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Vibrio alginolyticus is the common pathogen affecting various species of marine organisms. It has been demonstrated that fliR is a necessary virulence factor to adhere and infect their hosts for pathogenic bacteria. Frequent disease outbreaks in aquaculture have highlighted the necessity of developing effective vaccines. In the present study, in order to investigate the function of fliR in V.alginolyticus, the fliR deletion mutant ΔfliR was constructed and its biological properties were evaluated, additionally, the differences in gene expression levels between wild-type and ΔfliR were analyzed by transcriptomics. Finally, ΔfliR was used as a live attenuated vaccine to immunize grouper via the intraperitoneal route to evaluate its protective effect. Results show that fliR gene of V. alginolyticus was identified as being 783 bp in length, encoding 260 amino acids, and showing significant similarity to homologs of other Vibrio species. The fliR-deletion mutant ΔfliR of V. alginolyticus was successfully constructed, and its biological phenotype analysis showed no significant differences in growth capacity and extracellular enzyme activity compared to the wild-type. However, a substantial reduction of motility ability was detected in ΔfliR. Transcriptomic analysis revealed that the absence of fliR gene is responsible for a significantly decreased expression of flagellar genes, including flaA, flaB, fliS, flhB and fliM. The fliR-deletion mainly affects the related pathways involved in cell motility, membrane transport, signal transduction, carbohydrate metabolism, and amino acid metabolism in V. alginolyticus. The efficacy of ΔfliR as a candidate of live attenuated vaccine were evaluated by intraperitoneal injection in grouper. The ΔfliR provided the RPS (Relative protection rate) of 67.2% against V. alginolyticus in groupers. The ΔfliR efficiently stimulated antibody production with specific IgM still detected at 42 d post-vaccination, and significantly elevated the activity of antioxidant enzymes like Catalase (CAT), Superoxide dismutase (SOD), and lactate dehydrogenase (LDH) in the serum. The higher expression levels of immune-related genes were observed in the immune tissues of inoculated grouper compared to the control. In conclusion, ΔfliR effectively improved the immunity of inoculated fish. The results suggest that ΔfliR is an effective live attenuated vaccine against vibriosis in in grouper.

Pearl gentian grouper (♀Epinephelus fuscoguttatus × ♂E. lanceolatus) is a marine fish with high nutritional value. At present, it has become one of the important reared species in aquaculture of China for its tender meat and high unsaturated fatty acids content. However, with the continuous improvement of intensive cultivation of marine aquaculture in recent years, outbreaks of vibriosis are more and more frequent and cause serious economic loss (Chen et al., 2019; Deng et al., 2020). Vibrio harveyi, the main causative agent of vibriosis, caused high mortality in pearl gentian grouper culture industry in China. In the past decades, antibiotics were used to control V. harveyi because of their good antibacterial efficiency. However, antibiotics abuse leads to the rapid increasing of drug-resistant bacterial strains and the emergence of drug residues (Wei et al., 2020a(Wei et al., , 2020b. In recent years, vaccines were found to be effective against pathogens challenge in fish. The inactivated vaccine, attenuated live vaccine, subunit vaccine and DNA vaccine have been used to control bacteria and virus infection in aquaculture. However, due to different vaccination administration rates or antigen types, poor antigen

Vibrio alginolyticus (V. alginolyticus) is one of the important epizootic pathogens in marine animals. VAGM001033 belongs to a diguanylate cyclase, responsible for the synthesis of dimeric guanosine monophosphate (c-di-GMP), a ubiquitous second messenger involved in the function of biofilm formation, motility, and virulence. This study confirmed that VAGM001033 was an active diguanylate cyclase by Congo red assay. The red-stained, dry, and rough form of colonies were observed with the increasing concentration of the L-arabinose on Congo red plates. Also, an in-frame deleted ΔVAGM001033 mutant was constructed and changes of ΔVAGM001033 mutant in physiology and pathogenicity were detected. The ΔVAGM001033 mutant displayed similar morphology and growth curve with the wild-type strain showing no significant differences. The swarming ability of the ΔVAGM001033 mutant was significantly enhanced showing bigger swarming circles, while the biofilm formation, extracellular proteases, and virulence were significantly attenuated. The results of the test for antibiotic susceptibility showed that the wild type and ΔVAGM001033 mutant had similar sensitivity or resistance to most antibiotics used in this study, except cefotaxime and nitrofurantoin. The mutant was sensitive to cefotaxime and nitrofurantoin, while the wild type was intermediate. A total of 756 differentially expressed genes (DEGs) were identified by RNA-seq, of which 109 were upregulated and 647 were downregulated. Flagellar assembly, two-component system, ATP-binding cassette (ABC) transporters, and peptidoglycan biosynthesis were significantly enriched in the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway. Moreover, the ΔVAGM001033 mutant induced high antibody titers and provided immune protectivity with a relative percent survival (RPS) of 82%. Immune-related genes of pearl gentian grouper (♀Epinephelus fuscoguttatus × ♂ Epinephelus lanceolatus), namely, IgM, MHC-Iα, interleukin-1β (IL-1β), interleukin-16 (IL-16), and tumor necrosis factor-α (TNF-α) were upregulated after vaccination. Overall, the results suggested that VAGM001033 plays a crucial role in V. alginolyticus. The ΔVAGM001033 mutant might be applied as an effective live vaccine candidate against V. alginolyticus.