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

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.IMPORTANCE Antibiotic resistance is a major public health concern. In Gram-negative bacteria, overexpression of the AcrAB-TolC multidrug efflux system confers resistance to clinically useful drugs. Here, we show that loss of AcrB efflux function causes loss of virulence in Salmonella enterica serovar Typhimurium. This is due to the reduction of bacterial factors necessary for infection, which is likely to be caused by the retention of noxious molecules inside the bacterium. We also show that, 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. Understanding the biological role of AcrB will help to assess the risks of targeting efflux pumps as a strategy to combat antibiotic resistance.

Wang-Kan X ，Blair JMA ，Chirullo B ，Betts J ，La Ragione RM ，Ivens A ，Ricci V ，Opperman TJ ，Piddock LJV ... -  《mBio》

Metabolomics Reveal Potential Natural Substrates of AcrB in Escherichia coli and Salmonella enterica Serovar Typhimurium.

In the fight against antibiotic resistance, drugs that target resistance mechanisms in bacteria can be used to restore the therapeutic effectiveness of antibiotics. The multidrug resistance efflux complex AcrAB-TolC is the most clinically relevant efflux pump in Enterobacterales and is a target for drug discovery. Inhibition of the pump protein AcrB allows the intracellular accumulation of a wide variety of antibiotics, effectively restoring their therapeutic potency. To facilitate the development of AcrB efflux inhibitors, it is desirable to discover the native substrates of the pump, as these could be chemically modified to become inhibitors. We analyzed the native substrate profile of AcrB in Escherichia coli MG1655 and Salmonella enterica serovar Typhimurium SL1344 using an untargeted metabolomics approach. We analyzed the endo- and exometabolome of the wild-type strain and their respective AcrB loss-of-function mutants (AcrB D408A) to determine the metabolites that are native substrates of AcrB. Although there is 95% homology between the AcrB proteins of S. Typhimurium and E. coli, we observed mostly different metabolic responses in the exometabolomes of the S. Typhimurium and E. coli AcrB D408A mutants relative to those in the wild type, potentially indicating a differential metabolic adaptation to the same mutation in these two species. Additionally, we uncovered metabolite classes that could be involved in virulence of S. Typhimurium and a potential natural substrate of AcrB common to both species.IMPORTANCE Multidrug-resistant Gram-negative bacteria pose a global threat to human health. The AcrB efflux pump confers inherent and evolved drug resistance to Enterobacterales, including Escherichia coli and Salmonella enterica serovar Typhimurium. We provide insights into the physiological role of AcrB: (i) we observe that loss of AcrB function in two highly related species, E. coli and S. Typhimurium, has different biological effects despite AcrB conferring drug resistance to the same groups of antibiotics in both species, and (ii) we identify potential natural substrates of AcrB, some of which are in metabolite classes implicated in the virulence of S. Typhimurium. Molecules that inhibit multidrug efflux potentiate the activity of old, licensed, and new antibiotics. The additional significance of our research is in providing data about the identity of potential natural substrates of AcrB in both species. Data on these will facilitate the discovery of, and/or could be chemically modified to become, new efflux inhibitors.

Wang-Kan X ，Rodríguez-Blanco G ，Southam AD ，Winder CL ，Dunn WB ，Ivens A ，Piddock LJV ... -  《mBio》

TolC, but not AcrB, is involved in the invasiveness of multidrug-resistant Salmonella enterica serovar Typhimurium by increasing type III secretion system-1 expression.

The AcrAB-TolC efflux system is involved in multidrug and bile salt resistances. In addition, this pump has recently been suggested to increase the invasion of Salmonella enterica serovar Typhimurium (S. Typhimurium) into host cells in vitro and could therefore have an important clinical relevance for multidrug-resistant strains. The aim of this study was to investigate the role of the TolC outer membrane channel and the AcrB transporter in the interaction of multidrug-resistant S. Typhimurium strains with eukaryotic cells, especially in relation to the expression of the type III secretion system-1 (TTSS-1) required for Salmonella invasion. Deletion of tolC led to a reduced transcription of the Salmonella pathogenicity island-1 genes sipA, invF and hilA, demonstrating that all genes required for TTSS-1 biosynthesis are down-regulated in this mutant. Consequently, tolC mutants secreted smaller amounts of the TTSS-1 effector proteins SipA and SipC, and invasion tests performed with one mutant showed that it was significantly less able to invade HT-29 epithelial cells than its parental strain. This control seems specific to the TTSS-1 among the three TTSS of Salmonella as no down-regulation of expression of TTSS-2 or flagella was observed in this mutant. By contrast, acrB mutants behaved as their parents except that they secrete a slightly greater amount of SipA and SipC proteins. These data indicate that TolC but not AcrB mediates the uptake of multidrug-resistant S. Typhimurium into target host cells. Therefore, this role of TolC in the invasion of the intestine in addition to its role in bile salt resistance reinforces the interest of targeting TolC for fighting multidrug-resistant Salmonella.

Virlogeux-Payant I ，Baucheron S ，Pelet J ，Trotereau J ，Bottreau E ，Velge P ，Cloeckaert A ... -  《-》

Periplasmic adaptor protein AcrA has a distinct role in the antibiotic resistance and virulence of Salmonella enterica serovar Typhimurium.

Blair JM ，La Ragione RM ，Woodward MJ ，Piddock LJ ... -  《-》

TolC but not AcrB is essential for multidrug-resistant Salmonella enterica serotype Typhimurium colonization of chicks.

To study the role of the multidrug efflux system AcrAB-TolC in resistance of multidrug-resistant Salmonella enterica serotype Typhimurium (S. Typhimurium) phage type DT104 and DT204 strains to detergents and bile salts. To evaluate the importance of the inner membrane transporter AcrB and the outer membrane component TolC of this efflux system in the colonization of two multidrug-resistant S. Typhimurium DT104 and one DT204 strain in chicks. acrB and tolC mutants of multidrug-resistant S. Typhimurium DT104 and DT204 strains were constructed by insertional inactivation of the acrB gene and deletion of the tolC gene. MICs of detergent and bile salts were determined for the wild-type strains, the acrB and the tolC mutant strains, in presence and in absence of the efflux pump inhibitor Phe-Arg beta-naphthylamide. The effect of sodium choleate on the in vitro growth of these strains was also evaluated. The LD50s of the strains were measured in a day-old chicken model, inoculated with several doses (1 x 10(3) to 1 x 10(8) cfu) by the oral route, for 7 days post-inoculation. The colonization levels were assessed at the sublethal dose 7 days post-inoculation by determining the number of cfu of Salmonella in the faeces, caecum, spleen and liver. The decrease in resistance levels to bile salts was 64- to 256-fold higher for the tolC mutants than for the acrB mutants relative to those of the parental strains. Addition of choleate in culture medium did not affect the growth of the wild-type strains or that of the acrB mutants, but inhibited completely the growth of the tolC mutants. The LD50s were 1.0 x 10(6) and 1.2 x 10(7) cfu for one wild-type S. Typhimurium DT104 strain and the acrB mutant, respectively, and were >1.0 x 10(8) for the tolC mutants or the S. Typhimurium DT204 strains. In contrast to the acrB mutants, the tolC mutants were unable to colonize the caecum, spleen and liver after 1 week of infection. Moreover, in most chicks, no intestinal excretion was detected for the tolC mutants. The colonization levels of the acrB mutants were not significantly different from those of the wild-type strains. TolC but not AcrB appears to be essential for multidrug-resistant S. Typhimurium DT104 and DT204 colonization of chicks, which is in accordance with their respective roles in resistance to detergents and bile salts. Therefore, TolC could be a better target than AcrB for the development of efflux pump inhibitors.

Baucheron S ，Mouline C ，Praud K ，Chaslus-Dancla E ，Cloeckaert A ... -  《JOURNAL OF ANTIMICROBIAL CHEMOTHERAPY》