Absence, loss-of-function, or inhibition of Escherichia coli AcrB does not increase expression of other efflux pump genes supporting the discovery of AcrB inhibitors as antibiotic adjuvants.

OBJECTIVES: To determine whether expression of efflux pumps and antibiotic susceptibility are altered in Escherichia coli in response to efflux inhibition. METHODS: The promoter regions of nine efflux pump genes (acrAB, acrD, acrEF, emrAB, macAB, cusCFBA, mdtK, mdtABC, mdfA) were fused to gfp in pMW82 and fluorescence from each reporter construct was used as a measure of the transcriptional response to conditions in which AcrB was inhibited, absent or made non-functional. Expression was also determined by RT-qPCR. Drug susceptibility of efflux pump mutants with missense mutations known or predicted to cause loss of function of the encoded efflux pump was investigated. RESULTS: Data from the GFP reporter constructs revealed that no increased expression of the tested efflux pump genes was observed when AcrB was absent, made non-functional, or inhibited by an efflux pump inhibitor/competitive substrate, such as PAßN or chlorpromazine. This was confirmed by RT-qPCR for PAßN and chlorpromazine; however, a small but significant increase in macB gene expression was seen when acrB is deleted. Efflux inhibitors only synergized with antibiotics in the presence of a functional AcrB. When AcrB was absent or non-functional, there was no impact on MICs when other efflux pumps were also made non-functional. CONCLUSIONS: Absence, loss-of-function, or inhibition of E. coli AcrB did not significantly increase expression of other efflux pump genes, which suggests there is no compensatory mechanism to overcome efflux inhibition and supports the discovery of inhibitors of AcrB as antibiotic adjuvants.

Overexpression of RamA, Which Regulates Production of the Multidrug Resistance Efflux Pump AcrAB-TolC, Increases Mutation Rate and Influences Drug Resistance Phenotype.

In Enterobacteriales, the AcrAB-TolC efflux pump exports substrates, including antimicrobials, from the cell. Overexpression of AcrAB-TolC can occur after exposure to fluoroquinolones, leading to multidrug resistance. The expression of AcrAB-TolC in Salmonella is primarily regulated by the transcriptional activator RamA. However, other transcriptional activators, such as MarA, SoxRS, and Rob, can influence AcrAB-TolC expression. This study determined whether the overproduction or absence of RamA influences the mutation rate or the phenotype of mutants selected in Salmonella enterica serovar Typhimurium SL1344 after ciprofloxacin exposure. The absence of RamA (SL1344 ramA::aph) resulted in mutation frequencies/rates similar to those of wild-type Salmonella Typhimurium SL1344. However, the overproduction of RamA (SL1344 ramR::aph) and, consequently, AcrB resulted in a significantly higher mutation frequency and rate than for wild-type Salmonella Typhimurium SL1344. Whole-genome sequencing revealed that in addition to selecting gyrA mutants resistant to quinolones, SL1344 and SL1344 ramA::aph also produced multidrug-resistant (MDR) mutants, associated with mutations in soxR Conversely, mutations in SL1344 ramR::aph occurred in gyrA only. Although transcriptional regulators such as SoxRS are believed to play a minor role in AcrAB-TolC regulation under antibiotic selective pressure, we show that soxR mutants can be selected after exposure to ciprofloxacin, including when RamA is absent. This demonstrates that under selective pressure, Salmonella can respond to increased efflux pump expression by mutating other AcrAB-TolC regulatory genes, allowing for the evolution of MDR. Understanding how Salmonella responds to antibiotic pressure in the absence/overproduction of RamA is important if targeting transcriptional regulators to alter efflux is to be considered an avenue for future drug discovery.

Chlorpromazine and Amitriptyline Are Substrates and Inhibitors of the AcrB Multidrug Efflux Pump.

Efflux is an important mechanism in Gram-negative bacteria conferring multidrug resistance. Inhibition of efflux is an encouraging strategy to restore the antibacterial activity of antibiotics. Chlorpromazine and amitriptyline have been shown to behave as efflux inhibitors. However, their mode of action is poorly understood. Exposure of Salmonella enterica serovar Typhimurium and Escherichia coli to chlorpromazine selected for mutations within genes encoding RamR and MarR, regulators of the multidrug tripartite efflux pump AcrAB-TolC. Further experiments with S. Typhimurium containing AcrB D408A (a nonfunctional efflux pump) and chlorpromazine or amitriptyline resulted in the reversion of the mutant acrB allele to the wild type. Together, this suggests these drugs are AcrB efflux substrates. Subsequent docking studies with AcrB from S. Typhimurium and E. coli, followed by molecular dynamics simulations and free energy calculations showed that chlorpromazine and amitriptyline bind at the hydrophobic trap, a preferred binding site for substrates and inhibitors within the distal binding pocket of AcrB. Based on these simulations, we suggest that chlorpromazine and amitriptyline inhibit AcrB-mediated efflux by interfering with substrate binding. Our findings provide evidence that these drugs are substrates and inhibitors of AcrB, yielding molecular details of their mechanism of action and informing drug discovery of new efflux inhibitors.IMPORTANCE Efflux pumps of the resistance nodulation-cell division (RND) superfamily are major contributors to multidrug resistance for most of the Gram-negative ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogens. The development of inhibitors of these pumps would be highly desirable; however, several issues have thus far hindered all efforts at designing new efflux inhibitory compounds devoid of adverse effects. An alternative route to de novo design relies on the use of marketed drugs, for which side effects on human health have been already assessed. In this work, we provide experimental evidence that the antipsychotic drugs chlorpromazine and amitriptyline are inhibitors of the AcrB transporter, the engine of the major RND efflux pumps in Escherichia coli and Salmonella enterica serovar Typhimurium. Furthermore, in silico calculations have provided a molecular-level picture of the inhibition mechanism, allowing rationalization of experimental data and paving the way for similar studies with other classes of marketed compounds.