The tripartite multi-drug efflux pump, MexAB-OprM in Pseudomonas aeruginosa confers resistance to many antibiotics. Work to investigate interactions between individual components of the MexAB-OprM complex is limited by their low affinity in vitro. To overcome this, genetically engineered fusions of component proteins of the homologous complex in E. coli, AcrAB-TolC have been prepared. We investigated whether the fusion proteins still retain the ability to efflux drugs and how drug transport compares to that of the native proteins.

Different arrangements of the bipartite fusions of AcrA-AcrB and AcrA-TolC were expressed in E.coli cells with deletions of the corresponding efflux pump component.

The ability of the fusion proteins to convey resistance to a variety of compounds, compared with wild type, was investigated using cytotoxicity assays followed by transport assays to directly observe efflux of fluorescent compounds.

The fusion proteins retained the ability to efflux drugs.

The novel fusion proteins overcame the issue of low affinity between AcrA, AcrB and TolC in vitro. This method of producing a fusion protein opens up new avenues for investigating interactions between components of protein complexes and may provide a means by which the crystal structure of the tripartite drug efflux complex can be determined.