Dataset Information

Evaluation of Immunogenicity, Protective Immunity on Aquaculture Pathogenic Vibrio and Fermentation of Vibrio alginolyticus Flagellin FlaC Protein.

ABSTRACT: Background:Vibrio are the main pathogenic bacteria in aquaculture. The flagellin protein C (FlaC) of Vibrio alginolyticus has good immunogenicity and the prospect of potential application in a vaccine. Objectives:We aimed to evaluate the immunogenicity, protective immunity, and prokaryotic expression fermentation of V. alginolyticus FlaC protein for the vaccine in aquaculture. Material and Methods:A molecular cloning method was used to construct the expression strain of FlaC protein, and the protein was purified with Ni-affinity chromatography. Polyclonal antiserum was prepared via mice immunized with the FlaC protein. The Western blot and enzyme-linked immunosorbent assay (ELISA) were used to check the specificity and titre of the antiserum. ELISA and pull-down assay detected the interaction between FlaC protein antiserum and Vibrio. The immune protection function of FlaC protein was detected with mice actively immunized with FlaC protein and challenged by V. alginolyticus and V. parahaemolyticus. The optimal expression conditions for FlaC protein were detected using an L9(34) orthogonal design model. Results:The expression strain of FlaC protein was obtained successfully, and purified FlaC protein was prepared using a mice polyclonal antibody. The FlaC protein antiserum held a high specificity, and the titre was 13200. The antiserum directly interacted with V. alginolyticus and V. parahaemolyticus, and the FlaC protein demonstrated a significant immune protection function (50%) against V. alginolyticus infection and some immune protection function (41.66%) against V. parahaemolyticus. The optimal expression conditions for FlaC protein included a strain OD600 value of 0.8, final isopropyl-?-d-thiogalactoside (IPTG) concentration of 0.1 mmol/L, an inducing time of 8 hours, and an inducing temperature of 28°C. Conclusions:This study showed that the FlaC protein possesses a significant immunogenicity and immune protection effect and obtained the optimal fermentation conditions. It is expected to be a potential vaccine against V. alginolyticus and V. parahaemolyticus.

SUBMITTER: Chen C

PROVIDER: S-EPMC7080974 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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Evaluation of Immunogenicity, Protective Immunity on Aquaculture Pathogenic <i>Vibrio</i> and Fermentation of <i>Vibrio alginolyticus</i> Flagellin FlaC Protein.

Chen Chen C Kang Chao C Rong Na N Wu Nana N Chen Chunlin C Wu Sanqiao S Zhang Xiaoying X Liu Xiang X

Iranian journal of biotechnology 20190901 3

<h4>Background</h4><i>Vibrio</i> are the main pathogenic bacteria in aquaculture. The flagellin protein C (FlaC) of <i>Vibrio alginolyticus</i> has good immunogenicity and the prospect of potential application in a vaccine.<h4>Objectives</h4>We aimed to evaluate the immunogenicity, protective immunity, and prokaryotic expression fermentation of <i>V. alginolyticus</i> FlaC protein for the vaccine in aquaculture.<h4>Material and methods</h4>A molecular cloning method was used to construct the exp ...[more]

PMID: 32195288

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Project description:In order to develop more effective immunological strategies to prevent vibriosis of farmed marine fish in Hong Kong and southern China, various vaccine preparations including formalin-, phenol-, chloroform- and heat-killed whole cell bacterins and subcellular lipopolysaccharides (LPS), as well as different administration routes, were investigated. Fish immunized with the subcellular LPS exhibited the best protection [Relative Percent of Survival (RPS) = 100], while fish immunized with whole cell bacterins displayed varying degrees of protection (RPS ranged from 28 to 80), in descending order: formalin-killed > phenol-killed > heat-killed > chloroform-killed bacterins. Regarding various administration routes, fish immunized with two intraperitoneal (i.p.) injections exhibited the best protection, and the RPS values were 100 or 85 upon higher or lower doses of pathogenic V. alginolyticus challenges. Both oral vaccination and a combination of injection/immersion trial were also effective, which achieved relatively high protection (the RPS values ranged from 45 to 64.3). However, two hyperosmotic immersions could not confer satisfactory protection, especially when fish were exposed to the severe pathogenic bacteria challenge. Marked elevations of serum agglutinating antibody titer were detected in all immunized fish. Macrophage phagocytosis was enhanced significantly, especially in the fish immunized by formalin- and phenol-killed bacterins through various administration routes. Both adaptive (specific antibody) and innate (phagocytic activity) immunity elicited by different immunization strategies were in parallel with the degree of protection offered by each of them. Although all vaccination trials had no significant effect on the serum hematocrit and hemoglobin levels, the circulating lymphocyte counts were significantly elevated in the fish immunized with LPS, formalin- and phenol-killed bacterins. Serum cortisol levels appeared to be reduced in all immunized fish except the trial of hyperosmotic immersion, which indicated the stressful impact on vaccinated fish.

Project description:The pathogenic bacterium Vibrio vulnificus exhibits the ability to form biofilm, for which initiation is dependent upon swimming motility by virtue of a polar flagellum. The filament of its flagellum is composed of multiple flagellin subunits, FlaA, -B, -C, and -D. In V. vulnificus genomes, however, open reading frames (ORFs) annotated by FlaE and -F are also present. Although neither FlaE nor FlaF is involved in filament formation and cellular motility, they are well expressed and secreted to the extracellular milieu through the secretion apparatus for flagellar assembly. In the extrapolymeric matrix of V. vulnificus biofilm, significant levels of FlaEF were detected. Mutants defective in both flaE and flaF formed significantly decreased biofilms compared to the wild-type biofilm. Thus, the potential role of FlaEF during the biofilm-forming process was investigated by exogenous addition of recombinant FlaEF (rFlaEF) to the biofilm assays. The added rFlaE and rFlaF were predominantly incorporated into the biofilm matrix formed by the wild type. However, biofilms formed by a mutant defective in exopolysaccharide (EPS) biosynthesis were not affected by added FlaEF. These results raised a possibility that FlaEF specifically interact with EPS within the biofilm matrix. In vitro pulldown assays using His-tagged rFlaEF or rFlaC revealed the specific binding of EPS to rFlaEF but not to rFlaC. Taken together, our results demonstrate that V. vulnificus FlaEF, flagellin-homologous proteins (FHPs), are crucial for biofilm formation by directly interacting with the essential determinant for biofilm maturation, EPS. Further analyses performed with other pathogenic Vibrio species demonstrated both the presence of FHPs and their important role in biofilm formation.IMPORTANCE Flagellar filaments of the pathogenic Vibrio species, including V. vulnificus, V. parahaemolyticus, and V. cholerae, are composed of multiple flagellin subunits. In their genomes, however, there are higher numbers of the ORFs encoding flagellin-like proteins than the numbers of flagellin subunits required for filament assembly. Since these flagellin-homologous proteins (FHPs) are well expressed and excreted to environments via a flagellin transport channel, their extracellular role in the pathogenic Vibrio has been enigmatic. Their biological significance, which is not related with flagellar functions, has been revealed to be in maturation of biofilm structures. Among various components of the extracellular polymeric matrix produced in the V. vulnificus biofilms, the exopolysaccharides (EPS) are dominant constituents and crucial in maturation of biofilms. The enhancing role of the V. vulnificus FHPs in biofilm formation requires the presence of EPS, as indicated by highly specific interactions among two FHPs and three EPS.

Dataset Information

Evaluation of Immunogenicity, Protective Immunity on Aquaculture Pathogenic Vibrio and Fermentation of Vibrio alginolyticus Flagellin FlaC Protein.

Publications

Evaluation of Immunogenicity, Protective Immunity on Aquaculture Pathogenic <i>Vibrio</i> and Fermentation of <i>Vibrio alginolyticus</i> Flagellin FlaC Protein.

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