Pathogenic function of the Lafa gene in Vibrio harveyi and its regulatory role in host immune response during infection of large yellow croaker (Larimichthys crocea)

Vibrio harveyi, a ubiquitous pathogen in aquaculture, poses significant threats to numerous farmed aquatic species. Previous studies have demonstrated that bacterial flagella play a crucial role in adhesion and infection mechanisms, with the lateral flagellin lafA identified as a key virulence factor. To investigate the functional role of lafA in V. harveyi, this study constructed a gene deletion mutant (ΔlafA) from the wild-type strain LcV6 (Wt LcV6) and characterized its biological properties. Concurrently, transcriptomic analyses were conducted to identify expression differences between Wt LcV6 and ΔlafA. Additionally, the protective efficacy of ΔlafA was evaluated through intraperitoneal vaccination of large yellow croaker (Larimichthys crocea). Key findings revealed that ΔlafA exhibited no significant changes in growth kinetics compared to Wt LcV6 but showed enhanced biofilm formation capacity. Notably, the mutant strain displayed a marked reduction in motility. Transcriptomic analysis revealed a multifaceted bacterial adaptation strategy: significant upregulation of flagellar assembly pathway genes (flhB, FliK, lafS, fliF, flhA, and flaE) alongside lafA downregulation compensated for lateral flagella loss and maintained environmental adaptability, while sulfur-related genes exhibited marked divergence, with dsrC and AsrA being significantly upregulated, whereas cysI, cysD, cysC, cysH, and cysN were downregulated (P < 0.05). Furthermore, vaccination with ΔlafA conferred a relative protection rate of 63.3 % against Wt LcV6 infection in large yellow croaker. The mutant also significantly increased specific IgM antibody titers and enhanced the enzymatic activities of catalase (CAT), alkaline phosphatase (AKP), and superoxide dismutase (SOD) in the livers of immunized fish. Moreover, it upregulated the expression of multiple immune-related genes, including IgM, IgD, TGF-β1, IL-1β, TNF-α, MHC IIα, CD4, and CD8 in the livers, spleen, and kidney tissues of immunized fish. Collectively, these results underscore lafA as an essential virulence factor in V. harveyi. The ΔlafA mutant not only enhances immune responses in vaccinated fish but also represents a promising candidate for vaccine development, providing novel insights into the design of attenuated vaccines targeting V. harveyi.