in its current form as an antibiotic adjuvant. However, we
believe that L1 represents a good lead fragment for the design of a more
potent inhibitor against WaaG. By expanding chemical space around L1, we
could identify an additional molecule, namely L8 (Figure 16), that binds to
WaaG in vitro. In a next optimization round, we plan to systematically
evaluate the chemical space around these two molecules to gain further
insight into the properties that are required for binding to and inhibiting
WaaG.
61
Populärvetenskaplig sammanfattning
Antibiotika anses vara en av de viktigaste upptäckterna inom
humanmedicinen. Sedan de infördes in i klinikerna på 1940-talet har de
framgångsrikt använts för att bota bakteriella infektioner. På grund av sin
omfattande användning och missbruk under de senaste decennierna har dock
många bakteriearter som inte längre är känsliga mot de flesta antibiotika
uppstått. Dessa så kallade multiresistenta bakterier har utvecklat olika
tillvägagångssätt för att inaktivera antibiotika. Följaktligen har vi ont om
behandlingsalternativ och är ofta tvungna att använda äldre antibiotika som
är mindre säkra.
Resistens mot antibiotika är ett allmänt problem. Gramnegativa bakterier är
dock arter som är särskilt bekymmersamma. Dessa bakterier dödar tusentals
människor årligen och anses därför vara ett stort hot mot folkhälsan. Ett av
de största problemen är att många antibiotika är ineffektiva mot
gramnegativa bakterier. Detta beror dels på en fysisk barriär, nämligen det
yttre membranet, som hämmar effektivt upptag av många antibiotika.
I den presenterade doktorsavhandlingen undersökte jag hur dessa fysiska
barriärer kunde försvagas. Det ultimata målet med min avhandling var att
hitta små molekyler som skulle kunna användas för att bryta denna fysiska
barriär. Vi resonerade att dessa små molekyler skulle kunna användas för att
förbättra upptagningen av de antibiotika som normalt inte effektivt passerar
yttermembranet.
62
Acknowledgements
I would like to take the opportunity to thank my supervisor Daniel Daley for his excellent supervision during the past 5 years. I really enjoyed it to be part of your group. Thanks mate! I also would like to thank our current group members Kiavash, Rageia, Zoe and Patrick, and the former group members Jörg, Stephen, Bill and Isolde. It was very pleasant to work with all of you and I think we have a really good ambience in the group. Special thanks to my co-supervisor Göran Widmalm and his current group members Jonas and Alessandro. It was fun to work together with you guys! Also a special thanks to our former collaborators Klaas, Spyridon, Amin, Olof and Åke. I am also grateful to my former students Lars Nilsson, Johanna Hultgren, Muna Amanuel, Andreas Dunge and Anja Blümler. Thanks to our neighbors from the Jan-Willem de Gier group: Thomas, Zhe, Henry and Alex. Special thanks to my table soccer friend Thomas for never giving up and Alex the Greek for all the insightful philosophical discussions. Thanks to the Rob Daniels group: Henrik, Hao, Dan, Rebecca and the former group members Johan and Diogo. It was always nice to see other people in the lab on the weekends. A big thanks to the Gunnar von Heijne and Ingmarie Nilsson group: Renuka, Grant, Felix, Ioanna, Luigi, Nir, José, Nina and Åsa. Special thanks to my bongo friend Mr. Felix – it was a pleasure to explore Africa with you! Thanks to the Tara Hessa group: Tara, Theresa, Genia and Anastasia. Cool group, good people!
63
Thanks to the Elzbieta Glaser group: Pedro and Beata for always being in a good mood and Elzbieta for never-ending but interesting discussions at the lunch table. Many thanks to Brzezinski and Ädelroth group members Jacob, Johan, Tobias, Camilla, Ingrid, Max, Nathalie and Pia. Great thanks to my climbing and sailing friend Max (aka sailor man) – I knew it was a good decision to have a friend with a sailing boat! We truly had some good laughs and I will never forget the large number of fish we caught - zero! Thanks to the David Drew group members Emmanuel, Mathieu, Aziz, Povilas, Pascal and Magnus. Bsunderä Dank äm bärtigä Päsci und sim Schätzi dr Regula - heb sorg zunnärä! Du bisch wahrhaftig en Glückspilz! Sogar dr Andi us Rümlang wär nidisch! Thanks to the Martin Ott group members Hannah, Mama, Magdalena, Tamara and Braulio for the discussions about the book exam and other stuff. Special thanks to Alex Tuuling, Maria Sallander, Anita Hjelm, Lotta Sturell, Karin Häggbom Sandberg, Malin Kamb, Håkan Thorén, Matthew Bennett, Ann-Britt Rönnell and Peter Nyberg for running DBB in the background. Last but not least, a very special thanks to my family and friends back home for their great support and visits to Stockholm. Looking forward to see you again!
64
References
1. Hall, B. G., Barlow, M. Evolution of the serine -lactamases: past, present and future. Drug Resist. Updat. 7, 111–123 (2004).
2. Baltz, R. A. Antibiotic discovery from Actinomycetes: Will a renaissance follow the decline and fall? SIM News 55, 188–96 (2005).
3. Udekwu, K. I., Parrish, N., Ankomah, P., Baquero, F., Levin, B. R. Functional relationship between bacterial cell density and the efficacy of antibiotics. J. Antimicrob. Chemother. 63, 745–757 (2009).
4. Li, J. et al. Antimicrobial Activity and Resistance: Influencing Factors. Front. Pharmacol. 8, (2017).
5. Scholar, E. M., Pratt, W. B. The Antimicrobial Drugs. 2nd edition. Oxford University Press, New York (2000).
6. Lewis, K. Platforms for antibiotic discovery. Nat. Rev. Drug Discov. 12, 371–387 (2013). 7. Kohanski, M. A., Dwyer, D. J., Collins, J. J. How antibiotics kill bacteria: from targets to
networks. Nat. Rev. Microbiol. 8, 423–435 (2010). 8. Kong, K.F., Schneper, L., Mathee, K. -lactam Antibiotics: From Antibiosis to Resistance and
Bacteriology. APMIS 118, 1–36 (2010). 9. Barlow, M. What antimicrobial resistance has taught us about horizontal gene transfer. Methods
Mol. Biol. 532, 397–411 (2009). 10. Coates, A., Hu, Y., Bax, R., Page, C. The future challenges facing the development of new
antimicrobial drugs. Nat. Rev. Drug Discov. 1, 895–910 (2002). 11. Fleming, A. On the Antibacterial Action of Cultures of a Penicillium, with Special Reference to
their Use in the Isolation of B. influenzae. Br. J. Exp. Pathol. 10, 226–236 (1929). 12. Lobanovska, M., Pilla, G. Penicillin’s Discovery and Antibiotic Resistance: Lessons for the
Future? Yale J. Biol. Med. 90, 135–145 (2017). 13. Coates, A. R., Halls, G., Hu, Y. Novel classes of antibiotics or more of the same? Br. J.
Pharmacol. 163, 184–194 (2011). 14. Silver, L. L. Challenges of antibacterial discovery. Clin. Microbiol. Rev. 24, 71–109 (2011). 15. Wenzel, R. P. The Antibiotic Pipeline – Challenges, Costs, and Values. N. Engl. J. Med. 351,
523–526 (2004). 16. Spellberg, B. The future of antibiotics. Crit. Care 18, 228 (2014). 17. Bakken, J. S. Antibiotic partners promote discovery. Nature 537, 167–167 (2016). 18. Piddock, L. J. V. The crisis of no new antibiotics – What is the way forward? Lancet Infect. Dis.
12, 249–253 (2012). 19. Michael, C. A., Dominey-Howes, D., Labbate, M. The Antimicrobial Resistance Crisis: Causes,
Consequences, and Management. Front. Public Health 2, (2014). 20. Payne, D. J., Gwynn, M. N., Holmes, D. J., Pompliano, D. L. Drugs for bad bugs: confronting the
challenges of antibacterial discovery. Nat. Rev. Drug Discov. 6, 29–40 (2007). 21. An antibiotic comeback ? Nat. Rev. Drug Discov. 13, 165–165 (2014). 22. Col, N. F., O’Connor, R. W. Estimating Worldwide Current Antibiotic Usage: Report of Task
Force 1. Rev. Infect. Dis. 9, S232–S243 (1987). 23. Barbosa, T. M., Levy, S. B. The impact of antibiotic use on resistance development and
persistence. Drug Resist. Updat. 3, 303–311 (2000).
65
24. Goossens, H., Ferech, M., Vander Stichele, R., Elseviers, M., ESAC Project Group. Outpatient antibiotic use in Europe and association with resistance: a cross-national database study. Lancet Lond. Engl. 365, 579–587 (2005).
25. Riedel, S. et al. Antimicrobial use in Europe and antimicrobial resistance in Streptococcus pneumoniae. Eur. J. Clin. Microbiol. 26, 485–490 (2007).
26. Centers for Disease Control and Prevention. Antibiotic Resistance Threats in the United States, 2013. Available at: http://www.cdc.gov/drugresistance/threat-report-2013/index.html, Accessed 20.09.2017
27. Ayukekbong, J. A., Ntemgwa, M., Atabe, A. N. The threat of antimicrobial resistance in developing countries: causes and control strategies. Antimicrob. Resist. Infect. Control 6, 47 (2017).
28. Witte, W. Selective pressure by antibiotic use in livestock. Int. J. Antimicrob. Agents 16, 19–24 (2000).
29. Bengtsson, B., Wierup, M. Antimicrobial Resistance in Scandinavia after a Ban of Antimicrobial Growth Promoters. Anim. Biotechnol. 17, 147–156 (2006).
30. Butaye, P., Devriese, L. A., Haesebrouck, F. Antimicrobial Growth Promoters Used in Animal Feed: Effects of Less Well Known Antibiotics on Gram-Positive Bacteria. Clin. Microbiol. Rev. 16, 175–188 (2003).
31. Larsson, D. G. J. Pollution from drug manufacturing: review and perspectives. Phil. Trans. R. Soc. B. 369, (2014).
32. Chemaly, R. F. et al. The role of the healthcare environment in the spread of multidrug-resistant organisms: update on current best practices for containment. Ther. Adv. Infect. Dis. 2, 79–90 (2014).
33. Sydnor, E. R. M., Perl, T. M. Hospital Epidemiology and Infection Control in Acute-Care Settings. Clin. Microbiol. Rev. 24, 141–173 (2011).
34. The evolving threat of antimicrobial resistance: options for action. World Health Organization: Geneva, Switzerland, 2012. Available at: http://apps.who.int/iris/bitstream/10665/44812/1/9789241503181_eng.pdf, Accessed: 23.09.2017
35. Allerberger, F., Gareis, R., Jindrák, V., Struelens, M. J. Antibiotic stewardship implementation in the EU: the way forward. Expert Rev. Anti Infect. Ther. 7, 1175–1183 (2009).
36. Allerberger, F., Lechner, A., Wechsler-Fördös, A., Gareis, R. Optimization of Antibiotic Use in Hospitals – Antimicrobial Stewardship and the EU Project ABS International. Chemotherapy 54, 260–267 (2008).
37. Barlam, T. F. et al. Implementing an Antibiotic Stewardship Program: Guidelines by the Infectious Diseases Society of America and the Society for Healthcare Epidemiology of America. Clin. Infect. Dis. 62, 51-77 (2016).
38. Waterer, G. W., Wunderink, R. G. Increasing threat of Gram-negative bacteria. Crit. Care Med. 29, 75-81 (2001).
39. Zowawi, H. M. et al. The emerging threat of multidrug-resistant Gram-negative bacteria in urology. Nat. Rev. Urol. 12, 570–584 (2015).
40. Boucher, H. W. et al. Bad bugs, no drugs: no ESKAPE! An update from the Infectious Diseases Society of America. Clin. Infect. Dis. 48, 1–12 (2009).
41. Peleg, A. Y., Hooper, D. C. Hospital-acquired infections due to gram-negative bacteria. N. Engl. J. Med. 362, 1804–1813 (2010).
42. Falagas, M. E., Kasiakou, S. K. Toxicity of polymyxins: a systematic review of the evidence from old and recent studies. Crit. Care 10, R27 (2006).
43. Silhavy, T. J., Kahne, D., Walker, S. The Bacterial Cell Envelope. Cold Spring Harb. Perspect. Biol. 2, a000414 (2010).
44. Brown, L., Wolf, J. M., Prados-Rosales, R., Casadevall, A. Through the wall: extracellular vesicles in Gram-positive bacteria, mycobacteria and fungi. Nat. Rev. Microbiol. 13, 620–630 (2015).
45. Morein, S., Andersson, A., Rilfors, L., Lindblom, G. Wild-type Escherichia coli cells regulate the membrane lipid composition in a ‘window’ between gel and non-lamellar structures. J. Biol. Chem. 271, 6801–6809 (1996).
66
46. Whitfield, C., Trent, M. S. Biosynthesis and export of bacterial lipopolysaccharides. Annu. Rev. Biochem. 83, 99–128 (2014).
47. Raetz, C. R. H., Whitfield, C. Lipopolysaccharide endotoxins. Annu. Rev. Biochem. 71, 635–700 (2002).
48. Clifton, L. A. et al. Asymmetric phospholipid: lipopolysaccharide bilayers; a Gram-negative bacterial outer membrane mimic. J. R. Soc. Interface 10, (2013).
49. Medzhitov, R., Janeway, C. J. Innate Immunity. N. Engl. J. Med. 343, 338–344 (2000). 50. Raetz, C. R., Reynolds, C. M., Trent, M. S., Bishop, R. E. Lipid A modification systems in gram-
negative bacteria. Ann. Rev. Biochem. 76, 295–329 (2007). 51. Wang, L., Wang, Q., Reeves, P. R. The variation of O-antigens in gram-negative bacteria.
Subcell. Biochem. 53, 123–152 (2010). 52. Koebnik, R., Locher, K. P., Van Gelder, P. Structure and function of bacterial outer membrane
proteins: barrels in a nutshell. Mol. Microbiol. 37, 239–253 (2000). 53. Nikaido, H. Molecular basis of bacterial outer membrane permeability revisited. Microbiol. Mol.
Biol. Rev. 67, 593–656 (2003). 54. Pagès, J. M., James, C. E., Winterhalter, M. The porin and the permeating antibiotic: a selective
diffusion barrier in Gram-negative bacteria. Nat. Rev. Microbiol. 6, 893–903 (2008). 55. Grabowicz, M., Silhavy, T. J. Redefining the essential trafficking pathway for outer membrane
lipoproteins. Proc. Natl. Acad. Sci. 114, 4769–4774 (2017). 56. Hirota, Y., Suzuki, H., Nishimura, Y., Yasuda, S. On the process of cellular division in
Escherichia coli: a mutant of E. coli lacking a murein-lipoprotein. Proc. Natl. Acad. Sci. 74, 1417–1420 (1977).
57. Suzuki, H. et al. Murein-lipoprotein of Escherichia coli: a protein involved in the stabilization of bacterial cell envelope. Mol. Gen. Genet. 167, 1–9 (1978).
58. Merdanovic, M., Clausen, T., Kaiser, M., Huber, R., Ehrmann, M. Protein quality control in the bacterial periplasm. Annu. Rev. Microbiol. 65, 149–168 (2011).
59. Schleifer, K. H., Kandler, O. Peptidoglycan types of bacterial cell walls and their taxonomic implications. Bacteriol. Rev. 36, 407–477 (1972).
60. Yamagami, A., Yoshioka, T., Kanemasa, Y. Differences in Phospholipid Composition between Wild Strains and Streptomycin Resistant Mutants of Certain Enteric Bacteria. Jpn. J. Microbiol. 14, 174–176 (1970).
61. Yasuhiro, K., Yuzuru, A., Shoshichi, N. Composition and turnover of the phospholipids in Escherichia coli. Biochim. Biophys. Acta 144, 382–390 (1967).
62. Facey, S. J., Kuhn, A. Biogenesis of bacterial inner-membrane proteins. Cell. Mol. Life Sci. 67, 2343–2362 (2010).
63. Okuda, S., Tokuda, H. Lipoprotein Sorting in Bacteria. Annu. Rev. Microbiol. 65, 239–259 (2011).
64. Delcour, A. H. Outer membrane permeability and antibiotic resistance. Biochim. Biophys. Acta 1794, 808–816 (2009).
65. Delcour, A. H. Solute uptake through general porins. Front. Biosci. 8, 1055–1071 (2003). 66. Masi, M., Pagès, J. M. Structure, Function and Regulation of Outer Membrane Proteins Involved
in Drug Transport in Enterobactericeae: the OmpF/C – TolC Case. Open Microbiol. J. 7, 22–33 (2013).
67. Li, X. Z., Plésiat, P., Nikaido, H. The challenge of efflux-mediated antibiotic resistance in Gram-negative bacteria. Clin. Microbiol. Rev. 28, 337–418 (2015).
68. Richter, M. F. et al. Predictive compound accumulation rules yield a broad-spectrum antibiotic. Nature 545, 299–304 (2017).
69. Hasdemir, U. O., Chevalier, J., Nordmann, P., Pagès, J. M. Detection and Prevalence of Active Drug Efflux Mechanism in Various Multidrug-Resistant Klebsiella pneumoniae Strains from Turkey. J. Clin. Microbiol. 42, 2701–2706 (2004).
70. Vaara, M., Plachy, W. Z., Nikaido, H. Partitioning of hydrophobic probes into lipopolysaccharide bilayers. Biochim. Biophys. Acta 1024, 152–158 (1990).
71. Plésiat, P., Nikaido, H. Outer membranes of gram-negative bacteria are permeable to steroid probes. Mol. Microbiol. 6, 1323–1333 (1992).
67
72. Krishnamoorthy, G. et al. Breaking the Permeability Barrier of Escherichia coli by Controlled Hyperporination of the Outer Membrane. Antimicrob. Agents Chemother. 60, 7372–7381 (2016).
73. Hancock, R. E. W. Alterations in Outer Membrane Permeability. Annu. Rev. Microbiol. 38, 237–264 (1984).
74. Vaara, M. Agents that increase the permeability of the outer membrane. Microbiol. Rev. 56, 395–411 (1992).
75. Liu, A. et al. Antibiotic sensitivity profiles determined with an Escherichia coli gene knockout collection: generating an antibiotic bar code. Antimicrob. Agents Chemother. 54, 1393–1403 (2010).
76. Hancock, R. E., Bell, A. Antibiotic uptake into gram-negative bacteria. Eur. J. Clin. Microbiol. 7, 713–720 (1988).
77. Zgurskaya, H. I., Löpez, C. A., Gnanakaran, S. Permeability Barrier of Gram-Negative Cell Envelopes and Approaches To Bypass It. ACS Infect. Dis. 1, 512–522 (2015).
78. Silver, L. L. A Gestalt approach to Gram-negative entry. Bioorg. Med. Chem. 24, 6379–6389 (2016).
79. Yang, N. J., Hinner, M. J. Getting Across the Cell Membrane: An Overview for Small Molecules, Peptides, and Proteins. Methods Mol. Biol. 1266, 29–53 (2015).
80. Wargel, R. J., Hadur, C. A., Neuhaus, F. C. Mechanism of D-cycloserine action: transport mutants for D-alanine, D-cycloserine, and glycine. J. Bacteriol. 105, 1028–1035 (1971).
81. Chopra, I., Ball, P. Transport of antibiotics into bacteria. Adv. Microb. Physiol. 23, 183–240 (1982).
82. Taber, H. W., Mueller, J. P., Miller, P. F., Arrow, A. S. Bacterial uptake of aminoglycoside antibiotics. Microbiol. Rev. 51, 439–457 (1987).
83. Chopra, I. Molecular mechanisms involved in the transport of antibiotics into bacteria. Parasitology 96 Suppl, 25-44 (1988).
84. Piddock, L. J. V. Clinically relevant chromosomally encoded multidrug resistance efflux pumps in bacteria. Clin. Microbiol. Rev. 19, 382–402 (2006).
85. Piddock, L. J. V. Multidrug-resistance efflux pumps not just for resistance. Nat. Rev. Microbiol. 4, 629–636 (2006).
87. Du, D., van Veen, H. W., Murakami, S., Pos, K. M., Luisi, B. F. Structure, mechanism and cooperation of bacterial multidrug transporters. Curr. Opin. Struct. Biol. 33, 76–91 (2015).
88. Nikaido, H., Pagès, J. M. Broad Specificity Efflux pumps and Their Role in Multidrug Resistance of Gram Negative Bacteria. FEMS Microbiol. Rev. 36, 340–363 (2012).
89. Okusu, H., Ma, D., Nikaido, H. AcrAB efflux pump plays a major role in the antibiotic resistance phenotype of Escherichia coli multiple-antibiotic-resistance (Mar) mutants. J. Bacteriol. 178, 306–308 (1996).
90. Nikaido, H. Multiple antibiotic resistance and efflux. Curr. Opin. Microbiol. 1, 516–523 (1998). 91. Rosenberg, E. Y., Ma, D., Nikaido, H. AcrD of Escherichia coli is an aminoglycoside efflux
pump. J. Bacteriol. 182, 1754–1756 (2000). 92. Zgurskaya, H. I., Nikaido, H. Multidrug resistance mechanisms: drug efflux across two
membranes. Mol. Microbiol. 37, 219–225 (2000). 93. Nishino, K., Yamaguchi, A. Analysis of a complete library of putative drug transporter genes in
Escherichia coli. J. Bacteriol. 183, 5803–5812 (2001). 94. Mazzariol, A., Tokue, Y., Kanegawa, T. M., Cornaglia, G., Nikaido, H. High-level
fluoroquinolone-resistant clinical isolates of Escherichia coli overproduce multidrug efflux protein AcrA. Antimicrob. Agents Chemother. 44, 3441–3443 (2000).
95. Oethinger, M., Kern, W. V., Jellen-Ritter, A. S., McMurry, L. M., Levy, S. B. Ineffectiveness of topoisomerase mutations in mediating clinically significant fluoroquinolone resistance in Escherichia coli in the absence of the AcrAB efflux pump. Antimicrob. Agents Chemother. 44, 10–13 (2000).
68
96. Webber, M. A., Piddock, L. J. V. Absence of Mutations in marRAB or soxRS in acrB-Overexpressing Fluoroquinolone-Resistant Clinical and Veterinary Isolates of Escherichia coli. Antimicrob. Agents Chemother. 45, 1550–1552 (2001).
97. Stoitsova, S. O., Braun, Y., Ullrich, M. S., Weingart, H. Characterization of the RND-Type Multidrug Efflux Pump MexAB-OprM of the Plant Pathogen Pseudomonas syringae. Appl. Environ. Microbiol. 74, 3387–3393 (2008).
98. Dreier, J., Ruggerone, P. Interaction of antibacterial compounds with RND e ux pumps in Pseudomonas aeruginosa. Front. Microbiol. 6, (2015).
99. Ziha-Zarifi, I., Llanes, C., Köhler, T., Pechere, J. C., Plesiat, P. In Vivo Emergence of Multidrug-Resistant Mutants of Pseudomonas aeruginosa Overexpressing the Active Efflux System MexA-MexB-OprM. Antimicrob. Agents Chemother. 43, 287–291 (1999).
100. Bernal, P., Molina-Santiago, C., Daddaoua, A., Llamas, M. A. Antibiotic adjuvants: identification and clinical use. Microb. Biotechnol. 6, 445–449 (2013).
101. Wright, G. D. Antibiotic Adjuvants: Rescuing Antibiotics from Resistance. Trends Microbiol. 24, 862–871 (2016).
102. Melander, R. J., Melander, C. Antibiotic adjuvants. In: Topics in Medicinal Chemistry. Springer, Berlin, Heidelberg. (2017).
103. White, A. R. et al. Augmentin (amoxicillin/clavulanate) in the treatment of community-acquired respiratory tract infection: a review of the continuing development of an innovative antimicrobial agent. J. Antimicrob. Chemother. 53, i3–20 (2004).
104. Leflon-Guibout, V., Speldooren, V., Heym, B., Nicolas-Chanoine, M. H. Epidemiological Survey of Amoxicillin-Clavulanate Resistance and Corresponding Molecular Mechanisms in Escherichia coli Isolates in France: New Genetic Features of blaTEM Genes. Antimicrob. Agents Chemother. 44, 2709–2714 (2000).
105. Butler, M. S., Blaskovich, M. A., Cooper, M. A. Antibiotics in the clinical pipeline at the end of 2015. J. Antibiot. 70, 3–24 (2017).
106. Venter, H., Mowla, R., Ohene-Agyei, T., Ma, S. RND-type drug e ux pumps from Gram-negative bacteria: molecular mechanism and inhibition. Front. Microbiol. 6, (2015).
107. Pagès, J. M., Masi, M., Barbe, J. Inhibitors of efflux pumps in Gram-negative bacteria. Trends Mol. Med. 11, 382–389 (2005).
108. Lomovskaya, O. et al. Identification and characterization of inhibitors of multidrug resistance efflux pumps in Pseudomonas aeruginosa: novel agents for combination therapy. Antimicrob. Agents Chemother. 45, 105–116 (2001).
109. Pagès, J. M. et al. Efflux Pump, the Masked Side of -Lactam Resistance in Klebsiella pneumoniae Clinical Isolates. PLOS ONE 4, e4817 (2009).
110. Lomovskaya, O. et al. Identification and characterization of inhibitors of multidrug resistance efflux pumps in Pseudomonas aeruginosa: novel agents for combination therapy. Antimicrob. Agents Chemother. 45, 105–116 (2001).
111. Ofek, I. et al. Antibacterial synergism of polymyxin B nonapeptide and hydrophobic antibiotics in experimental gram-negative infections in mice. Antimicrob. Agents Chemother. 38, 374–377 (1994).
112. Pagès, J. M., Peslier, S., Keating, T. A., Lavigne, J. P., Nichols, W. W. Role of the Outer Membrane and Porins in Susceptibility of -Lactamase-Producing Enterobacteriaceae to Ceftazidime-Avibactam. Antimicrob. Agents Chemother. 60, 1349–1359 (2015).
113. Zabawa, T. P., Pucci, M. J., Parr, T. R., Lister, T. Treatment of Gram-negative bacterial infections by potentiation of antibiotics. Curr. Opin. Microbiol. 33, 7–12 (2016).
114. Ejim, L. et al. Combinations of antibiotics and nonantibiotic drugs enhance antimicrobial efficacy. Nat. Chem. Biol. 7, 348–350 (2011).
115. Taylor, P. L., Rossi, L., De Pascale, G., Wright, G. D. A forward chemical screen identifies antibiotic adjuvants in Escherichia coli. ACS Chem. Biol. 7, 1547–1555 (2012).
116. Gomez, M. J., Neyfakh, A. A. Genes Involved in Intrinsic Antibiotic Resistance of Acinetobacter baylyi. Antimicrob. Agents Chemother. 50, 3562–3567 (2006).
117. Lee, S. et al. Targeting a bacterial stress response to enhance antibiotic action. Proc. Natl. Acad. Sci. 106, 14570–14575 (2009).
69
118. Breidenstein, E. B. M., Khaira, B. K., Wiegand, I., Overhage, J., Hancock, R. E. W. Complex Ciprofloxacin Resistome Revealed by Screening a Pseudomonas aeruginosa Mutant Library for Altered Susceptibility. Antimicrob. Agents Chemother. 52, 4486–4491 (2008).
119. Fajardo, A. et al. The Neglected Intrinsic Resistome of Bacterial Pathogens. PLOS ONE 3, e1619 (2008).
120. Tamae, C. et al. Determination of antibiotic hypersensitivity among 4,000 single-gene-knockout mutants of Escherichia coli. J. Bacteriol. 190, 5981–5988 (2008).
121. Lazar, S. W., Kolter, R. SurA assists the folding of Escherichia coli outer membrane proteins. J. Bacteriol. 178, 1770–1773 (1996).
122. Rouvière, P. E., Gross, C. A. SurA, a periplasmic protein with peptidyl-prolyl isomerase activity, participates in the assembly of outer membrane porins. Genes Dev. 10, 3170–3182 (1996).
123. Hagan, C. L., Silhavy, T. J., Kahne, D. -Barrel membrane protein assembly by the Bam complex. Annu. Rev. Biochem. 80, 189–210 (2011).
124. Ruiz, N., Falcone, B., Kahne, D., Silhavy, T. J. Chemical conditionality: a genetic strategy to probe organelle assembly. Cell 121, 307–317 (2005).
125. Sklar, J. G. et al. Lipoprotein SmpA is a component of the YaeT complex that assembles outer membrane proteins in Escherichia coli. Proc. Natl. Acad. Sci. 104, 6400–6405 (2007).
126. Vorachek-Warren, M. K., Ramirez, S., Cotter, R. J., Raetz, C. R. A triple mutant of Escherichia coli lacking secondary acyl chains on lipid A. J. Biol. Chem. 277, 14194–14205 (2002).
127. Kadrmas, J. L., Raetz, C. R. Enzymatic synthesis of lipopolysaccharide in Escherichia coli. Purification and properties of heptosyltransferase i. J. Biol. Chem. 273, 2799–2807 (1998).
128. Mahalakshmi, S., Sunayana, M. R., SaiSree, L., Reddy, M. yciM is an essential gene required for regulation of lipopolysaccharide synthesis in Escherichia coli. Mol. Microbiol. 91, 145–157 (2014).
129. Tsirigotaki, A., De Geyter, J., Šoštaric, N., Economou, A., Karamanou, S. Protein export through the bacterial Sec pathway. Nat. Rev. Microbiol. 15, 21–36 (2017).
130. Sklar, J. G., Wu, T., Kahne, D., Silhavy, T. J. Defining the roles of the periplasmic chaperones SurA, Skp, and DegP in Escherichia coli. Genes Dev. 21, 2473–2484 (2007).
131. Schäfer, U., Beck, K., Müller, M. Skp, a Molecular Chaperone of Gram-negative Bacteria, Is Required for the Formation of Soluble Periplasmic Intermediates of Outer Membrane Proteins. J. Biol. Chem. 274, 24567–24574 (1999).
132. Grabowicz, M., Silhavy, T. J. Envelope Stress Responses: An Interconnected Safety Net. Trends Biochem. Sci. 42, 232–242 (2017).
133. Antonoaea, R., Fürst, M., Nishiyama, K. & Müller, M. The Periplasmic Chaperone PpiD Interacts with Secretory Proteins Exiting from the SecYEG Translocon. Biochemistry 47, 5649–5656 (2008).
134. Matern, Y., Barion, B., Behrens-Kneip, S. PpiD is a player in the network of periplasmic chaperones in Escherichia coli. BMC Microbiol. 10, 251 (2010).
135. Götzke, H. et al. YfgM is an ancillary subunit of the SecYEG translocon in Escherichia coli. J. Biol. Chem. 289, 19089–19097 (2014).
136. Pathania, R. et al. Chemical genomics in Escherichia coli identifies an inhibitor of bacterial lipoprotein targeting. Nat. Chem. Biol. 5, 849–856 (2009).
137. Loh, B., Grant, C., Hancock, R. E. Use of the fluorescent probe 1-N-phenylnaphthylamine to study the interactions of aminoglycoside antibiotics with the outer membrane of Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 26, 546–551 (1984).
138. Bean, G. J. et al. A22 disrupts the bacterial actin cytoskeleton by directly binding and inducing a low-affinity state in MreB. Biochemistry 48, 4852–4857 (2009).
139. Landström, J. et al. Small molecules containing hetero-bicyclic ring systems compete with UDP-Glc for binding to WaaG glycosyltransferase. Glycoconj. J. 29, 491–502 (2012).
140. Yethon, J. A., Vinogradov, E., Perry, M. B., Whitfield, C. Mutation of the lipopolysaccharide core glycosyltransferase encoded by waaG destabilizes the outer membrane of Escherichia coli by interfering with core phosphorylation. J. Bacteriol. 182, 5620–5623 (2000).
141. Patel, D. S. et al. Dynamics and Interactions of OmpF and LPS: Influence on Pore Accessibility and Ion Permeability. Biophys. J. 110, 930–938 (2016).
70
142. Muheim, C. et al. Identification of a Fragment-Based Scaffold that Inhibits the Glycosyltransferase WaaG from Escherichia coli. Antibiot. 5, (2016).
143. Hajduk, P. J., Greer, J. A decade of fragment-based drug design: strategic advances and lessons learned. Nat. Rev. Drug Discov. 6, 211–219 (2007).
144. de Kloe, G. E., Bailey, D., Leurs, R., de Esch, I. J. P. Transforming fragments into candidates: small becomes big in medicinal chemistry. Drug Discov. Today 14, 630–646 (2009).
145. Cerveny, L. et al. Tetratricopeptide Repeat Motifs in the World of Bacterial Pathogens: Role in Virulence Mechanisms. Infect. Immun. 81, 629–635 (2013).
146. Harms, N. et al. The early interaction of the outer membrane protein PhoE with the periplasmic chaperone Skp occurs at the cytoplasmic membrane. J. Biol. Chem. 276, 18804–18811 (2001).
147. Maurer, L. M., Yohannes, E., Bondurant, S. S., Radmacher, M., Slonczewski, J. L. pH Regulates Genes for Flagellar Motility, Catabolism, and Oxidative Stress in Escherichia coli K12. J. Bacteriol. 187, 304–319 (2005).
148. House, B. et al. Acid-stress-induced changes in enterohaemorrhagic Escherichia coli O157:H7 virulence. Microbiol. Read. Engl. 155, 2907–2918 (2009).
149. Stincone, A. et al. A systems biology approach sheds new light on Escherichia coli acid resistance. Nucleic Acids Res. 39, 7512–7528 (2011).
150. Kannan, G. et al. Rapid acid treatment of Escherichia coli: transcriptomic response and recovery. BMC Microbiol. 8, 37 (2008).
151. Barker, C. A. et al. Degradation of MAC13243 and studies of the interaction of resulting thiourea compounds with the lipoprotein targeting chaperone LolA. Bioorg. Med. Chem. Lett. 23, 2426–2431 (2013).