Functional investigation of the efflux pump MexA–MexB-OprM of Pseudomonas aeruginosa

Abstract : Among the various mechanisms developed by the bacteria to counter to the effect of antibiotics, active efflux is on the front line. In Pseudomonas aeruginosa, a Gram negative bacteria, efflux transporters are organized as multicomponent systems where MexB, the pump located in the inner membrane, works in conjunction with MexA, a periplasmic protein, and OprM, an outer membrane protein. MexB is a proton motive force-dependent pump with broad substrate specificity. During my PhD, I have designed an original activity assay for MexB and MexA. The pump is coreconstituted into proteoliposomes together with bacteriorhodopsin (BR), a light-activated proton pump. In this system, upon illumination with visible light, the photo-induced proton gradient created by the BR is shown to be coupled to the active transport of substrates through the pump. The activity of MexB is monitored indirectly. Since MexB uses the protomotive force to transport antibiotics, one can determine substrate transport though MexB by monitoring the pH inside the liposomes. For that purpose, pyranine, a fluorescent probe whose fluorescence yield increases with increasing pH, is encapsulated inside the liposomes. This test makes the investigation of the pump possible. In the absence of MexA, MexB has a basal activity which is not substrate-dependent. Once MexB is reconstituted together with MexA, its activity is specific and substrate-dependent. Then I worked on the reconstitution of the whole efflux pump. For this, I prepare two different kinds of liposomes: i) Liposomes with reconstituted MexA and MexB in which pyranine and a nucleic acid intercalating agent are encapsulated, ii) Liposomes with reconstituted OprM and encapsulated RNA. The activity of MexB is monitored thanks to the addition of EthB, a substrate of MexB, that is poorly fluorescent in aqueous medium and highly fluorescent when intercalated into RNA. Upon generation of a pH gradient, I observe two concomitant phenomena: the decrease of pyranine fluorescence, as MexB is using protons to transport the substrate, and the increase of the fluorescence of the RNA intercalating agent as a result of its interaction with RNA. I have successfully assembled the efflux pump and monitored transport through it from one liposome to the other. I have demonstrated that OprM needs to interact with MexA and MexB in order to open and that MexB activity is accelerated when the pump is assembled.
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Alice Verchère. Functional investigation of the efflux pump MexA–MexB-OprM of Pseudomonas aeruginosa. Biomolecules [q-bio.BM]. Université René Descartes - Paris V, 2014. English. ⟨NNT : 2014PA05P622⟩. ⟨tel-01160389⟩

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