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 a r esistance- n odulation-cell d ivision (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. IMPORTANCE Drug efflux protein complexes or efflux pumps are considered as the major determinants of multiple antimicrobial resistance by exporting a wide range of structurally diverse antibiotics in bacterial pathogens. Despite the clinical significance of the increased expression of the efflux pumps, their substrate specificity and regulation mechanisms are poorly understood. Here, we demonstrated that VexAB-TolC, a r esistance- n odulation-cell d ivision (RND) efflux pump of V. cholerae , is responsible for the resistance to artemisinin (ARS), an antimalarial drug with bactericidal activity. Furthermore, we newly identified AtrR, a TetR family repressor, as a global regulator for VexRAB and the common outer membrane channel, TolC, where VexR functions as the pathway-specific regulator of the vexAB operon. Our findings will help improve our insight into a broad range of substrate specificity of the VexAB-TolC system and highlight the complex regulatory networks of the multiple RND efflux systems during V. cholerae pathogenesis.