The Vibrio cholerae VexGH RND Efflux System Maintains Cellular Homeostasis by Effluxing Vibriobactin.

Resistance-nodulation-division (RND) superfamily efflux systems have been widely studied for their role in antibiotic resistance, but their native biological functions remain poorly understood. We previously showed that loss of RND-mediated efflux in Vibrio cholerae resulted in activation of the Cpx two-component regulatory system, which mediates adaptation to stress resulting from misfolded membrane proteins. Here, we investigated the mechanism linking RND-mediated efflux to the Cpx response. We performed transposon mutagenesis screening of RND-deficient V. cholerae to identify Cpx suppressors. Suppressor mutations mapped to genes involved in the biosynthesis of the catechol siderophore vibriobactin. We subsequently demonstrated that vibriobactin secretion is impaired in mutants lacking the VexGH RND efflux system and that impaired vibriobactin secretion is responsible for Cpx system activation, suggesting that VexGH secretes vibriobactin. This conclusion was bolstered by results showing that vexGH expression is induced by iron limitation and that vexH -deficient cells exhibit reduced fitness during growth under iron-limiting conditions. Our results support a model where VexGH contributes to cellular homeostasis by effluxing vibriobactin. In the absence of vexGH , retained vibriobactin appears to chelate iron from iron-rich components of the respiratory chain, with the deferrated proteins functioning to activate the Cpx response. Our collective results demonstrate that a native function of the V. cholerae VexGH RND efflux system is in vibriobactin secretion and that vibriobactin efflux is critical for maintenance of cellular homeostasis. IMPORTANCE RND efflux systems are ubiquitous Gram-negative transporters that play critical roles in antimicrobial resistance. In addition to antimicrobial resistance, RND transporters also affect the expression of diverse phenotypes, including virulence, cell metabolism, and stress responses. The latter observations suggest that RND transporters fulfill unknown physiological functions in the cell independently of their role in antimicrobial resistance. Vibrio cholerae is representative of many Gram-negative bacteria in encoding multiple RND transporters that are redundant in antimicrobial resistance and affect multiple phenotypes. Here we describe a novel function of the V. cholerae VexGH RND transporter in vibriobactin secretion. We show that vibriobactin production in VexGH-deficient cells impacts cell homeostasis, leading to activation of the Cpx stress response and reduced fitness under iron-limiting conditions. Our results highlight a native physiological function of an RND transporter and provide insight into the selective forces that maintain what was thought to be a redundant multidrug transporter.