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Substrate-dependent activation of the Vibrio cholerae vexAB RND efflux system requires vexR.

ABSTRACT: Vibrio cholerae encodes six resistance-nodulation-division (RND) efflux systems which function in antimicrobial resistance, virulence factor production, and intestinal colonization. Among the six RND efflux systems, VexAB exhibited broad substrate specificity and played a predominant role in intrinsic antimicrobial resistance. The VexAB system was encoded in an apparent three gene operon that included vexR; which encodes an uncharacterized TetR family regulator. In this work we examined the role of vexR in vexRAB expression. We found that VexR bound to the vexRAB promoter and vexR deletion resulted in decreased vexRAB expression and increased susceptibility to VexAB antimicrobial substrates. Substrate-dependent induction of vexRAB was dependent on vexR and episomal vexR expression provided a growth advantage in the presence of the VexAB substrate deoxycholate. The expression of vexRAB increased, in a vexR-dependent manner, in response to the loss of RND efflux activity. This suggested that VexAB may function to export intracellular metabolites. Support for this hypothesis was provided by data showing that vexRAB was upregulated in several metabolic mutants including tryptophan biosynthetic mutants that were predicted to accumulate indole. In addition, vexRAB was found to be upregulated in response to exogenous indole and to contribute to indole resistance. The collective results indicate that vexR is required for vexRAB expression in response to VexAB substrates and that the VexAB RND efflux system modulates the intracellular levels of metabolites that could otherwise accumulate to toxic levels.

SUBMITTER: Taylor DL

PROVIDER: S-EPMC4335029 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Substrate-dependent activation of the Vibrio cholerae vexAB RND efflux system requires vexR.

Taylor Dawn L DL Ante Vanessa M VM Bina X Renee XR Howard Mondraya F MF Bina James E JE

PloS one 20150219 2

Vibrio cholerae encodes six resistance-nodulation-division (RND) efflux systems which function in antimicrobial resistance, virulence factor production, and intestinal colonization. Among the six RND efflux systems, VexAB exhibited broad substrate specificity and played a predominant role in intrinsic antimicrobial resistance. The VexAB system was encoded in an apparent three gene operon that included vexR; which encodes an uncharacterized TetR family regulator. In this work we examined the role ...[more]

PMID: 25695834

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Project description:Antibiotic-resistant Acinetobacter baumannii causes infections that are extremely difficult to treat. A significant role in these resistance profiles is attributed to multidrug efflux pumps, especially those belonging to the resistance-nodulation-cell division (RND) superfamily of transporters. In this study, we analyzed functions and properties of RND efflux pumps in A. baumannii ATCC 17978. This strain is susceptible to antibiotics and does not contain mutations that are commonly selected upon exposure to high concentrations of antibiotics. We constructed derivatives of ATCC 17978 lacking chromosomally encoded RND pumps and complemented these strains by the plasmid-borne genes. We analyzed the substrate selectivities and efficiencies of the individual pumps in the context of native outer membranes and their hyperporinated variants. Our results show that inactivation of AdeIJK provides the strongest potentiation of antibiotic activities, whereas inactivation of AdeFGH triggers the overexpression of AdeAB. The plasmid-borne overproduction complements the hypersusceptible phenotypes of the efflux deletion mutants to the levels of the parental ATCC 17978. Only a few antibiotics strongly benefitted from the overproduction of efflux pumps and antibacterial activities of some of those depended on the synergistic interaction with the low permeability barrier of the outer membrane. Either overproduction or inactivation of efflux pumps change dramatically the lipidome of ATCC 17978. We conclude that efflux pumps of A. baumannii are tightly integrated into physiology of this bacterium and that clinical levels of antibiotic resistance in A. baumannii isolates are unlikely to be reached solely due to the overproduction of RND efflux pumps.IMPORTANCE RND-type efflux pumps are important contributors in development of clinical antibiotic resistance in A. baumannii However, their specific roles and the extent of contribution to antibiotic resistance remain unclear. We analyzed antibacterial activities of antibiotics in strains with different permeability barriers and found that the role of active efflux in antibiotic resistance of A. baumannii is limited to a few select antibiotics. Our results further show that the impact of efflux pump overproduction on antibiotic susceptibility is significantly lower than the previously reported for clinical isolates. Additional mechanisms of resistance, in particular those that improve the permeability barriers of bacterial cells and act synergistically with active efflux pumps are likely involved in antibiotic resistance of clinical A. baumannii isolates.

Project description:Enteric pathogens have developed several resistance mechanisms to survive the antimicrobial action of bile. We investigated the transcriptional profile of Vibrio cholerae O1 El Tor strain C6706 under virulence gene inducing conditions, in the presence and absence of bile. Microarray analysis revealed that the expression of 119 genes was affected by bile. The mRNA levels of genes encoding proteins involved in transport was increased in the presence of bile, whereas mRNA levels of genes encoding proteins involved in pathogenesis and chemotaxis was decreased. This study identified genes encoding transcriptional regulators from the TetR family (vexR and breR) and multidrug efflux pumps from the RND superfamily (vexB and vexD [here renamed breB]) that were induced in response to bile. Further analysis regarding vexAB and breAB expression in the presence of various antimicrobial compounds established that vexAB was induced in the presence of bile, SDS or novobiocin, whereas induction of breAB was specific to bile. BreR is a direct repressor of the breAB promoter and is able to autoregulate its own expression, as demonstrated by transcriptional and electrophoretic mobility shift assays (EMSA). Expression of breR and breAB is induced in the presence of the bile salts cholate, deoxycholate or chenodeoxycholate and EMSA showed that deoxycholate is able to abolish formation of BreR-PbreR complexes. We propose that deoxycholate is able to interact with BreR and induce a conformational change that will interfere with its DNA binding ability resulting in breAB and breR expression. These results provide new insight into a transcriptional regulator and a transport system that likely play an essential role in the ability of Vibrio cholerae to resist the action of bile in the host. Keywords: Vibrio cholerae response to bile Three independent experiments were performed for the microarray analysis. From each experiment, RNA was obtained from four different time points and was prepared for hybridization, therefore, a total of 3 slides were analyzed per time point. The growth conditions were as follows; C6706 str2 was grown 15 h in LB medium at 30 degrees C with aeration. The cultures were then diluted 100-fold into AKI medium with, or without, 0.4% crude bile (high bile concentration) and were grown at 37degrees C for 3.5 h stationary followed by 2 h with aeration to induce virulence gene expression. Then the cultures were diluted 100-fold into AKI medium with, or without, 0.02% crude bile (low bile concentration) and were grown at 37 degrees C for 2 h stationary. Samples were obtained from 4 different time points: 2, 4, and 5.5 h from the high bile cultures, and at 2 h from the low bile cultures.

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Substrate-dependent activation of the Vibrio cholerae vexAB RND efflux system requires vexR.

Publications

Substrate-dependent activation of the Vibrio cholerae vexAB RND efflux system requires vexR.

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