The CpxR–GlvR regulatory axis of Edwardsiella piscicida: a novel virulence regulatory mechanism integrating glycerol metabolism and oxidative stress adaptation

Abstract

Edwardsiella piscicida is a devastating bacterial pathogen in aquaculture, and in-depth elucidation of its pathogenic mechanisms is essential for establishing effective control strategies; the identification of novel regulatory factors can deepen our understanding of bacterial pathogenesis. Our previous work identified the functionally uncharacterized protein ETAE_2516 as a potential target of the response regulator CpxR of the CpxRA two-component system, a key virulence regulator in this pathogen. Bioinformatic analysis and functional prediction indicated that ETAE_2516 is an AraC-family transcription factor that is potentially involved in glycerol metabolism; it was therefore designated GlvR (Glycerol-linked Virulence Regulator). In this study, an in-frame glvR deletion mutant (Δ glvR ) and its complemented strain (Δ glvR C) were constructed by gene knockout, and their phenotypes in metabolism, stress resistance, biofilm formation, and pathogenicity were systematically characterized. Deletion of glvR did not affect basal bacterial growth but significantly increased sensitivity to oxidative stress and led to intracellular accumulation of reactive oxygen species. The Δ glvR mutant exhibited markedly impaired growth in glycerol-supplemented medium, and the expression of glycerol metabolism-associated genes was significantly downregulated, confirming that GlvR positively regulates glycerol metabolism. Loss of GlvR also repressed the expression of flhC , the master regulator of the flagellar regulon, thereby impairing bacterial motility. Infection assays using tilapia epithelial (TSE-04) cells, RAW 264.7 macrophages, and a tilapia ( Oreochromis niloticus ) infection model demonstrated that GlvR is essential for bacterial adhesion to host epithelial cells, resistance to host serum killing, intracellular survival within macrophages, and tissue colonization, and all of these defects were restored to wild-type levels in the complemented strain, verifying that they are specifically attributable to the loss of glvR . Analysis of the upstream regulatory mechanism further revealed that CpxR binds directly to the glvR promoter region and positively activates its transcription. Collectively, these findings demonstrate that the CpxR–GlvR regulatory axis functions as a central hub integrating carbon metabolism, oxidative stress adaptation, and virulence expression, thereby providing a novel perspective for understanding the host adaptation mechanisms of E. piscicida and a potential target for anti-virulence strategies against edwardsiellosis.