The aquatic pathogen Aeromonas salmonicida SRW-OG1 is responsible for substantial mortality in Epinephelus coioides and many other cultured fish species. This study investigates the contribution of trpE to the pathogenicity of A. salmonicida SRW-OG1 in E. coioides. Specifically, trpE encodes a key enzyme in tryptophan biosynthesis; however, its function in regulating virulence in A. salmonicida remains uncertain. Phenotypic assays indicate that deletion of trpE markedly impaired bacterial growth and significantly reduced multiple virulence-associated traits, including swimming motility, biofilm formation, chemotaxis, hemolytic activity, and extracellular enzyme activity. Transmission electron microscopy revealed that the ΔtrpE mutant lacked flagella, indicating that trpE is involved in flagellar formation or maintenance. In an E. coioides infection model, fish infected with ΔtrpE showed a cumulative survival rate of 48.3%, whereas those infected with the wild-type SRW-OG1 suffered 100% mortality. The ΔtrpE mutant also exhibited reduced bacterial loads in target organs (liver, head kidney, and spleen), while pretreatment with ΔtrpE provided 53.3% protection against a subsequent wild-type challenge. Transcriptomic analysis identified 616 differentially expressed genes, mainly enriched in pathways including flagellar assembly, bacterial chemotaxis, two-component systems, quorum sensing, and biofilm formation. Metabolomic analysis further showed a significant decrease in N-butyryl-L-homoserine lactone, an acyl-homoserine lactone quorum-sensing signal in the ΔtrpE mutant. These findings suggest that trpE contributes to mesophilic A. salmonicida SRW-OG1’s virulence by partially affecting quorum-sensing signal production and motility-related pathways. The attenuated phenotype and protective effect of ΔtrpE indicate its potential as a live attenuated vaccine candidate for controlling A. salmonicida infection in aquaculture.

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.