Mémoire
| Résumé : | The study of multidrug-resistant (MDR) transport proteins, also known as multidrug efflux pumps isimportant to understand the molecular basis underlying the emergence of bacterial antibioticresistance. For this purpose, the development of in vitro experiments allowing to observe andquantify the transport of several different substrates is essential but is an experimental challenge. Inthis work, we set out to develop a transport assay for a multidrug efflux pump with the objective toquantify the coupled transport of protons, to be able to quantify transport in a subtree-independentway, thereby circumventing the conundrum of substrate polyspecificity.The development of such a “universal” transport assay consists of several steps. The first is theproduction and purification of the FoF1 ATPase. This ATPase is necessary for generating a pH gradientthat can be used by the MDR protein as a source of energy to transport its substrates that areantibiotics.The purification steps have been optimized. The type of detergent or lipid that should be used forthe ATPase solubilization was also looked at. The best result was given with the detergent digitonin.Then the physio-chemical properties of the liposome, that will be used as the simplified model of acell, was looked at. The parameters that were analysed were the solubilization concentration (Rsol)of the liposome with Triton X-100. We found an Rsol value of 1.2 mM. The size distribution of theliposomes, after their formation, was looked at. A leak test was also done, to observe if the liposomeswere tight enough to be able to sustain a pH gradient, generated by the ATPase, with a lower pH inthe inside than the outside environment.The final step is the reconstitution of the MDR transport protein and the FoF1 ATPase into aproteoliposome. It can be confirmed that both proteins were integrated into the liposome.At the end, we managed to create a functional universal transport assay with ATPase for creating apH gradient with an MDR transport protein. Several different aspects were looked at for the creationof this transport assay and aspects could be further optimized, but the foundations are now knownand can be used for further research in MDR transport proteins. |
