Bacterial Pathogenicity

Group

Permanent staff :

• Viviana Job, Ph.D. (CEA researcher)
• Alexandre Dos Santos Martins (CEA technical staff)

Introduction

Our group is interested in the mechanisms of bacterial pathogenicity and tackles structure-function studies of proteins and macromolecular complexes involved in two main systems:

• the type III secretion system (T3SS) of major human pathogens, such as Pseudomonas aeruginosa and Escherichia coli,
• proteins involved in cell division and surface-exposed virulence factors of Streptococcus pneumoniae.

We employ techniques ranging from molecular biology (site specific mutagenesis, polycistronic cloning) and classical biochemistry (recombinant protein expression and purification, cross-linking, enzymatic analyses) to X-ray crystallography, in order to correlate the structure of a protein or a multi-protein complex with its function within the cell or in the infection process.

The type III secretion system (T3SS)

The T3SS is a macromolecular complex that is used as a weapon to inject toxins directly into the cytoplasm of eukaryotic cells. The complex is anchored on both bacterial membranes, and is terminated by a hollow needle formed by a single polymerized protein (PscF in P. aeruginosa) through which toxins and effectors are injected (Fig. 1). Recently, our group has shown that needle biogenesis in P. aeruginosa is regulated by the formation of a ternary complex between proteins Psc, PscF, and PscG in the bacterial cytoplasm, which could represent a novel, potential antibiotherapy target (collaboration: I. Attree group, iRTSV Grenoble).

Fig. 1: (A) the T3SS (from Troisfontaines and Cornelis, 2006); (B), in P. aeruginosa, premature polymerization of the needle protein (PscF) is blocked by formation of a complex with PscG and PscE (Quinaud et al. (2007) Proc. Natl. Acad. Sci. USA 104).

Related publications

• Quinaud, M., Ple, S., Job, V., Contreras-Martel, C., Simorre, J.-P., Attree, I., and Dessen, A. (2007) Structure of the heterotrimeric complex that regulates type III secretion needle formation. Proc. Natl. Acad. Sci. USA 104, 7803-7808
• Faudry, E., Job, V., Dessen, A., Attree, I., and Forge, V. (2007) type III secretion system translocator has a molten globule conformation both in its free and chaperone-bound forms. FEBS J. 274, 3601-3610
• Quinaud, M., Chabert, J., Faudry, E., Neumann, e., Lemaire, D., Pastor, A., Elsen, S., Dessen, A., and Attree, I. (2005) The PscE-PscF-PscG complex controls type III secretion needle biogenesis in Pseudomonas aeruginosa. J. Biol Chem. 280, 36293-36300.
• Gouré, J., Pastor, A., Faudry, E., Brochier, G., Chabert, J., Dessen, A., and Attree, I. (2004). The V antigen of Pseudomonas aeruginosa is required for assembly of the functional type III translocation pore in host cell membranes. Infect. Immun 72, 4741-4750.
• Schoehn, G., DiGuilmi, A.M.., LeMaire, D., Attree, I., Weissenhorn, W., and Dessen, A. (2003). Oligomerization of type III secretion proteins PopB and PopD precedes pore formation in Pseudomonas. EMBO J. 22, 4957-4967

Streptococcus pneumoniae: Penicillin-Binding Proteins (PBPs) and cell division

Penicillin-Binding Proteins (PBPs) play key roles in formation of peptidoglycan, a key component of the bacterial cell wall. They are also the targets of beta-lactam antibiotics such as penicillin. Thanks to a grant from the EU (EUR-INTAFAR), our group has solved the crystal structure of PBP1b in complex with lactivicin, the only non-beta lactam antibiotic which interacts with PBPs. Lactivicin and its analogs have shown to be active against different drug-resistant bacterial strains and could prove to be good starting points for novel drug development (collaboration: C. Schofield group, Univ. Oxford; T. Vernet and A.M. Di Guilmi, LIM, IBS Grenoble).

Fig. 3: Lactivicin makes a covalent complex within the PBP active site (Macheboeuf et al. (2007) Nature Chem. Biol. 3)

Related publications

Job, V., Carapito, R., Vernet, T., Dessen, A., and Zapun, A. (2007) Common alterations in PBP1a from resistantStreptococcus pneumoniae decrease its reactivity towards b-lactams: structural insights. J. Biol. Chem., in press.

Macheboeuf, P., Lemaire, Dos Santos Martins, A., Dideberg, O., Jamin, M., and Dessen, A. (2007) Trapping of an acyl-enzyme intermediate in a Penicillin-Bing Protein (PBP)-catalyzed reaction.J. Mol. Biol., in press.

Macheboeuf, P., Fischer, D., Zervosen, A., Luxen, A., Joris, B., Dessen, A. and Schofield, C.* (2007) Structural and mechanistic basis of penicillin-binding protein inhibition by lactivicins. Nature Chem. Biol. 3, 565-569.

Contreras-Martel, C., Job, V., Di Guilmi, A.M., Vernet, T., Dideberg, O., and Dessen, A. (2006) Structural basis for b-lactam resistance by a class A penicillin-binding protein from Streptococcus pneumoniae. J. Mol. Biol. 544, 684-696.

Macheboeuf, P., Di Guilmi, A.M., Job, V., Vernet, T., Dideberg, O., and Dessen, A. (2005). Active site restructuring regulates ligand recognition in class A penicillin-binding proteins (PBPs). Proc. Natl. Acad. Sci. USA 102, 577-582.