Nouvelle approche dans la lutte contre la résistance aux antibiotiques des bactéries colonisant les poumons des patients atteints de mucoviscidose : reconstitution d'une pompe d'efflux de Pseudomonas aeruginosa

Abstract : Multidrug efflux systems are membrane transport proteins that are used to translocate a wide variety of drugs across the inner and the outer membranes of Gram-negative bacteria, leading to natural and acquired antimicrobial resistances.Most of the multidrug transporters of P. aeruginosa belong to the resistance-nodulation-cell division (RND) superfamily.They function as three-component assemblies made of an inner membrane transporter (RND), a periplasmic membrane fusion protein (MFP) and an outer membrane factor channel (OMF). Along with functional studies, many crystal structures of the individual components of the pump have been solved but the interactions underlying the complex assembly and the opening mechanism of the outer channel remain unclear. In this study, we investigated structural and functional insights of P. aeruginosa efflux pumps. We solved the crystal structure of the MexEF-OprN efflux pump outer membrane channel OprN mainly involved in fluoroquinolones resistance. Our new structure highlights the differences between P. aeruginosa and other Gram-negative bacteria OMFs that could explain their specific interaction with the cognate MFP partners. We also purified and characterized the inner membrane transporter MexY from the MexXY-OprM efflux pump, which is the major determinant of aminoglycosides resistance in P. aeruginosa. Besides, we solved the crystal structure of a mutatedform of the outer membrane channel OprM in order to understand its opening mechanism. We also investigated in vivo effect of the OprM mutants in antibiotics resistance by MIC measurements and tried to correlate with the observed structural modifications leading to the open state. Moreover, we set up a new in vitro test allowing the investigation of the assembly of the MexAB-OprM efflux pump. Our results showed the importance of MexA and its lipid anchor in promoting and stabilizing the complex assembly. In addition, as a side project with the group of Pr Plésiat (laboratoire de Bactériologie de Besançon), we built different structural models of AmpC mutants from overproducing clinical isolates,showing the possible conformational changes that lead to the resistance increase.
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Véronique Yvette Ntsogo Enguene. Nouvelle approche dans la lutte contre la résistance aux antibiotiques des bactéries colonisant les poumons des patients atteints de mucoviscidose : reconstitution d'une pompe d'efflux de Pseudomonas aeruginosa. Biochimie [q-bio.BM]. Université Sorbonne Paris Cité, 2016. Français. ⟨NNT : 2016USPCB146⟩. ⟨tel-01977986⟩

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