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Dissecting the molecular mechanism of an antibiotic efflux pump (H/F)

This offer is available in the following languages:
Français - Anglais

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General information

Reference : UMR5086-CEDORE-002
Workplace : LYON 07
Date of publication : Friday, September 06, 2019
Scientific Responsible name : Cédric Orelle
Type of Contract : PhD Student contract / Thesis offer
Contract Period : 36 months
Start date of the thesis : 1 November 2019
Proportion of work : Full time
Remuneration : 2 135,00 € gross monthly

Description of the thesis topic

Antibiotic resistance is becoming a major threat for human health. It represents a difficult challenge to address, especially with the outcome of multidrug resistance (MDR), when bacteria become resistant to multiple structurally and functionally unrelated molecules. Drug efflux pumps contribute to this phenomenon and can promote bacterial adaptation by favoring the emergence of other resistance mechanisms. An increasing number of bacterial ABC transporters, which utilize the energy of ATP hydrolysis to expel drugs, have been implicated in multidrug resistance mechanisms. Because their functioning mechanism remains unclear, we propose to decrypt the molecular mechanism of one of this pump, BmrA from Bacillus subtilis, by using a combination of structural and conformational dynamics approaches. This thesis is financed by an ANR grant (BIOTIFLUX) in collaboration with 3 other teams, two being in our MMSB unit and one in Marseille.

Work Context

Our team « Bacterial ATP/GTPases: antibiotic resistance and new targets » identified BmrA and has been working for many years to understand its functioning mechanism. Recently, collaborators from the consortium determined the structure de BmrA in an ATP-bound conformation. To understand its transport mechanism, we will characterize its conformational changes and protein dynamics, and stabilize some catalytic cycle intermediates (use of mutants and/or combination of appropriate ligands) for further structural studies with our collaborators.
The candidate will produce and purify BmrA (wild-type and and various mutants), according to established procedures. Classical biochemical techniques will be performed to measure transport, ligand binding and ATPase assays. After reconstituting BmrA in various lipidic environments, we will study its conformational changes with several approaches: hydrogen/deuterium exchanged coupled with mass spectrometry (collaboration with a team in Toulouse), disulfide cross-linking, and EPR spectroscopy (collaboration with a team in Marseille).

Candidate skills:

- Biochemistry (purification et characterization) of proteins, ideally membrane proteins
- Good level of english

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