Bacterial cell wall

From basic biology to antibiotic targeting

Diagram of Peptidoglycan sacculus and location between outer membrane and cytoplasmic membraneBacteria build a robust cell envelope that enables them to inhabit all ecosystems on earth. Some disease-causing bacteria modify their cell wall to resist antibacterial components of our immune system. My group studies the bacterial cell envelope. For example, we want to gain understanding of how bacteria build and remodel their essential peptidoglycan sacculus (the "cell wall"), a net-like, stress-bearing layer in the cell envelope (1,2). In Gram-negative bacteria, the sacculus is sandwiched between the cytoplasmic membrane and an outer membrane, which protects the cell from many toxic molecules and antibacterial enzymes (Figure 1). Our research covers cell envelope topics in in model bacteria, for example Escherichia coli, and many other bacteria, including important pathogens, bacteria living in the environment and biotechnologically important bacteria.

An E. coli cell lysed by penicillinOur most successful antibiotics (e.g., beta-lactams like Pencillin) kill bacteria by blocking the synthesis of the peptidoglycan sacculus (PG). Figure 2 shows an E. coli cell that lysed by the action of Penicillin. We also develop assays to screen for novel antibiotics that target peptidoglycan synthesis and cell division, and we discover mechanisms of antimicrobial tolerance and resistance. We are also interested in how bacteria coordinate PG synthesis with the biogenesis of the other cell envelope layers and how bacteria maintain a robust cell envelope in different environments and under stress conditions. 

Group leader

Professor Waldemar Vollmer

Professor Waldemar Vollmer

Group Leader, Bacterial cell wall

  +61 7 334 62055
  w.vollmer@imb.uq.edu.au
  UQ Researcher Profile

Molecular mechanisms of PG synthesis

My laboratory has identified molecular interactions between different types of PG synthases (3,4). Synthases utilize lipid II precursor to polymerize glycan chains (GTase) and cross-link peptides (TPase), forming the net-like PG polymer. We have established in vitro PG synthesis assay, demonstrating the glycan chain polymerising and peptide cross-linking activities by key PG synthases (5,6). We recently developed a continuous PG synthesis assay for the analysis of membrane-reconstituted PG synthases (Figure 3) (7). 

We have discovered with Nassos Typas and Carol Gross the lipoproteins LpoA and LpoB which activate major PG synthases (8). With Jean-Pierre Simorre we determined the structures of the activators and the interface between LpoB and PBP1B, providing the first model of a PG synthase - activator complex (9,10,11). We also discovered a mechanism by which the cell coordinates PG synthesis and outer membrane constriction in cell division (12) and showed how synthases are controlled by members of the divisome during pre-septal PG synthesis (13).

Robust cell envelope maintenance

E. coli has 18 known periplasmic autolysins (PG hydrolases) active during growth and cell division, and at least 6 PG carboxypeptidases for the regulation of PG synthesis. In 2016 we discovered a PG hydrolase that is required to maintain proper cell shape at acidic pH (14). With Tanneke den Blaauwen, Manuel Banzhaf and Nassos Typas we then discovered a network of interactions between different PG hydrolases and the adaptor protein NlpI (15) and with Petra Levin and Elizabeth Mueller deciphered the role of different PG synthases and hydrolases for growth under a range of environmental conditions (16,17). These findings support our model according to which the cell maintains sets of seemingly redundant PG enzymes to ensure that sufficient activity is available and regulated at a range of conditions the poorly buffered periplasm may encounter, allowing for robust cell envelope growth (18). 

We recently began to study a novel research direction, the coordination between the biogenesis of the outer membrane with PG growth. Already our initial work with the group of Alessandra Polissi discovered a PG repair mechanism involving LD-transpeptidases that becomes essential when the biogenesis of the outer membrane is compromised (19) and showed that key LD-transpeptidases are inactivated by copper, preventing a PBP-bypass mechanisms for beta-lactam resistance (20). In 2022 we published with Colin Kleanthous and Gideon Mamou that the cell controls the essential outer membrane beta-barrel assembly machine (BAM) by the maturation state of the PG (21). Pentapeptide-rich PG, which occurs transiently at sites of PG growth, allows for BAM activity, whilst mature, tetrapeptide-rich PG inhibits BAM (Figure 4). This mechanism couples the BAM-mediated insertion of outer membrane (OM) proteins to PG growth sites, coordinating OM biogenesis with PG growth and maintaining a stable, functional OM around the PG layer. 

Bacterial cell shape and cell division

We are fortunate to have been collaborating with many other research groups on growth and cell shape of Gram-negative bacteria. For example, with Christine Jacobs-Wagner (Stanford) we have discovered FtsZ-dependent pre-septal PG synthesis (22) and a mechanism of mechanical control to generate cell curvature in Caulobacter crescentus (23), and established the mode of PG growth in Borrelia burgdorferi (24). With Nina Salama (Seattle) we have identified PG hydrolases required for helical cell shape and efficient stomach colonization in Helicobacter pylori (25). Subsequent work identified additional PG hydrolases participating in cell shape-generating pathways in H. pylori and, in collaboration with Erin Gaynor, Campylobacter jejuni (e.g., 26, 27). My group also contributed to the discovery of PG in chlamydia in collaboration with Grant Jensen and Martin Pilhofer (Caltech) (28). We have collaborated with the groups of Stephen Trent, Joe Boll and Mario Feldman on Acinetobacter baumannii and together we established the PG profile, discovered new PG modifications in the stationary phase, and showed the importance of LD-transpeptidases in colistin-resistant strains and PG recycling in carbapenem-resistance (e.g., 29, 30, 31).

The cell wall as target for antimicrobials and lysins

In the recent years, and based on our results from parts A-C, the focus of my group began to shift towards antimicrobial drug discovery. In collaboration with Cuong Voung (AiCuris Anti-infectives Cures GmbH, Wuppertal (Germany), and within a shared PhD studentship project, we identified lead inhibitors of the essential peptidoglycan synthase PBP3 (32). With Joen Luirink (Amsterdam) we identified novel inhibitors of the beta-barrel assembly machine (BAM) (33). We also work on antibacterial enzymes (lysins). For example, we collaborated with Joseph Mougous (Seattle) and Seemay Chou (San Francisco) on PG hydrolases that are secreted by type VI secretion system to target rival bacteria (34). Our subsequent work showed the wide distribution of these systems in bacteria (35) and provided a structural rationale for the high activity of these 'attacking hydrolases' (36). Some ticks and mites acquired PG hydrolase genes from bacterial origin during evolution (37) and use these hydrolases to control host skin bacteria during their blood meal (38). We have collaborated with Liz Sockett (Nottingham) and Andy Lovering (Birmingham) and identified PG hydrolases that Bdellovibrio uses to attack Gram-negative prey cells, showing these hydrolases are controlled by interaction with ankryn-like proteins and identified PG editing enzymes that generate the helical cell curvature (e.g., 39). With Jorge Galan (Yale) we discovered an LD-transpeptidase required for the secretion of typhoid toxin (40). 

Novel cell wall modifiations, the cell wall architecture and new chemical tools

Within the last two decades and often within collaborations with others we discovered the founding genes encoding important cell wall enzymes in pathogenic bacteria including PG deacetylase (41), PG O-acetyltransferases (42), PG amidotransferase (43), several teichoic acid enzymes, e.g. the long-searched for enzyme that attaches anionic cell wall polymers (teichoic acids, capsular polysaccharides) to peptidoglycan (44) or phosphocholine esterase (45), and the first enzyme that is capable of detaching a protein from PG (46); homologues of all of these enzymes have been subsequently identified in many bacteria. Our expertise on the biochemistry of the cell wall structure has also helped our collaborators to characterise new chemical tools to study bacterial cell wall structure and biogenesis (e.g., 47, 48).

  1. Typas A, Banzhaf M, Gross CA, Vollmer W. 2012. From the regulation of peptidoglycan synthesis to bacterial growth and morphology. Nature Reviews Microbiology 10, 123-136.
  2. Egan AJF, Errington J, Vollmer W. 2020. Regulation of peptidoglycan synthesis and remodelling. Nature Reviews Microbiology 18, 446–460.
  3. Typas A, Banzhaf M, Gross CA, Vollmer W. 2012. From the regulation of peptidoglycan synthesis to bacterial growth and morphology. Nature Reviews Microbiology 10, 123-136.
  4. Egan AJF, Errington J, Vollmer W. 2020. Regulation of peptidoglycan synthesis and remodelling. Nature Reviews Microbiology 18, 446–460.
  5. Bertsche U, Kast T, Fraipont C, Wolf B, Aarsman MEG, Kannenberg K, v.Rechenberg M, Nguyen-Distèche M, den Blaauwen T, Höltje J-V, Vollmer W. 2006. Interaction between two murein (peptidoglycan) synthases, PBP3 and PBP1B, in Escherichia coli.  Molecular Microbiology 61, 675-690.
  6. Banzhaf M, van den Berg van Saparoea B, Terrak M, Fraipont C, Egan AJF, Philippe J, Zapun A, Breukink E, Nguyen-Distèche M, den Blaauwen T, Vollmer W. 2012. Cooperativity of peptidoglycan synthases active in bacterial cell elongation. Molecular Microbiology 85, 179-194.
  7. Bertsche U, Breukink E, Kast T, Vollmer W. 2005. In vitro murein (peptidoglycan) synthesis by dimers of the bifunctional transglycosylase-transpeptidase PBP1B from Escherichia coli. Journal of Biological Chemistry 280, 38096-38101.
  8. Born P, Breukink E, Vollmer W. 2006. In vitro synthesis of cross-linked murein (peptidoglycan) and its attachment to sacculi by PBP1A from Escherichia coli. Journal of Biological Chemistry 281, 26985-26993.
  9. Hernández-Rocamora VM, Baranova N, Peters K, Breukink E, Loose M, Vollmer W. 2021. Real time monitoring of peptidoglycan synthesis by membrane-reconstituted penicillin binding proteins. eLife 10:e61525.
  10. Typas A, Banzhaf M, van den Berg van Saparoea B, Verheul J, Biboy J, Nichols RJ, Zietek M, Beilharz K, Kannenberg K, von Rechenberg M, Breukink B, den Blaauwen T, Gross CA, Vollmer W. 2010. Regulation of peptidoglycan synthesis by outer-membrane proteins. Cell 143, 1097-1109.
  11. Egan AJF, Jean NL, Koumoutsi A, Bougault CM, Biboy J, Sassine J, Solovyova     AS, Breukink E, Typas A, Vollmer W, Simorre JP. 2014. Outer-membrane lipoprotein LpoB spans the periplasm to stimulate the peptidoglycan synthase PBP1B. Proceedings of the National Academy of Sciences USA 111, 8197–8202.
  12. Jean NL, Bougault CM, Lodge A, Derouaux A, Callens G, Egan AJF, Lewis RJ, Vollmer W, Simorre J-P. 2014. Elongated structure of the outer-membrane activator of peptidoglycan synthesis LpoA: Implications for PBP1A-stimulation. Structure 22, 1047-1054.
  13. Egan AJF, Maya-Martinez R, Ayala I, Bougault CM, Banzhaf M, Breukink E, Vollmer W, Simorre JP. 2018. Induced conformational changes activate the peptidoglycan synthase PBP1B. Molecular Microbiology 110, 335-356.
  14. Typas A, Banzhaf M, Gross CA, Vollmer W. 2012. From the regulation of peptidoglycan synthesis to bacterial growth and morphology. Nature Reviews Microbiology 10, 123-136.
  15. Egan AJF, Errington J, Vollmer W. 2020. Regulation of peptidoglycan synthesis and remodelling. Nature Reviews Microbiology 18, 446–460.
  16. Bertsche U, Kast T, Fraipont C, Wolf B, Aarsman MEG, Kannenberg K, v.Rechenberg M, Nguyen-Distèche M, den Blaauwen T, Höltje J-V, Vollmer W. 2006. Interaction between two murein (peptidoglycan) synthases, PBP3 and PBP1B, in Escherichia coli.  Molecular Microbiology 61, 675-690.
  17. Banzhaf M, van den Berg van Saparoea B, Terrak M, Fraipont C, Egan AJF, Philippe J, Zapun A, Breukink E, Nguyen-Distèche M, den Blaauwen T, Vollmer W. 2012. Cooperativity of peptidoglycan synthases active in bacterial cell elongation. Molecular Microbiology 85, 179-194.
  18. Bertsche U, Breukink E, Kast T, Vollmer W. 2005. In vitro murein (peptidoglycan) synthesis by dimers of the bifunctional transglycosylase-transpeptidase PBP1B from Escherichia coli. Journal of Biological Chemistry 280, 38096-38101.
  19. Born P, Breukink E, Vollmer W. 2006. In vitro synthesis of cross-linked murein (peptidoglycan) and its attachment to sacculi by PBP1A from Escherichia coli. Journal of Biological Chemistry 281, 26985-26993.
  20. Hernández-Rocamora VM, Baranova N, Peters K, Breukink E, Loose M, Vollmer W. 2021. Real time monitoring of peptidoglycan synthesis by membrane-reconstituted penicillin binding proteins. eLife 10:e61525.
  21. Typas A, Banzhaf M, Gross CA, Vollmer W. 2012. From the regulation of peptidoglycan synthesis to bacterial growth and morphology. Nature Reviews Microbiology 10, 123-136.
  22. Typas A, Banzhaf M, Gross CA, Vollmer W. 2012. From the regulation of peptidoglycan synthesis to bacterial growth and morphology. Nature Reviews Microbiology 10, 123-136.
  23. Egan AJF, Errington J, Vollmer W. 2020. Regulation of peptidoglycan synthesis and remodelling. Nature Reviews Microbiology 18, 446–460.
  24. Bertsche U, Kast T, Fraipont C, Wolf B, Aarsman MEG, Kannenberg K, v.Rechenberg M, Nguyen-Distèche M, den Blaauwen T, Höltje J-V, Vollmer W. 2006. Interaction between two murein (peptidoglycan) synthases, PBP3 and PBP1B, in Escherichia coli.  Molecular Microbiology 61, 675-690.
  25. Banzhaf M, van den Berg van Saparoea B, Terrak M, Fraipont C, Egan AJF, Philippe J, Zapun A, Breukink E, Nguyen-Distèche M, den Blaauwen T, Vollmer W. 2012. Cooperativity of peptidoglycan synthases active in bacterial cell elongation. Molecular Microbiology 85, 179-194.
  26. Bertsche U, Breukink E, Kast T, Vollmer W. 2005. In vitro murein (peptidoglycan) synthesis by dimers of the bifunctional transglycosylase-transpeptidase PBP1B from Escherichia coli. Journal of Biological Chemistry 280, 38096-38101.
  27. Born P, Breukink E, Vollmer W. 2006. In vitro synthesis of cross-linked murein (peptidoglycan) and its attachment to sacculi by PBP1A from Escherichia coli. Journal of Biological Chemistry 281, 26985-26993.
  28. Hernández-Rocamora VM, Baranova N, Peters K, Breukink E, Loose M, Vollmer W. 2021. Real time monitoring of peptidoglycan synthesis by membrane-reconstituted penicillin binding proteins. eLife 10:e61525.
  29. Typas A, Banzhaf M, van den Berg van Saparoea B, Verheul J, Biboy J, Nichols RJ, Zietek M, Beilharz K, Kannenberg K, von Rechenberg M, Breukink B, den Blaauwen T, Gross CA, Vollmer W. 2010. Regulation of peptidoglycan synthesis by outer-membrane proteins. Cell 143, 1097-1109.
  30. Egan AJF, Jean NL, Koumoutsi A, Bougault CM, Biboy J, Sassine J, Solovyova     AS, Breukink E, Typas A, Vollmer W, Simorre JP. 2014. Outer-membrane lipoprotein LpoB spans the periplasm to stimulate the peptidoglycan synthase PBP1B. Proceedings of the National Academy of Sciences USA 111, 8197–8202.
  31. Jean NL, Bougault CM, Lodge A, Derouaux A, Callens G, Egan AJF, Lewis RJ, Vollmer W, Simorre J-P. 2014. Elongated structure of the outer-membrane activator of peptidoglycan synthesis LpoA: Implications for PBP1A-stimulation. Structure 22, 1047-1054.
  32. Typas A, Banzhaf M, Gross CA, Vollmer W. 2012. From the regulation of peptidoglycan synthesis to bacterial growth and morphology. Nature Reviews Microbiology 10, 123-136.
  33. Egan AJF, Errington J, Vollmer W. 2020. Regulation of peptidoglycan synthesis and remodelling. Nature Reviews Microbiology 18, 446–460.
  34. Bertsche U, Kast T, Fraipont C, Wolf B, Aarsman MEG, Kannenberg K, v.Rechenberg M, Nguyen-Distèche M, den Blaauwen T, Höltje J-V, Vollmer W. 2006. Interaction between two murein (peptidoglycan) synthases, PBP3 and PBP1B, in Escherichia coli.  Molecular Microbiology 61, 675-690.
  35. Banzhaf M, van den Berg van Saparoea B, Terrak M, Fraipont C, Egan AJF, Philippe J, Zapun A, Breukink E, Nguyen-Distèche M, den Blaauwen T, Vollmer W. 2012. Cooperativity of peptidoglycan synthases active in bacterial cell elongation. Molecular Microbiology 85, 179-194.
  36. Bertsche U, Breukink E, Kast T, Vollmer W. 2005. In vitro murein (peptidoglycan) synthesis by dimers of the bifunctional transglycosylase-transpeptidase PBP1B from Escherichia coli. Journal of Biological Chemistry 280, 38096-38101.
  37. Born P, Breukink E, Vollmer W. 2006. In vitro synthesis of cross-linked murein (peptidoglycan) and its attachment to sacculi by PBP1A from Escherichia coli. Journal of Biological Chemistry 281, 26985-26993.
  38. Hernández-Rocamora VM, Baranova N, Peters K, Breukink E, Loose M, Vollmer W. 2021. Real time monitoring of peptidoglycan synthesis by membrane-reconstituted penicillin binding proteins. eLife 10:e61525.
  39. Typas A, Banzhaf M, van den Berg van Saparoea B, Verheul J, Biboy J, Nichols RJ, Zietek M, Beilharz K, Kannenberg K, von Rechenberg M, Breukink B, den Blaauwen T, Gross CA, Vollmer W. 2010. Regulation of peptidoglycan synthesis by outer-membrane proteins. Cell 143, 1097-1109.
  40. Egan AJF, Jean NL, Koumoutsi A, Bougault CM, Biboy J, Sassine J, Solovyova     AS, Breukink E, Typas A, Vollmer W, Simorre JP. 2014. Outer-membrane lipoprotein LpoB spans the periplasm to stimulate the peptidoglycan synthase PBP1B. Proceedings of the National Academy of Sciences USA 111, 8197–8202.
  41. Vollmer W, Tomasz A. 2000. The pgdA gene encodes for a peptidoglycan N-acetylglucosamine deacetylase in Streptococcus pneumoniae. Journal of Biological Chemistry 275, 20496-20501.
  42. Bera A, Herbert S, Jakob A, Vollmer W, Götz F. 2005. Why are pathogenic staphylococci so lysozyme resistant? The peptidoglycan O-acetyltransferase OatA is the major determinant for lysozyme resistance of Staphylococcus aureus. Molecular Microbiology 55, 778-787.
  43. Figueiredo TA, Sobral RG, Ludovice AM, de Almeida JMF, Bui NK, Vollmer W, de Lencastre H, Tomasz A. 2012. Identification of genetic determinants and enzymes involved with the amidation of glutamic acid residues in the peptidoglycan of Staphylococcus aureus. PLOS Pathogens 8(1): e1002508.
  44. Kawai Y, Marles-Wright J, Cleverly RM, Emmins R, Ishikawa S, Kuwano M, Heinz N, Bui NK, Hoyland CN, Ogasawara N, Lewis RJ, Vollmer W, Daniel RA, Errington J. 2011. A widespread family of bacterial cell wall assembly proteins. EMBO Journal 30, 4931-4941.
  45. Vollmer W, Tomasz A. 2001. Identification of the teichoic acid phosphorylcholine esterase in Streptococcus pneumoniae. Molecular Microbiology 39, 1610-1622.
  46. Winkle M, Hernández-Rocamora VM, Pullela K, Goodall ECA, Martorana AM, Gray J, Henderson IR, Polissi A*, Vollmer W*. 2021. DpaA detaches Braun's lipoprotein from peptidoglycan. mBio 12:e00836-21.
  47. Kuru E, Radkov A, Meng X, Egan A, Alvarez L, Dowson AD, Booher G, Breukink E, Roper D, Cava F, Vollmer W, Brun YV, VanNieuwenhze MS. 2019. Mechanisms of incorporation for D-amino acid probes that target peptidoglycan biosynthesis. ACS Chemical Biology 14, 2745-2756.
  48. Kern T, Giffard M, Hediger S, Amoroso A, Giustini C, Bui NK, Joris B, Bougault C, Vollmer W, Simorre J-P. 2010. Dynamics characterization of fully hydrated bacterial cell walls by solid-state NMR - evidence for cooperative binding of metal ions. Journal of the American Chemical Society 132, 10911-10919.
     

Our Approach

We discover molecular mechanisms in cell envelope biogenesis and use innovative biochemical assays to identify inhibitors of these. Our goal is to identify lead molecules for development of novel antibiotics that can cure infections caused by multi-resistant bacterial pathogens. 

Research areas

Our research is focused on the bacterial cell wall and aims to answer the the following questions:
•    How do bacteria enlarge their cell wall?
•    How do they strengthen their cell envelope under stress conditions?
•    How do antibiotics or components of our immune system attack the bacterial cell wall and how do some pathogenic bacteria resist these attacks?
 

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