Functional study of SEDS glycosyltransferases (RodA, FtsW) in Enterococcus faecium
Quertemont, Mathias
Promotor(s) : Terrak, Mohammed
Date of defense : 4-Sep-2024 • Permalink : http://hdl.handle.net/2268.2/21011
Details
Title : | Functional study of SEDS glycosyltransferases (RodA, FtsW) in Enterococcus faecium |
Translated title : | [fr] Etude fonctionnelle des glycosyltransférases SEDS (RodA, FtsW) chez Enterococcus faecium |
Author : | Quertemont, Mathias |
Date of defense : | 4-Sep-2024 |
Advisor(s) : | Terrak, Mohammed |
Committee's member(s) : | Dumoulin, Mireille
Kerff, Frédéric Bouché, Frédéric |
Language : | English |
Number of pages : | 65 |
Keywords : | [en] SEDS, [en] Enterococcus [en] PBP [en] glycosyltransferase [en] in vitro [en] purification |
Discipline(s) : | Life sciences > Biochemistry, biophysics & molecular biology |
Research unit : | Center for Protein Engineering (CIP) |
Target public : | Researchers Professionals of domain Student |
Institution(s) : | Université de Liège, Liège, Belgique |
Degree: | Master en biochimie et biologie moléculaire et cellulaire, à finalité approfondie |
Faculty: | Master thesis of the Faculté des Sciences |
Abstract
[en] 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.
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