Mémoire

Abstract

Antibiotic resistance is an increasing threat to human society. Among multi-resistant strains, E. faecalis and E. faecium are frequent nosocomial opportunistic pathogens. These intestinal-dwelling bacteria can enter the bloodstream, causing life-threatening diseases in humans. CC17, the high-virulence E. faecium clinical isolate, is nearing a panresistance phenotype. The study of the peptidoglycan biosynthesis pathway is promising for discovering novel therapies against CC17. Peptidoglycan synthesis involves many regulatory proteins, which may also be antibiotic targets. These proteins are gathered in two molecular machines, the elongasome and the divisome, to coordinate the pathway with cell division. Peptidoglycan is synthesized by class A or class B PBPs. The latter are monofunctional transpeptidases and thus catalyze peptidoglycan synthesis with a SEDS glycosyltransferase partner. E. faecium possesses three SEDS-bPBP couples: FtsW1-PBPB and FtsW2-PBP5 in the divisome, and RodA-PBPA in the elongasome. The latter two are responsible for innate cephalosporin resistance. The aim of this study is to gain a deeper understanding of E. faecium SEDS-bPBP function and regulation. SEDS and their bPBP partners were produced in fusion in E. coli C43. Fusion proteins were consecutively isolated from the cell membrane by DDM treatment and purified by Ni²+ affinity chromatography. Each fusion protein activity was tested in vitro and monitored by fluorescence-revealed SDS-PAGE. A shuttle vector was also constructed with upstream and downstream regions of E. faecium seds. This vector will be used to inactivate seds by homologous recombination in the future. Merging FtsW1 with its cognate bPBP is found to activate the SEDS. This is not the case for the other two glycosyltransferases. No tested medium modifications affect SEDS activities in vitro. Some parameters, most likely regulatory factors from the bacteria, are missing to reach optimal SEDS catalysis in vitro. Even if the methods employed need improvement, this study constitutes a first step to functionally characterize E. faecium SEDS.