Redundancy of AcrA and AcrE to support efflux of antibiotics through AcrD in Salmonella Typhimurium

RND efflux pumps are important mediators of antibiotic resistance. RND efflux pumps including AcrB, are organized as tripartite systems, consisting of an inner membrane RND transporter, a periplasmic adaptor protein (PAP) and an outer membrane factor. We have previously identified the residues required for binding of the major PAP AcrA to the major RND pump AcrB and shown that there is promiscuity between the PAPs such that they can function with non-cognate pumps. AcrE is a PAP homologous to AcrA and AcrD and AcrF are RND pumps homologous to AcrB. This study aimed to determine whether the PAP AcrE can function with AcrD, which does not have its own PAP, to mediate efflux and whether the previously identified RND binding residues in AcrA and AcrE were also required for AcrD binding. The acrD and acrE genes were cloned into compatible vectors and co-transformed into a strain lacking acrAB, acrD and acrEF. When expressed together, acrDand acrE significantly decreased susceptibility of the efflux mutant strain to AcrD substrates including aztreonam, carbenicillin, cloxacillin, fusidic acid, nafcillin, novobiocin, oxacillin and ticarcillin, indicating that AcrE can also form a functional complex with AcrD. These experiments also highlighted that the substrate profile of AcrD in S. entericaand E. coli are different. Point mutations in the previously defined RND binding residues of AcrA and AcrE impaired AcrD-mediated efflux of substrate drugs which validates the interchangeability of AcrA and AcrE and highlights these residues as ideal drug targets for efflux inhibition to combat antimicrobial resistance.