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Lack of AcrB efflux function confers loss of virulence to Salmonella Typhimurium

Wang-Kan, X, Blair, JMA, Chirullo, B, Betts, Jonathan, La Ragione, Roberto, Ivens, A, Ricci, V, Opperman, TJ and Piddock, LJV (2017) Lack of AcrB efflux function confers loss of virulence to Salmonella Typhimurium mBio, 8 (4). e00968-17.

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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 non-functional AcrB mutant by substituting 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 media. However, compared with wild-type SL1344, the mutant had increased accumulation of the Hoechst 33342 dye, decreased efflux of ethidium bromide and was multidrug hyper-susceptible. The D408A mutant was attenuated in vivo in a mouse model 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. RNAseq revealed down-regulation 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 up-regulated, 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 suggests 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.

Item Type: Article
Divisions : Faculty of Health and Medical Sciences > School of Veterinary Medicine
Authors :
Wang-Kan, X
Blair, JMA
Chirullo, B
La Ragione,
Ivens, A
Ricci, V
Opperman, TJ
Piddock, LJV
Date : 18 July 2017
DOI : 10.1128/mBio.00968-17
Copyright Disclaimer : Copyright © 2017 Wang-Kan et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.
Uncontrolled Keywords : AcrB; SPI; Salmonella; Salmonella pathogenicity island; efflux; motility; transcriptome; virulence
Depositing User : Clive Harris
Date Deposited : 01 Aug 2017 15:08
Last Modified : 16 Jan 2019 18:54

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