mesnages

Dr Stéphane Mesnage

Room: F22b
0114 222 4405
s.mesnage@sheffield.ac.uk

General

Career History

  • 2012 - present: Lecturer, Dept. of Molecular Biology and Biotechnology, University of Sheffield, UK
  • 2010 - 2012: Marie Curie (IEF) Fellow, Dept. of Molecular Biology and Biotechnology, University of Sheffield, UK
  • 2002 - 2010: INSERM Research Assistant, University of Paris 6, France
  • 2000 - 2002: EMBO Research assistant, John Innes Centre, Norwich, UK
  • 1996 - 2000: PhD student, Pasteur Institute, Paris, France

Research Keywords

Microbiology, Enterococcus, Staphylococcus, cell wall, peptidoglycan

Research

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.


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.

fig1

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


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.

fig2

Figure 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

Figure 3: analysis of peptidoglycan digestion by autolysins using rp-HPLC and mass spectrophotometry.


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.

fig4

Figure 4: Lysozyme resistance in Enterococcus faecalis.

People

Postdoc

Nicola Galley Postdoc (BBSRC funding)

PhD students

Bartlomiej Salamaga (MBB funding; 2013-)
Fathe Elsarmane (Lybian government funding; 2013-)
Robert Smith (BBSRC funding; 2016-)
Luz Selene Gonzalez (Conacyt funding; 2016-)

Postgraduate students

Isabel Diez (Molecular Medicine)
Heloise Cahuzac (ENSCM, France)

Teaching

Level 3 Modules

Level 1 Modules

PhD Opportunities

I welcome applications from international students who are either self-funded or who have scholarships from their governments; see examples of possible projects below.

You can apply for a PhD position in MBB here.

Contact me at s.mesnage@sheffield.ac.uk for further information.



Analysis of the mechanisms controlling cell separation in Enterococcus faecalis
Molecular basis underpinning protein-peptidoglycan interactions in Gram-positive bacteria
Molecular basis for lysozyme resistance in Enterococcus faecalis








































Publications

Journal articles

Conference proceedings papers

  • Bern MW, Beniston R & Mesnage S (2016) Automated Analysis of Bacterial Peptidoglycan Structure. GLYCOBIOLOGY, Vol. 26(12) (pp 1457-1457)