The main goals of our research activity reflect our common interests and complementary approaches aimed at elucidating new pathways/mechanisms involved in the pathogenesis of low GC% Gram-positive bacteria, using the major human pathogens Staphylococcus aureus and Streptococcus agalactiae as model organisms. Our research topics include the study of bacterial surface components involved in interactions with the host, of relationships between metabolic adaptation and virulence, and gene regulation in relation to host environment and adaptation to stress responses.

Our research also includes Streptomyces spp. that are filamentous Gram-positive bacteria producing various secondary metabolites. Our studies centred on pupylation: a system of protein modification similar to eukaryote ubiquitination, which appears to be involved in the control of Streptomyces differentiation cycle and antibiotic production.

Surface proteins play important roles during bacterial adhesion and colonization of host epithelia and escape from host immunity. Bioinformatic genome analysis of S. agalactiae (Group B Streptococci or GBS) strain NEM316 identifies 41 putative lipoproteins and 35 LPXTG proteins. We showed that inactivation of sortase A, the enzyme required for anchoring LPXTG proteins to the cell wall, strongly impaired GBS adherence to epithelial cells. Two major LPXTG-adhesins in S. agalactiae, PilA, which is part of a pilus structure, and Srr1, a glycosylated Ser-rich repeat protein exported by a dedicated accessory secretion pathway, were characterized and shown to contribute to virulence. We previously reported the polymorphic structure of two GBS LPXTG proteins designated BibA and HvgA. Importantly, we demonstrated that HvgA is specific for the “highly virulent” ST17 clones and have patented an assay for the rapid detection of this clone in vaginal swabs. We have demonstrate that HvgA, but not BibA, mediates GBS neonatal intestinal colonization and crossing of the intestinal and blood-brain barriers, leading to meningitis, making it a promising target for the development of novel antibacterial strategies. Determination of the structure of HvgA and characterization of its cellular receptor(s) are currently being investigated.

Cell envelope signaling and pleiotropic regulation in Staphylococcus aureus

The S. aureus GraS/GraR antimicrobial peptide resistance pathway

S. aureus is able to resist Cationic Anti-Microbial Peptides (CAMPs) by increasing its positive cell surface charges through D-alanylation of wall teichoic acids and lysylination of phospholipids. Synthesis of the major enzymes involved in these mechanisms (DltA, MprF) is positively controlled by the GraS/GraR two-component system (TCS). Mutations were constructed inactivating the graSR, graS, graX, vraFG, and dltA genes allowing us to show that both GraX and the VraFG ABC transporter play a crucial role in this signal transduction pathway, and that both likely act by signaling through the GraS histidine kinase. A bacterial two-hybrid approach designed to identify interactions between GraS/GraR/GraX/VraF and VraG was carried out in collaboration with Gouzel Karimova (Biochemistry of Macromolecular Interactions Unit), showing that GraS interacts with both GraX and the VraG permease, indicating the existence of a multicomponent membrane-linked signaling complex involved in CAMP sensing and signal transduction. We recently discovered that S. aureus produces massive amounts of a giant extracellular structure (Figure 2) when grown under specific nutritional conditions. Preliminary results suggest this giant matrix is proteinaceous in nature and linked to the presence/modification of teichoic acids in the cell envelope.

BraS/BraR: a novel bacitracin resistance pathway in S. aureus

We recently discovered a novel TCS involved in bacitracin resistance in S. aureus, BraS/BraR (Bacitracin resistance associated) and showed that it activates transcription of two operons encoding ABC transporters: braDB and vraDE. We identified a highly conserved imperfect palindromic sequence upstream from the braDB and vraDE promoter sequences, and showed it is essential for transcriptional activation by BraS/BraR, suggesting it is the BraR binding site. The BraS histidine kinase lacks an extracellular sensing domain and we demonstrated that the two ABC transporters play distinct and original roles in bacitracin resistance: BraDB is only involved in bacitracin sensing and signaling through BraS/BraR, whereas VraDE acts specifically as a detoxification module and is sufficient to confer bacitracin resistance when expressed on its own. This is the first example of a TCS associated with two ABC transporters involved in separate functions in signal transduction and antibiotic resistance.

The CymR pleiotropic regulator of sulfur metabolism in S. aureus

In collaboration with the group of Isabelle Martin-Verstraete (Anaerobe Bacteria and Toxins Unit, Institut Pasteur, PTR N°256), we have characterized a novel pleiotropic role for CymR, the master regulator of cysteine metabolism and shown that it plays an important role in stress response, biofilm formation and virulence of S. aureus. Our results indicate that the deletion of cymR promotes survival of S. aureus inside macrophages, consistent with the observed derepression of oxidative stress response genes, whereas virulence of the ∆cymR mutant is highly impaired in a murine infection model. These data indicate that CymR plays a major role in virulence and adaptation of S. aureus for survival within the host.

Pupylation in Streptomyces

In continuation of our interest on proteolysis, we are now studying the proteasome of Streptomyces. The discovery in Mycobacterium (Science, 2008) of Pup (Prokaryotic Ubiquitin-like Protein) and pupylation a system addressing protein to the proteasome, provides further interest to this topic. Although new results come out frequently in Mycobacterium, there are specific issues in Streptomyces. Indeed pupylation appears to be involved in the control of Streptomyces differentiation cycle and antibiotic production.

Keywords: Neonatal and nosocomial infections, antibiotic production and resistance, Gram-positive cocci, streptomycetes, surface proteins, cell envelope, pupylation, metabolism, and regulation

1. Chapot-Chartier, M. P., E. Vinogradov, I. Sadovskaya, G. Andre, M. Y. Mistou, P. Trieu-Cuot, S. Furlan, E. Bidnenko, P. Courtin, C. Pechoux, P. Hols, Y. F. Dufrene, and S. Kulakauskas. 2010. Cell surface of Lactococcus lactis is covered by a protective polysaccharide pellicle. J. Biol. Chem. 285:10464-10471.

2. Fiocco, D., V. Capozzi, M. Collins, A. Gallone, P. Hols, J. Guzzo, S. Weidmann, A. Rieu, T. Msadek, and G. Spano. 2010. Characterization of the CtsR stress response regulon in Lactobacillus plantarum. J. Bacteriol. 192:896-900.

3. Gotoh, Y., A. Doi, E. Furuta, S. Dubrac, Y. Ishizaki, M. Okada, M. Igarashi, N. Misawa, H. Yoshikawa, T. Okajima, T. Msadek, and R. Utsumi. 2010. Novel antibacterial compounds specifically targeting the essential WalR response regulator. J Antibiot (Tokyo) 63:127-134.

4. Gouin, E., M. Adib-Conquy, D. Balestrino, M. A. Nahori, V. Villiers, F. Colland, S. Dramsi, O. Dussurget, and P. Cossart. 2010. The Listeria monocytogenes InlC protein interferes with innate immune responses by targeting the I{kappa}B kinase subunit IKK{alpha}. Proc Natl Acad Sci U S A 107:17333-17338.

5. Le Bouguenec, C., and C. Schouler. 2011. Sugar metabolism, an additional virulence factor in enterobacteria. Int. J. Med. Microbiol. 301:1-6.

6. Lechardeur, D., A. Fernandez, B. Robert, P. Gaudu, P. Trieu-Cuot, G. Lamberet, and A. Gruss. 2010. The 2-Cys peroxiredoxin alkyl hydroperoxide reductase c binds heme and participates in its intracellular availability in Streptococcus agalactiae. J. Biol. Chem. 285:16032-16041.

7. Morikawa, K., R. L. Ohniwa, T. Ohta, Y. Tanaka, K. Takeyasu, and T. Msadek. 2010. Adaptation beyond the Stress Response: Cell Structure Dynamics and Population Heterogeneity in Staphylococcus aureus. Microbes Environ. 25:75-82.

8. Rusniok, C., E. Couve, V. Da Cunha, R. El Gana, N. Zidane, C. Bouchier, C. Poyart, R. Leclercq, P. Trieu-Cuot, and P. Glaser. 2010. Genome sequence of Streptococcus gallolyticus: insights into its adaptation to the bovine rumen and its ability to cause endocarditis. J. Bacteriol. 192:2266-2276.

9. Soutourina, O., S. Dubrac, O. Poupel, T. Msadek, and I. Martin-Verstraete. 2010. The pleiotropic CymR regulator of Staphylococcus aureus plays an important role in virulence and stress response. PLoS Pathog 6:e1000894.

10. Tazi, A., O. Disson, S. Bellais, A. Bouaboud, N. Dmytruk, S. Dramsi, M. Y. Mistou, H. Khun, C. Mechler, I. Tardieux, P. Trieu-Cuot, M. Lecuit, and C. Poyart. 2010. The surface protein HvgA mediates group B streptococcus hypervirulence and meningeal tropism in neonates. J. Exp. Med. 207:2313-2322.