Functional Analysis Of Sialic Acid Cetabolism System Of Vibrio Mimicus That Isolated From Catfish

Vibrio mimicus is a bacterium that infects both humans and aquatic animals.The V.mimicus has a close relationship with V.cholerae and were thought to be a biochemically atypical V.cholerae.V.mimicus SCCF01 is a highly pathogenic strain that isolated from catfish.By genomic analysing,we found that the chromosome 2 of V.mimicus SCCF01encodes a full-length sialic acid catabolism system(gene location:C2?100484?C2?112247).The sialic acid catabolism system is the virulence gene that has been proved play an important role in bacterial growth,adhesion,capsular formation,biofilm formation and pathogenicity of V.cholerae,B.fragilis,H.influenzae and P.gingivalis,etc.In order to figure out the relationship between this catabolism system and bacterial phenotypic characteristics of V.mimicus SCCF01,this study used natural transformation technology and successfully constructed a sialic acid catabolic system deficiency strain?PI-SCCF01.There was no significant chage between wt-SCCF01 and?PI-SCCF01 when growing in the LB medium,while?PI-SCCF01 growing barely satisfactory in DMEM medium which is lacking protein components.Sialic acid catabolism do affect the cell and colony morphology.Average cell length of?PI-SCCF01 was 1.42 times longer than that of wt-SCCF01,and the colonies of?PI-SCCF01 formed on LB and TCBS agar medium are both smaller than that of wt-SCCF01.Using 2E+08 CFU wt-SCCF01 and?PI-SCCF01 to incubate EPC cells for 30 minutes showed that the adhesion ability of?PI-SCCF01 was enhanced by 3-6 times than that of wt-SCCF01.There was no obvious change in biochemical characteristics and aggregation ability between two strains.In order to investigate the pathogenicity of the two strains,different concentrations of?PI-SCCF01 and Wt-SCCF01were intraperitoneally injected to the zebrafish and hybrid catfish.The LD₅₀ of?PI-SCCF01 and Wt-SCCF01 infecting zebrafish were 3.69×10⁵ and 1.86×10⁴,respectively,which means the pathogenicity of?PI-SCCF01 was decreased by 19.84 times;the LD₅₀ of?PI-SCCF01 and Wt-SCCF01 infecting hybrid catfish were 1.21×10⁵and 4.47×10³,respectively,which means the pathogenicity of?PI-SCCF01 was decreased by 26.99 times.Those results showed that the sialic acid catabolism system mediates pathogenisity of V.mimicus SCCF01.Using Lable-free technology to quantitative and qualitative analyse the secretome of?PI-SCCF01 and wt-SCCF01,total of 1480 proteins were identified,in which 212 proteins were significantly up-regulated and 191 proteins were significantly down-regulated.?PI-SCCF01 cannot secret at least 32 proteins compared with wt-SCCF01,which includs sialidase,N-acetylneuraminic lyase,potential virulence factor neutral metalloproteinase,bacterial flagella system components(Flg C,Flg F,Flg J,Flg G)and bacterial IX secretion system components Por P/Spr F,etc.Those results indicate that the sialic acid catabolism system take part in the regulation of proteins secreting and the significantly changed proteins may be the key virulence factors for V.mimicus SCCF01 to become a highly pathogenic strain which deserve further verification.