Roles of virulence regulator ToxR in viable but non-culturable formation by controlling reactive oxygen species resistance in pathogen Vibrio alginolyticus

Abstract

Under adverse circumstances, bacteria enter the viable but non-culturable (VBNC) state, a dormancy-like state for survival. The altered gene regulation underlying the entry of the VBNC state has not yet been well elucidated. Here, we reported that a subpopulation of cells (23.8 %) in Vibrio alginolyticus cultures enters the VBNC state in response to nutrient limitation at alkaline pH. The proteolysis of pivotal virulence regulator ToxR at these conditions is associated with VBNC formation. Meantime, ToxR abrogation impaired the mobility and the expression of virulence-associated genes, resulting in attenuated virulence in V. alginolyticus. RNA-seq and ChIP-seq analyses of the cells grown in VBNC-inducing conditions revealed that ToxR directly controls the expression of ∼8 genes including ahpC and dps involved in reactive oxygen species (ROS) resistance. ToxR binds to the promoter regions of kdgR, ppiC, ahpC, and dps and further controls their respective expression under oxidative stress conditions. The cells with impaired ToxR accumulated detrimental intracellular ROS. Moreover, these genes contribute to bacterial culturability as their in-frame deletion strains exhibiting severely decreased plate counts and the complementary strain showed rescued viability. Collectively, this study revealed the role of ToxR in switching on the VBNC state by sensing unfavorable environmental signals such as endogenous ROS (hydrogen peroxide, H2O2) in V. alginolyticus and provided mechanistic insights into Vibrio lifestyle adaptation in the marine environment.