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

Abstract

AcrAB-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.

Efflux-Mediated Resistance to New Oxazolidinones and Pleuromutilin Derivatives in Escherichia coli with Class Specificities in the Resistance-Nodulation-Cell Division-Type Drug Transport Pathways

“…To exclude efflux-independent effects due to smaller amounts of integral membrane proteins in a deletion mutant, we engineered an AcrB mutant D408A (Table 1). This mutation has been shown to prevent PMF generation without compromising the incorporation of functionless AcrB proteins (16,17). We proved similar susceptibilities of the D408A and ΔAcrB mutants and the triple-knockout (TKO) mutant additionally lacking AcrF and YhiV ( Table 2).…”

Adaptation of Salmonella enterica to bile: essential role of AcrAB‐mediated efflux

“…Not surprisingly, S. enterica mutants lacking AcrAB‐TolC are avirulent upon infection of immunodeficient mice, which involves colonization of the hepatobiliary tract (Stone and Miller, ; Lacroix et al ., ; Nishino et al ., ). However, sensitivity to bile is not only cause of attenuation as the AcrAB‐TolC system plays multiple roles in bacterial physiology and virulence (Piddock, ; Sun et al ., ; Buckner et al ., ; Wang‐Kan et al ., ).…”

“…The observation that lack of AcrB function impairs Salmonella enterica virulence in vivo and in cell cultures (Wang‐Kan et al . ) therefore suggests that AcrAB‐mediated activity may be necessary inside the animal host not only for efflux of bile and other obnoxious compounds but also for the secretion of virulence effectors.…”

Identification of binding residues between periplasmic adapter protein (PAP) and RND efflux pumps explains PAP-pump promiscuity and roles in antimicrobial resistance

“…There is some reported redundancy between AcrB and AcrF with increased expression of one system to compensate for loss of the other [8,55]. However, making inactive AcrB protein rather than deleting the gene did not result in this compensatory expression [56]. The regulation of the different efflux systems is complex and it is possible that this compensatory expression is also dependent on other factors.…”

“…RNA isolation and qRT-PCR were performed as previously described [56] except that the Total RNA Purification Plus Kit (Norgen) was used, cDNA was synthesized from RNA samples using FastGene 55-Scriptase (Nippon genetics) and 16S rRNA was used as a housekeeping gene for data normalisation.…”