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Vibrio alginolyticus, a bacterial pathogen in fish and humans, expresses a type III secretion system (T3SS) that is critical for pathogen virulence and disease development. In this study, the T3SS chaperone protein gene vscB was cloned from V. alginolyticus wild-type strain HY9901and the vscB gene has a length of 429 bp and encodes 142 amino acids. The amino acid sequence homology comparison results showed that VscB had the highest similarity with that of Vibrio parahaemolyticus VscB, reaching 91%. The mutant strain HY9901ΔvscB was constructed by the in-frame deletion method. The HY9901ΔvscB showed an attenuated swarming phenotype and a 23-fold decrease in virulence to pearl gentian grouper (♀Epinephelus fuscoguttatus × ♂Epinephelus lanceolatu). However, the HY9901ΔvscB showed no difference in morphology, growth, biofilm formation and extracellular protease (ECPase) activity. The results of antibiotic susceptibility testing showed that the wild-type HY9901 is more sensitive to Doxycycline, Minocyline and Kanamycin than in the HY9901ΔvscB. A total of 95 differentially expressed genes were screened by transcriptome sequencing analysis of HY9901 and strain ΔvscB, revealing 57 genes up-regulated and 38 genes were down-regulated in the ΔvscB compared to HY9901. qRT-PCR was employed to analyze the transcription levels of T3SS-related genes showing that ΔvscB had decreased expression of vopN and vscO and increased expression of hop, vscN, vscK, vscL and vopS compared to HY9901. Finally, grouper vaccinated via intraperitoneal (IP) injection with HY9901ΔvscB had a relative percent survival (RPS) of 77.6% following challenge with HY9901. Real-time qPCR assays showed that vaccination with HY9901ΔvscB enhanced the expression of immune-related genes, including MHC-I, IgM, and CD8α both in the liver and spleen, indicating that HY9901ΔvscB is able to induce humoral and cell-mediated immune responses in pearl gentian grouper. These results demonstrate that the HY9901ΔvscB mutant could be used as an effective live attenuated vaccine to combat V. alginolyticus infection in pearl gentian grouper.

The survival and immune responses of Litopenaeus vannamei were evaluated during white spot syndrome virus (WSSV) or Vibrio parahaemolyticus single and concurrent infections. The mortality, WSSV load, activities of 4 immune enzymes: acid phosphatase (ACP), alkaline phosphatase (AKP), peroxidase (POD) and superoxide dismutase (SOD), and the transcription of Evolutionarily Conserved Signaling Intermediate in Toll pathways of L.vannamei (LvECSIT) were quantified at 0, 3, 6, 12, 24, 48, 72 and 96 h post-infection (pi). The results showed: (i) the cumulative mortality of the co-infection group (WSSV and V. Parahaemolyticus 83%) was significantly lower than the WSSV infection group (97%) (P < 0.05) at 96 hpi; (ii) copies of WSSV in the co-infection group were significantly lower than that of the single infection group from 24 to 96 hpi (P < 0.05); (iii) ACP, AKP,POD and SOD activity in the gills of the co-infection group was higher than that of the WSSV group at12, 48 and 96 hpi (P < 0.05).The expression of LvECSIT mRNA in the co-infection group was significantly higher than in the WSSV infection group from 12 to 72 hpi (P < 0.05).The results indicate that proliferation of WSSV is inhibited by V.parahaemolyticus infection. In addition, infection with WSSV alone causes a significant reduction in some immune responses of shrimp than co-infection with WSSV and V.parahaemolyticus occurs at 26 °C. Third, LvECSIT, an essential member of TLR signaling pathway might play a crucial role in shrimp defense against WSSV – Vibrio co-infection.

Vibrio alginolyticus is a major cause of Vibriosis in farmed marine aquatic animals and has caused large economic losses to the Asian aquaculture industry in recent years. Therefore, it is necessary to control V. alginolyticus effectively. The virulence mechanism of V. alginolyticus, the Type III secretion system (T3SS), is closely related to its pathogenicity. In this study, the T3SS gene tyeA was cloned from V. alginolyticus wild-type strain HY9901 and the results showed that the deduced amino acid sequence of V. alginolyticus tyeA shared 75-83% homology with other Vibrio spp. The mutant strain HY9901ΔtyeA was constructed by Overlap-PCR and homologous recombination techniques. The HY9901ΔtyeA mutant exhibited an attenuated swarming phenotype and an ~40-fold reduction in virulence to zebrafish. However, the HY9901ΔtyeA mutant showed no difference in growth, biofilm formation and ECPase activity. Antibiotic susceptibility test was observed that wild and mutant strains were extremely susceptible to Amikacin, Minocycline, Gentamicin, Cefperazone; and resistant to oxacillin, clindamycin, ceftazidime. In contrast wild strains are sensitive to tetracycline, chloramphenicol, kanamycin, doxycycline, while mutant strains are resistant to them. qRT-PCR was employed to analyze the transcription levels of T3SS-related genes, the results showed that compared with HY9901 wild type, ΔtyeA had increased expression of vscL, vscK, vscO, vopS, vopN, vscN, and hop. Following vaccination with the mutant strain, zebrafish had significantly higher survival than controls following infection with the wild-type HY9901 (71.2% relative percent survival; RPS). Analysis of immune gene expression by qPCR showed that vaccination with HY9901ΔtyeA increased the expression of IgM, IL-1β, IL-6, and TNF-α in zebrafish. This study provides evidence of protective efficacy of a live attenuated vaccine targeting the T3SS of V. alginolyticus which may be facilitated by up-regulated pro-inflammatory and immunoglobulin-related genes.

The present study was conducted to evaluate the effects of marine polysaccharides from seaweed Enteromorpha on growth performance, immune responses, intestinal morphology and microbial community in the banana shrimp Fenneropenaeus merguiensis. Two thousand and four hundred juvenile shrimps with an average body weight of 2.18 ± 0.06 g were fed for 42 d with diets containing different levels of Enteromorpha polysaccharides (EPS): 0 (control), 1, 2 and 3 g/kg as treatment groups, each of group was replicated three times with two hundred shrimps per replicate. Dietary supplementation of 1 g/kg EPS showed a consistent improvement in the final weight, weight gain, average daily gain rate (ADGR) and specific growth rate (SGR) (P

Vibrio alginolyticus, a Gram-negative bacterium, has been recognized as an opportunistic pathogen in marine animals as well as humans. Type III secretion system (T3SS) is critical for pathogen virulence and disease development. However, no more information is known about the C-ring component VscQ and its physiological role. In this study, gene vscQ was cloned from V. alginolyticus wild-type strain HY9901 and the mutant strain HY9901ΔvscQ was constructed by the in-frame deletion method. The HY9901ΔvscQ mutant showed an attenuated swarming phenotype and a closely 4.6-fold decrease in the virulence to Danio rerio. However, the HY9901ΔvscQ mutant showed no difference in growth, biofilm formation and ECPase activity. HY9901ΔvscQ reduces the release of LDH, NO and caspase-3 activity of infected FHM cell, which are involved in fish cell apoptosis. Deletion of gene vscQ downregulates the expression level of T3SS-related genes including vscL, vopB, hop, vscO, vscK, vopD, vcrV and vopS and flagellum-related genes (flaA and fliG). And Danio rerio vaccinated via i.m injection with HY9901ΔvscQ induced a relative percent survival (RPS) value of 71% after challenging with the wild-type HY9901. Real-time PCR assays showed that vaccination with HY9901ΔvscQ enhanced the expression of immune-related genes, including TNF-α, TLR5, IL-6R, IgM and c/ebpβ in liver and spleen after vaccination, indicating that it is able to induce humoral and cell-mediated immune response in zebrafish. These results demonstrate that the HY9901ΔvscQ mutant could be used as an effective live vaccine to combat V. alginolyticus infection.

It is well known that lysine acetylation (Kace) modification is a common post-translational modification (PTM) that plays an important role in multiple biological and pathological functions in bacteria. However, few studies have focused on lysine acetylation modification in aquatic pathogens to date. In this study, the acetylome profiling of fish pathogen, Vibrio alginolyticus was investigated by combining affinity enrichment with LC MS/MS. A total of 2883 acetylation modification sites on 1178 proteins in this pathogen were identified. The Kace modification of several selected proteins were further validated by Co-immunocoprecipitation combined with Western blotting. Bioinformatics analysis showed that seven conserved motifs can be enriched among Kace peptides, and many of them were significantly enriched in metabolic processes such as biosynthesis of secondary metabolites, microbial metabolism in diverse environments, and biosynthesis of amino acids, which was similar to data previously published for V. parahaemolyticus. Moreover, we found at least 102 acetylation modified proteins predicted as virulence factors, which indicate the important role of PTM on bacterial virulence. In general, our results provide a promising starting point for further investigations of the biological role of lysine acetylation on bacterial virulence in V. alginolyticus. BIOLOGICAL SIGNIFICANCE: Lysine acetylation (Kace) modification, is well known to play important roles on diverse biological functions in prokaryotic cell, whereas few studies focused on aquatic pathogens to date. In this study, the acetylome profiling of fish pathogen, Vibrio alginolyticus was investigated by combining affinity enrichment with LC MS/MS. A total of 2883 acetylation modification sites on 1178 proteins in this pathogen were identified. The further bioinformatics analysis showed that seven conserved motifs can be enriched among Kace peptides, and many of them were significantly enriched in metabolic processes, which was similar to data previously published for V. parahemolyticus. Moreover, we found at least 102 acetylation modified proteins predicted as virulence factors, which indicate the important role of PTM on bacterial virulence. In general, our results provide a promising starting point for further investigations of the biological role of lysine acetylation on bacterial virulence in V. alginolyticus.