Expression of homologous RND efflux pump genes is dependent upon AcrB expression: implications for efflux and virulence inhibitor design.

Jessica M A Blair,Vito Ricci,Helen E Smith,Amelia J Lawler,Louisa J Thompson,Laura J V Piddock

doi:10.1093/jac/dku380

Jessica M A Blair, Vito Ricci + Show 4 more

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https://doi.org/10.1093/jac/dku380

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Abstract

ObjectivesEnterobacteriaceae have multiple efflux pumps that confer intrinsic resistance to antibiotics. AcrB mediates clinically relevant multidrug resistance and is required for virulence and biofilm formation, making it an attractive target for the design of inhibitors. The aim of this study was to assess the viability of single transporters as a target for efflux inhibition using Salmonella Typhimurium as the model pathogen.MethodsThe expression of resistance–nodulation–division (RND) efflux pump genes in response to the inactivation of single or multiple homologues was measured using real-time RT–PCR. Phenotypes of mutants were characterized by measuring antimicrobial susceptibility, dye accumulation and the ability to cause infection in vitro.ResultsThe expression of all RND efflux pump genes was increased when single or multiple acr genes were inactivated, suggesting a feedback mechanism that activates the transcription of homologous efflux pump genes. When two or three acr genes were inactivated, the mutants had further reduced efflux, altered susceptibility to antimicrobials (including increased susceptibility to some, but conversely and counterintuitively, decreased susceptibility to some others) and were more attenuated in the tissue culture model than mutants lacking single pumps were.ConclusionsThese data indicate that it is critical to understand which pumps an inhibitor is active against and the effect of this on the expression of homologous systems. For some antimicrobials, an inhibitor with activity against multiple pumps will have a greater impact on susceptibility, but an unintended consequence of this may be decreased susceptibility to other drugs, such as aminoglycosides.

Highlights

Efflux is an important mechanism of multidrug resistance in bacteria, conferring decreased susceptibility to a wide range of substrates including antibiotics, dyes, detergents and biocides.[1]
We previously showed that inactivation of acrB led to increased accumulation of the dye Hoechst 33342.20 Compared to SL1344, inactivation of acrD (L132) or acrF (L131) or inactivation of both acrD and acrF (L1395) did not significantly alter the accumulation of Hoechst 33342 (Figure 2)
We show that single gene inactivation of either acrD (L132) or acrF (L131) significantly attenuate the virulence of Salmonella

Summary

Introduction

Efflux is an important mechanism of multidrug resistance in bacteria, conferring decreased susceptibility to a wide range of substrates including antibiotics, dyes, detergents and biocides.[1] This makes them an attractive target for the design of inhibitors which could be used to potentiate the use of existing antimicrobials. Resistance nodulation division (RND) efflux transporters are found in the inner membrane of. Gram-negative bacteria and form a complex with an outer membrane channel and a periplasmic adaptor protein (PAP) to form a tri-partite efflux pump system spanning both the inner and outer membrane.[2], structurally diverse and include antibiotics, biocides, dyes, detergents and host-derived molecules. Active efflux of substrates by RND systems is responsible for the intrinsic resistance of Gram negative bacteria to multiple classes of structurally distinct antimicrobials.[1].

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