The sRNA vvrr1 Modulates the VarS/VarA‐Csr System via LeuO to Regulate Biofilm Formation and Virulence in Vibrio alginolyticus

Abstract

Biofilm formation is a pivotal virulence trait in Vibrio alginolyticus , providing protection against antimicrobial agents and host defenses. However, the regulatory mechanisms governing biofilm development remain incompletely elucidated. This study uncovers a novel regulatory circuit in which the small RNA vvrr1 governs biofilm formation and pathogenicity by modulating the transcriptional regulator LeuO, which in turn coordinates the VarS/VarA two‐component system and the downstream Csr cascade. We demonstrate that vvrr1 post‐transcriptionally represses LeuO by targeting leuO mRNA and accelerating its decay. The interaction interface is evolutionarily conserved among pathogenic Vibrio species, highlighting its functional importance. Phenotypic analyses established LeuO as a positive regulator of both biofilm formation and virulence. Genome‐wide profiling of LeuO binding sites revealed its direct repression of varS , which encodes the sensor kinase of the VarS/VarA system. This repression initiates a regulatory cascade: Reduced VarS/VarA activity alleviates repression of the Csr sRNAs ( csrB , csrC , and csrD ), leading to sequestration of the global regulator CsrA and subsequent derepression of the quorum‐sensing regulator LuxO. This coordinated regulation ultimately modulates exopolysaccharide biosynthesis, shaping biofilm architecture and virulence. By employing an integrated approach combining molecular simulations, genetic manipulations, and high‐throughput sequencing, we define a multilayered regulatory network centered on the vvrr1 –LeuO axis. Our findings provide mechanistic insight into the sophisticated control of biofilm‐mediated virulence in a major aquaculture pathogen and identify potential targets for anti‐biofilm strategies.