Molecular Microbiology

Dr Stéphane Mesnage

Cell surface organisation of Gram-positive pathogens

• Sheffield 224 405
• s.mesnage@sheffield.ac.uk

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Career history

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Research Interests

My work is focused on the study of the bacterial cell wall using essentially two Gram-positive pathogens (Enterococcus faecalis and Staphylococcus aureus) as models. I am interested in three major areas of research.

1. Analysis of protein-cell wall interactions

The essential cell wall peptidoglycan polymer network forms a scaffold for the display of a large variety of proteins involved in cell division, metabolism, cell-cell communication and in pathogen interaction with the host. Although several multimodular cell wall binding domains have been identified to date in bacteria, both their structural organisation and binding mechanism (binding motif, subcellular localisation) have remained elusive.
The aim of this project is to understand the molecular mechanisms underpinning protein-bacterial cell wall interactions and subcellular localisation of surface proteins. Beyond these fundamental questions, the aim is to develop a translational research program to (i) develop peptidoglycan binding domains as new functional probes to provide a nanoscale topological map of the cell surface; (ii) provide an experimental rationale to engineer and target therapeutic molecules against pathogens.
This project involves multidisciplinary approaches across several disciplines including molecular biology, biochemistry, biophysics, structural biology and state-of-the-art super resolution microscopy. Some examples of the experimental strategies developed are described in Fig. 1.

Fig.1 some examples of the methods used to study protein-cell wall interactions

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2. Control of peptidoglycan hydrolysis during growth

During growth, the insertion of new precursors and separation of daughter cells requires limited cleavage of the peptidoglycan molecule by peptidoglycan hydrolases.

Depending on the bond they cleave, these enzymes are classified as N-acetylmuramidases, N-acetylglucosaminidases, N-acetylmuramoyl L-alanine amidases, endopeptidases or carboxypeptidases (Fig. 2). In addition to their contribution to cell growth and division, some peptidoglycan hydrolases play a role in adhesion and in amplification of the inflammatory response by releasing muramyl-peptides. The lytic activity of some peptidoglycan hydrolases (called autolysins) is potentially lethal and can lead to cell lysis and death. How bacteria control autolytic activities is poorly understood.

Fig.2 site of cleavage of peptidoglycan hydrolases

I am studying several mechanisms that may be responsible for the control of autolytic activities: (i) the subcellular targeting of autolysins; (ii) the impact of peptidoglycan structural properties on autolytic activities and (iii) the contribution of the electrochemical gradient across the cytoplasmic membrane to the control of peptidoglycan hydrolysis.
We routinely analyse peptidoglycan hydrolase substrate specificity by a combination of rp-HPLC and mass spectrometry (Fig. 3). The enzymatic activity of autolysins is measured spectrophotometrically.

Fig3: analysis of peptidoglycan digestion by autolysins using rp-HPLC and mass spectrophotometry

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3. Role of Enterococcus faecalis cell wall metabolism in the interaction with the host

Lysozyme is the major component of the innate immune system and represents the first line of defense against pathogens. E. faecalis is highly resistant to lysozyme, with a minimal inhibitory concentration (MIC) above 50 mg/ml. This extreme resistance has been partly explained by an additive effect of peptidoglycan O-acetylation and de-N-acetylation, as well as teichoic acid D-alanylation. It was also shown that full lysozyme resistance requires a signalling cascade involving the extracytoplasmic function (ECF) sigma factor SigV. Interestingly, E. faecalis strains harbouring multiple deletions in these pathways are still resistant to lysozyme.
The identification of novel factors involved in lysozyme resistance is currently underway using random transposon mutagenesis. The mutants isolated will be further characterised in vivo.

Fig. 4 Lysozyme resistance in Enterococcus faecalis

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Selected Publications

1. Benachour A, Ladjouzi R, Le Jeune A, Hébert L, Thorpe S, Courtin P, Chapot-Chartier MP, Prajsnar TK, Foster SJ, Mesnage S. The peptidoglycan N-acetylglucosamine deacetylase PgdA (EF1843), produced upon exposure to lysozyme, contributes to Enterococcus faecalis virulence. J. Bacteriol. 2012. 194:6066-6073

2. Wheeler R*, Mesnage S*, Boneca IG, Hobbs JK, Foster SJ. Super-resolution microscopy reveals cell wall dynamics and peptidoglycan architecture in ovococcal bacteria. Mol. Microbiol. 2011, 82: 1096-1109. (*, joint first)

3. de Roca FR, Duché C, Dong S, Rincé A, Dubost L, Pritchard DG, Baker JR, Arthur M, Mesnage S. Cleavage specificity of Enterococcus faecalis EnpA (EF1473), a peptidoglycan endopeptidase related to the LytM/lysostaphin family of metallopeptidases. J. Mol Biol. 2010. 398: 507-517.

4. Emirian A, Fromentin S, Eckert C, Chau F, Dubost L, Delepierre M, Gutmann L, Arthur M, Mesnage S. Impact of peptidoglycan O-acetylation on the Enterococcus faecalis N-acetylglucosaminidase AtlA and N-acetylmuramidase AtlB. FEBS Letters, 2009, 3033-3038.

5. Bravetti AL, Mesnage S, Lefort A, Chau F, Eckert C, Garry L, Arthur M, Fantin B. Contribution of the AtlA autolysin to the bactericidal activity of amoxicillin against Enterococcus faecalis JH2-2. Antimicrobiol. Agent Chemother. 2009. 53: 1667-1669.

6. Mesnage S, Chau F, Dubost L, Arthur M. Role of N-acetylglucosaminidase and N-acetylmuramidase activities in Enterococcus faecalis PG metabolism. J. Biol. Chem., 2008. 283: 19845-19853.

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Google scholar webpage:

scholar.google.co.uk/citations

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Joining the lab

If you are interested in joining the lab, please see details below and send an email (s.mesnage@sheffield.ac.uk)!

PhD Students. PhD studentships are available each year from different funding sources. These are usually awarded competitively. Details of funded PhD projects will be listed here in the near future, but I also encourage talented students with their own project ideas to get in touch!.

Post-Doctoral Research Fellows. If you are interested in joining the group as a post-doc, one option is to apply for a fellowship (EMBO, Human Frontier Science Program, FEBS, Marie Curie…). As long as the timing is favourable, I am happy to help with this process. Funded post-doc positions will be available in the future, please keep an eye on the website.

Project Students and Internships. Post-graduate students are welcome to apply for research projects. Those applying from outside of Sheffield University should send a CV (s.mesnage@sheffield.ac.uk) describing their motivations and interests.