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A TetR-family regulator of an RND efflux system that directs artemisinin resistance in Vibrio cholerae

ABSTRACT: Artemisinin (ARS) displayed bactericidal activity against Vibrio cholerae. To assess the mechanistic details of its antibacterial action, we have isolated V. cholerae mutants with enhanced ARS resistance and identified a gene (VCA0767), whose loss-of-function resulted in the ARS resistance phenotypes. This gene (atrR) encodes a TetR family transcriptional regulator, and its deletion mutant displayed the reduction in ARS-induced ROS formation and DNA damage. Transcriptomic analysis revealed that the genes encoding an RND efflux pump operon (vexRAB) and the outer membrane component (tolC) were highly upregulated in the artR mutant, suggesting that AtrR might act as a negative regulator of this operon and tolC. Gene deletion of vexR, vexB or tolC abrogated the ARS resistance of the atrR mutant and, more importantly, the ectopic expression of VexAB-TolC was sufficient for the ARS resistance, indicating that the increased expression of the VexAB-TolC efflux system is necessary and sufficient for the ARS resistance of the atrR mutant. The cytoplasmic accumulation of ARS was compromised in the vexBtolC mutant, suggesting that the VexAB-TolC might be the primary efflux system exporting ARS to reduce its toxicity inside of the bacterial cells. The atrR mutant displayed resistance to erythromycin as well in a VexR-dependent manner. This result suggests that AtrR may act as a global regulator responsible for preventing intracellular accumulation of toxic chemicals by enhancing the RND efflux system.

ORGANISM(S): Vibrio cholerae

PROVIDER: GSE245142 | GEO | 2023/10/16

REPOSITORIES: GEO

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Project description:We identified the mutated gene locus in a pigment overproducing Vibrio cholerae mutant of strain A1552. The deduced gene product is suggested to be a oxidoreductase based on partial homology to putative homogentisate 1,2-dioxygenase in Pseudomonas aeruginosa and Mesorhizobium loti and we propose that the gene VC1345 in the V. cholerae genome be denoted hmgA in accordance with the nomenclature for other species. The hmgA::miniTn5 mutant showed a non-pigmented phenotype after complementation with a plasmid clone carrying the wild type hmgA+ locus. Microarray transcription analysis revealed that expression of hmgA, and the neighboring genes encoding a postulated two component sensor system, was growth phase dependent. Results from qRT-PCR analysis showed that hmgA operon expression was reduced in the rpoS mutant, but pigment production by the wild type V. cholerae or the hmgA mutant was not detectably influenced by the stationary phase regulator RpoS. The pigmented mutant showed increased UV resistance in comparison with the wild-type strain. Interestingly, the pigment producing mutant expressed more toxin co-regulated pili and cholera toxin in comparison with wild type V. cholerae. Moreover, the hmgA mutant showed a 5-fold increase in the ability to colonize the intestines of infant mice. A possible mechanism by which pigment production might cause induction of the ToxR regulon due to generation of hydrogen peroxide was supported by results from tests showing that externally supplied H2O2 led to higher TcpA levels. Taken together, our findings suggest that melanin pigment formation may play a role in V. cholerae virulence factor expression. Groups of assays that are related as part of a time series. Elapsed Time: Wild type V. cholerae strain A1552 bacteria were cultured statically for 4 h in AKI medium at 37C and then shifted to aerobic growth for 6 h using shaken culture flasks. Samples were taken at hourly during the this process. Keywords: time_series_design Using regression correlation

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A TetR-family regulator of an RND efflux system that directs artemisinin resistance in Vibrio cholerae

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