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

Abstract

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 Gramnegative 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. Vibrio cholerae is a Gram-negative bacterium and the causative agent of the lifethreatening diarrheal disease cholera. V. cholerae is a native inhabitant of aquatic environments from which humans acquire cholera through the ingestion of V. choleraecontaminated food or wa...

A Bacterial Stress Response Regulates Respiratory Protein Complexes To Control Envelope Stress Adaptation

“…In this regard, it is known that electron flow leads to the generation of damaging reactive oxygen species (48), which could theoretically result in damage to respiratory complexes or other protein assemblies and a Cpx-inducing signal. In agreement with this model, Bina and coworkers recently showed in Vibrio cholerae that the inability to efflux the siderophore vibriobactin, as well as the oxidative-stress-inducing agent paraquat, resulted in Cpx pathway induction in an oxygendependent manner (49). Additionally, Chao and Vogel (50) demonstrated that the Cpx response is activated by the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) in Salmonella, suggesting that disruption of the proton-pumping activity associated with these complexes could also potentially be the source of a Cpx-activating signal.…”

Proteomic profiling of integral membrane proteins associated to pathogenicity in Vibrio parahaemolyticus strains

“…Transport proteins that showed differential accumulation included anaerobic C4‐dicarboxylate transporter‐DcuC, which participates in the succinate flow system during anaerobic fermentation of glucose ; efflux RND‐transporter periplasmic adaptor subunit, which carries factors of resistance, nodulation and cell division ; long‐chain fatty acid transporter, which drives mobilization of long chain fatty acids, either outside or inside the cell, or between cells ; MFS transporter, which facilitates transport of ions, sugar phosphates, drugs, neurotransmitters, nucleosides, amino acids and peptides ; multidrug efflux RND‐transporter permease subunit, which is responsible for transporting cadmium, zinc and cobalt ; and multidrug transporter‐AcrB, which cooperates with a membrane fusion protein, AcrA, and a TolC outer membrane channel .…”

Vibrio responses to extracytoplasmic stress

“…Vibriobactin, a siderophore, is produced by V. cholerae and is secreted out of the cell to bind iron and bring it back to the cytoplasm through the RND system (Griffiths et al ., ). In the absence of RND efflux, vibriobactin is unable to exit the cell and chelates iron within the cell to generate an activation signal for Cpx (Kunkle et al ., ). While the role of Cpx in iron scavenging has only been examined under virulence inducing conditions (Taylor et al ., ), it would be interesting to determine if the Cpx response is induced when Vibrio colonizes aquatic hosts and/or in seawater.…”