Lack of AcrB Efflux Function Confers Loss of Virulence on Salmonella enterica Serovar Typhimurium.

Xuan Wang-Kan,Jessica M A Blair,Timothy J Opperman,Alasdair Ivens,Roberto M La Ragione,Jonathan Betts,Barbara Chirullo,Vito Ricci,Laura J V Piddock,Julian E Davies

doi:10.1128/mbio.00968-17

Abstract

ABSTRACTAcrAB-TolC is the paradigm resistance-nodulation-division (RND) multidrug resistance efflux system in Gram-negative bacteria, with AcrB being the pump protein in this complex. We constructed a nonfunctional AcrB mutant by replacing D408, a highly conserved residue essential for proton translocation. Western blotting confirmed that the AcrB D408A mutant had the same native level of expression of AcrB as the parental strain. The mutant had no growth deficiencies in rich or minimal medium. However, compared with wild-type SL1344, the mutant had increased accumulation of Hoechst 33342 dye and decreased efflux of ethidium bromide and was multidrug hypersusceptible. The D408A mutant was attenuated in vivo in mouse and Galleria mellonella models and showed significantly reduced invasion into intestinal epithelial cells and macrophages in vitro. A dose-dependent inhibition of invasion was also observed when two different efflux pump inhibitors were added to the wild-type strain during infection of epithelial cells. RNA sequencing (RNA-seq) revealed downregulation of bacterial factors necessary for infection, including those in the Salmonella pathogenicity islands 1, 2, and 4; quorum sensing genes; and phoPQ. Several general stress response genes were upregulated, probably due to retention of noxious molecules inside the bacterium. Unlike loss of AcrB protein, loss of efflux function did not induce overexpression of other RND efflux pumps. Our data suggest that gene deletion mutants are unsuitable for studying membrane transporters and, importantly, that inhibitors of AcrB efflux function will not induce expression of other RND pumps.

Highlights

The presence of a T¡G substitution at position 530662 in the chromosome of the D408A mutant was confirmed by whole-genome sequencing (WGS)
We focused on investigating the biological function of the AcrB multidrug resistance (MDR) efflux pump and the impact of loss of efflux function on the virulence of Salmonella Typhimurium
Several other authors have observed loss of virulence in RND efflux pump deletion mutants across a range of bacterial species, but none have shown evidence of the phenotype being a consequence of loss of efflux function [13, 15,16,17,18, 20, 28, 29]

Summary

Introduction

Our data suggest that gene deletion mutants are unsuitable for studying membrane transporters and, importantly, that inhibitors of AcrB efflux function will not induce expression of other RND pumps. In contrast to loss of AcrB protein, loss of efflux does not induce overexpression of other efflux pumps from the same family This indicates that there are differences between loss of efflux protein and loss of efflux that make gene deletion mutants unsuitable for studying the biological function of membrane transporters. They associate with an outer membrane protein and a periplasmic adapter protein to form a tripartite MDR efflux system [8].

Methods

Results

Conclusion