T2SS Components GspJ, GspL, and GspM Regulate Virulence in Vibrio alginolyticus.

Vibrio alginolyticus is a major aquaculture pathogen whose virulence is largely mediated by the type II secretion system (T2SS). However, the functions of key T2SS components, including gspJ, gspL, and gspM, remain unclear. Earlier research indicated that a Scutellaria baicalensis extract at 8 mg/mL markedly suppressed the growth, motility, and biofilm-forming capacity of V. alginolyticus ND-01 (OD₆₀₀ = 0.5) following 12 h of treatment, with the resulting stress substantially altering the expression of multiple genes, including gspJ, gspL, and gspM. The gspJ-RNAi, gspL-RNAi, and gspM-RNAi strains were constructed using RNAi. Biological functional assays demonstrated that stable silencing of gspJ, gspL, and gspM markedly decreased biofilm formation ability (84.01%, 24.99%, and 78.26%, respectively) and host cell adhesion (73.57%, 75.65%, and 79.61%, respectively); gspJ and gspM silencing reduced motility (54.82% and 57.42%, respectively), whereas gspL silencing enhanced motility by 38.49%, indicating their essential roles in virulence-associated phenotypes. Notably, in vivo infection of Epinephelus coioides revealed that gspL-RNAi significantly attenuated pathogenicity, as evidenced by reduced host mortality (50%) and decreased bacterial colonization in liver and spleen tissues, compared with the wild-type group. Consistently, Label-Free Proteomic Analysis of the gspL-RNAi strain identified 189 differentially expressed proteins in the gspL-silenced strain, with enrichment in two-component signaling, ABC transport systems, and amino acid metabolism. Network analysis further positioned gspL as a central regulatory hub linking T2SS activity to signal transduction, transport, and metabolic pathways. In addition, the expression of gspJ-RNAi, gspL-RNAi, and gspM-RNAi exhibited distinct responses to environmental stresses, including pH and temperature variation. Collectively, our findings highlight that gspJ, gspL, and gspM are critical regulators of virulence in V. alginolyticus, with gspL playing a central role in coordinating T2SS function with global regulatory networks. This study advances our understanding of the pathogenic mechanisms of V. alginolyticus and provides potential targets for the development of anti-virulence aquaculture strategies.