RavA-ViaA antibiotic response is linked to Cpx and Zra2 envelope stress systems in Vibrio cholerae

RavA-ViaA were reported to play a role in aminoglycoside (AG) sensitivity, but the mechanisms remain elusive. Here, we performed competition and survival experiments to confirm that deletion of ravA-viaA increases tolerance of the Gram-negative pathogen Vibrio cholerae to low and high AG concentrations during aerobic growth. Using high-throughput strategies in this species, we identify Cpx and Zra2 two-component systems as new partners of RavA-ViaA. We show that the AG tolerance of Delta ravvia requires the presence of these membrane stress sensing two-component systems. We propose that deletion of the RavA-ViaA function facilitates the response AGs because of a pre-activated state of Cpx and Zra2 membrane stress response systems. We also find an impact of these genes on vancomycin resistance, and we show that simultaneous inactivation of ravvia function together with envelope stress response systems leads to outer membrane permeabilization. Vancomycin is mostly used for Gram-positive because of its low efficiency for crossing the Gram-negative outer membrane. Targeting of the ravA-viaA operon for inactivation could be a future strategy to allow uptake of vancomycin into multidrug-resistant Gram-negative bacteria. IMPORTANCE The RavA-ViaA complex was previously found to sensitize Escherichia coli to aminoglycosides (AGs) in anaerobic conditions, but the mechanism is unknown. AGs are antibiotics known for their high efficiency against Gram-negative bacteria. In order to elucidate how the expression of the ravA-viaA genes increases bacterial susceptibility to aminoglycosides, we aimed at identifying partner functions necessary for increased tolerance in the absence of RavA-ViaA, in Vibrio cholerae. We show that membrane stress response systems Cpx and Zra2 are required in the absence of RavA-ViaA, for the tolerance to AGs and for outer membrane integrity. In the absence of these systems, the ravvia strain's membrane becomes permeable to external agents such as the antibiotic vancomycin.