The research of our group is focused on the bacterial cell wall and in particular around peptidoglycan (PGN) metabolism with two main aims being pursued: 1) use Helicobacter pylori has an alternative model to study PGN assembly, 2) study how bacteria adapt their PGN metabolism, among other cell envelope components, to modulate host-microbe interactions and their consequences on the host innate response mediated by the NLR and TLR proteins.

a) peptidoglycan assembly

We focus on the enzymes required for PGN assembly and maturation in the periplasmic space. These include the PGN synthetases or also known as penicillin-binding proteins (PBPs) and the PGN hydrolases. H. pylorihas the advantage to have a minimal set of those enzymes (3 PBPs and 3 hydrolases) clearly identified from the genome analysis. All three PBPs were shown to be essential. Interestingly, H. pylori only encodes high-molecular weigth PBPs and is missing entirely low-molecular weight PBPs that have essential maturating functions such as carboxypeptidase or endopeptidase. We recently identified a new family of hydrolases of the zinc-metallopeptidase M23 family that fulfill this function in H. pylori. We have characterized the role of one of the four enzymes of this family, HdpA (HP0506) and shown it regulates the cell-shape and the number of poles of H. pylori furthermore, we show that the activity is essential for optimal colonization of the mouse gastric mucosa as a mutant is highly attenuated.

Of the three hydrolases homologues previously identified in the H. pylori genome, the lytic transglycosylases have also an important role in the functionality/assembly of surface appendices such as flagella and type 4 secretion systems. Usually, there are dedicated lytic transglycosylases for the assembly of these surface structures. However, H. pyloridoes not have any PGN hydrolase dedicated for the flagellum. We are addressing the role of Slt and MltD in the assembly and functionality of H. pyloriflagellum.

b) host-microbe interactions

PGN is a major pathogen-associated molecular pattern (PAMP) that is sensed by the host innate immune system. In mammals, PGN detection occurs by the intracellular pattern recognition receptors (PRRs) Nod1 and Nod2. These proteins detect degradation fragments of PGN that are generated by the endogenous bacterial PGN hydrolases or host PGN hydrolases. We have been studying several mechanisms that bacteria use to modulate either their own PGN metabolism or the host PGN hydrolases as an innate immune stealth mechanism. Hence, we have shown that H. pylori uses its morphological transformation into coccoïd bacteria to escape Nod1 detection by gastric epithelial cells. Alternatively, we have shown that Listeria monocytogenes modifies its PGN by deacetylating the N-acetylglucosamine moieties to confer resistance to host lysozyme and modulate Nod response.

The Nod proteins have a central role in detecting bacteria particularly in the gut. The first evidence for a central role of Nod proteins in the gut physiology came from epidemiological studies that clearly established Nod2 as involved in the development of Crohn’s disease. In collaboration with the group of Gérard Eberl (Institut Pasteur), we have shown that Nod1 is essential for the homeostasis of the instestinal lymphoid tissue. In fact, the homeastasis can be recapitulated in germ-free mice by feeding them with pure Nod1 ligand. This highlights the importance for better understanding the Nod proteins and Nod signaling during interactions of bacteria and the host. For example, we have shown that ubiquitin-editing enzyme A20, a general negative regulator of the TNF and NF-kB pathways, is needed to regulate Nod signaling.

Leptospirosis is a widespread zoonosis caused by pathogenic Leptospira interrogans that is transmitted by asymptomatic infected rodents. Leptospiral lipoproteins and lipopolysaccharide (LPS) have been shown to stimulate murine cells via Toll-like receptors (TLRs) 2 and 4. We showed that double (TLR2 and TLR4) knockout (DKO) mice rapidly died from severe hepatic and renal failure following Leptospira inoculation, reproducing the features of human acute leptospirosis. Strikingly, the severe pro-inflammatory response detected in the liver and kidney from Leptospira-infected DKO mice appears to be independent of MyD88, the main adaptor of TLRs. We identified B-cells as the crucial lymphocyte subset responsible for the clearance of Leptospira, through the early production of specific TLR4-dependent anti Leptospira IgMs elicited against the leptospiral LPS. Altogether these findings demonstrate that TLR2 and TLR4 play a key role in the early control of leptospirosis, but do not directly trigger the inflammation induced by pathogenic Leptospira.

Keywords: Peptidoglycan – antibiotics – penicillin-binding proteins – autolysins -peptidoglycan hydrolases – inflammation – Leptospira - Helicobacter – ulcer – gastric carcinoma – MALT – Nod1 – Nod2 – innate immunity – Toll-like receptor

Bonis M, Ecobichon C, Guadagnini S, Prévost MC, Boneca IG. 2010. A M23B family metallopeptidase of Helicobacter pylori required for cell shape, pole formation and virulence. Mol Microbiol. 2010 Nov;78(4):809-19. (PMID: 20815828).

Chassin C, Picardeau M, Goujon JM, Bourhy P, Quellard N, Darche S, Badell E, d'Andon MF, Winter N, Lacroix-Lamandé S, Buzoni-Gatel D, Vandewalle A, Werts C. 2009. TLR4- and TLR2-mediated B cell responses control the clearance of the bacterial pathogen, Leptospira interrogans. J Immunol. 183(4):2669-77. (PMID: 19635914).

Bouskra D, Brézillon C, Bérard M,Werts C, Varona R, Boneca IG, Eberl G. 2008. Lymphoid tissue genesis induced by commensals through NOD1 regulates intestinal homeostasis.Nature.456(7221):507-10. (PMID: 18987631).

Hitotsumatsu O, Ahmad RC, Tavares R, Wang M, Philpott D, Turer EE, Lee BL, Shiffin N, Advincula R, Malynn BA,Werts C, Ma A. 2008. The ubiquitin-editing enzyme A20 restricts nucleotide-binding oligomerization domain containing 2-triggered signals.Immunity.28.(3):381-90. (PMID: 18342009).

Boneca IG, Dussurget O, Cabanes D, Nahori MA, Sousa S, Lecuit M, Psylinakis E, Bouriotis V, Giovannini M, Hugot JP, Coyle A, Bertin J, Namane A, Rousselle JC, Cayet N, Prévost MC, Balloy V, Chignard M, Philpott DJ, Cossart P and Girardin SE. 2007. A critical role for peptidoglycan N-deacetylationin Listeria evasion from the host innate immune system. Proc Natl Acad Sci U S A.104.(3):997-1002. (PMID: 17215377).