Redundancy in the periplasmic adaptor proteins AcrA and AcrE provides resilience and an ability to export substrates of multidrug efflux

Objectives: The components of the AcrAB–TolC efflux pump function as a tripartite efflux system conferring resist-ance to multiple antibiotics and the individual components can also function in conjunction with other efflux pumps. This study aimed to establish whether redundancy exists between the homologous periplasmic adaptor proteins (PAPs) AcrA and AcrE and to measure the impact of this redundancy on antimicrobial resistance and the potential efficacy of inhibitor molecules. Methods: The acrEgene was inactivated in Salmonella enterica serovar Typhimurium SL1344 and aDacrAmutant by insertion of the aphgene. The mutants were complemented with plasmids carrying acrAoracrE. The antimicro-bial susceptibility of the mutants to various antimicrobials was determined and the accumulation or efflux of various substrates was measured. Results: Inactivation of acrEalone had no phenotypic effect. However, the effect of inactivation of PAPs was addi-tive; theacrAacrEmutant was more susceptible to certain antimicrobials and accumulated more Hoechst dye than single acrA, acrE or acrB mutants. In addition, the double mutant invaded human intestinal epithelial cells poorly. The phenotypic defects of the acrA acrE mutant were ameliorated by expression of either acrA or acrE, but the proteins exhibited some substrate specificity. Conclusions: These data show for the first time the level of redundancy between the PAPs AcrA and AcrE, and highlight the PAPs as excellent targets for inhibitor molecules that could be used to potentiate the action of clinical antimicrobials. However, the redundancy that exists between AcrA and AcrE means potential inhibitors must act on both targets to be effective