Functional Investigation of the MexA-MexB-OprM Efflux Pump of Pseudomonas Aeruginosa

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.We describe an original activity assay for MexB and MexA. The pump is co-reconstituted into proteoliposomes together with bacteriorhodopsin (BR), a light-activated proton pump [1]. 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. 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.We are working on the reconstitution of the whole efflux pump. To that purpose we reconstitute MexB and OprM in respective proteoliposomes. MexA will be reconstituted either in proteoliposomes (together with MexB) or will be present in the buffer as we have purified a soluble version of this lipid-anchored periplamsic protein. Upon mixing, the tripartite protein complex should form, making it possible to study transport through the whole efflux pump by use of carefully chosen fluorescent reporters.[1] Verchere, Broutin & Picard, Scientific reports, 2012