Construction And Biological Characterization Of Vibrio Mimicus Metalloproteinase Gene PrtV Deletion Strain

Vibrio mimicus is commonly found in various aquatic environments.It is an important pathogen of vibriosis in aquatic organisms,and can also infect humans,causing diarrhea and sepsis.PrtV encodes metalloproteinases in the Polycystic Kidney Disease(PKD)domain,it is found in V.cholerae,V.parahaemolyticus,V.anguillarum and V.coralliilyticus,which mediate cytotoxicity and hemolysis.It also plays an important role in the invasion and transfer of bacteria.In the whole genome analysis of SCCF01,a virulent strain of V.mimicus from yellow catfish,PrtV gene,which is 83% similar to the PrtV gene of V.cholerae,was found to exist.In order to reveal its role in the infection and pathogenesis of V.mimicus,the following studies were conducted:Using the wild strain of V.mimicus SCCF01 as the starting strain,4600 bp target fragment containing Kana resistance gene was obtained by overlapping extended PCR,and the recombinant strain SCCF01/p KD46 was constructed by electrical transformation.Then natural transformation combined with λ-Red recombinant enzyme system was used to obtain PrtV gene deletion positive transformants,and single colony with Kana resistance were selected for PCR verification.Finally,the p CP20 plasmid FLP/FRT(flippase recombination enzyme/recognition target)site-specific recombination system was used to eliminate the Kana resistance gene and successfully construct the PrtV gene deletion strain of V.mimicus(ΔPrtV).The full length of 2757 bp PrtV gene was amplified by PCR,and the high-copy recombinant expression plasmid p BAD24 was constructed by one-step homologous recombination.The plasmid was introduced into the deletion strain by electrical transformation and verified by colony PCR,and the gene complement strains(ΔPrtV/p PrtV)were successfully constructed,which laid the foundation for the subsequent gene functional analysis.In order to explore the effect of PrtV gene on the phenotype of V.mimicus,colony morphology,morphological characteristics,growth curve,physiological and biochemical characteristics,biofilm formation and self-aggregation ability of SCCF01 wild strain,deletion strain ΔPrtV and complementation strain ΔPrtV/p PrtV were compared and analyzed.It was found that compared with SCCF01 wild strain and complement strain ΔPrtV/p PrtV,the physicochemical properties of the deletion strain ΔPrtV in the decomposition of glycine,coumarate,ornithine and lysine were changed.There were no significant changes in colony morphology,morphological characteristics,growth characteristics,biofilm formation and self-aggregation ability.In order to reveal the effect of PrtV gene on pathogenic characteristics of V.mimicus,we analyzed the differences of cell adhesion characteristics,hemolytic activity,cytotoxicity,extracellular product enzyme activity,influence on host cytokine expression,pathogenicity and pathological damage of SCCF01 wild strain,deletion strain ΔPrtV and complement strain ΔPrtV/p PrtV.It was found that compared with SCCF01 wild strain ΔPrtV/p PrtV,deletion strain ΔPrtV had no significant difference in adhesion to EPC cells,but the hemolytic activity of EPC and CCK cells was significantly decreased(P<0.05),and the activities of protease and urease were significantly decreased(P<0.05).There was no significant difference in lecithin enzyme activity.ELISA results showed that the expression of TNF-α and IL-8 in CCK cells was significantly lower than that in SCCF01 wild strain and complement strain ΔPrtV/p PrtV after the extracellular product of deletion strain ΔPrtV(P<0.05).The expressions of IL-6,TGF-β1,IL-1α and IL-1β were not significantly different from those of wild strain SCCF01 and wild strain ΔPrtV/p PrtV.In the pathogenicity test of hybrid catfish,the median lethal weight(LD50)of SCCF01 wild strain and deletion strainΔPrtV for hybrid catfish in 14 days was 5.32E+03 CFU/m L and 5.32E+04 CFU/m L,respectively,and the virulence of deletion strain ΔPrtV decreased by 10 times.Masson staining was performed on the skin tissues of hybrid catfish with the concentration of1.00E+04 SCCF01 wild strain and ΔPrtV deletion strain,and it was found that the destruction of skin collagen fibers by the SCCF01 wild strain was more serious,indicating that the metalloproteinase encoded by the PrtV gene of V.mimicus was related to the skin damage caused by infection.In conclusion,PrtV gene plays an important role in the virulence of V.mimicus,mainly mediating virulence by regulating hemolytic,cytotoxicity,enzyme activity and secretion of inflammatory factors.