Clonning And Primary Functional Analysis Of T6SS Cluster Of Vibrio Anguillarum M3 Strain

Bacteria T6SS(TypeⅥSecretion System) is a novel protein secretion pathway which reported recently. Homologous gene of T6SS was found according to Fosmid library of partial genome information of Vibrio anguillarum M3 strain. T6SS gene cluster was obtained by homologous clone and genomic walking. The base length is 22879 and encoding 16 ORF.GC contents is 46.93%. Homeology of gene sequence is 87% compared with Vibrio cholerae. Pairs of primers were designed according to the sequence that locates on region stepping across two coutinous genes. These primers were used to assure whether transcription of M3 T6SS is controlled by one operon. A promoter was found in the upstream region of gene vaa108.To identify functions of Vibrio anguillarum T6SS genes. Seven mutation strains were constructed according to the method of In-frame-deletion. We studied the differences of growth condition, swimming motility and extracellular enzyme activity between wild type and mutations. Growth variance only occured in exponential phase. Compared to the wild type, four of seven mutation strains showed obviously growth acceleration (p<0.05),one was slower and two displayed no difference. Three mutation strains showed obviously difference in swimming motility compared to wild type(p<0.05). Colony was resuspended by physiological saline and enzymes activity was tested using API ZYM kits. Only the acitivity ofα-glucoamylase of lanH mutation strain seems to be different among the samples. Result of bacterial virulence chanllege on Trichogaster trichopterus showed that LD50 of four mutation strains were similar to wild type strain. Three mutation strains'virulence were decreasing and LD50 was 37~223 multiple of wild type.Biofilm formation was downregulated by vaa117 gene in Vibrio anguillarum M3 strain. Obviously different of biofilm formation occoured in 48h. Moreover, the biofilm formation of D117 was faster and higher than wild type. RT-PCR and Fluorescence quantitative PCR was applied to find regulation function of vaa117. In vaa117 mutation strain strains the expression of vaa017 (hcp) gene was significant down, compared to the wild type.The result indicate that vaa117 regulate the expression of vaa017.This research proves that Vibrio anguillarum M3 strain possesses T6SS firstly. This secretion system will affect the growth, swimming motility and extracellular enzyme activity of Vibrio anguillarum M3 strain. Gene vaa117 regulates the expression of vaa017.To sum up, Vibrio anguillarum is opportunistic pathogen. It spreads over the whole world oceans and causes great loss to the world aquaculture industry when it breaks out. T6SS is found in large amount of known pathogens and associating with virulence, interaction with host. The mechanisms of T6SS and functions of secreted factors are still uncovered. This research gets some basic information of Vibrio anguillarum M3 strain T6SS on purpose of further research and vaccine production.