拟态弧菌金属蛋白酶基因PrtV缺失株的构建及生物学特性分析

Construction and biological characterization of Vibrio mimicus’s metalloprotease gene PrtV-deletion strain

【背景】研究发现PrtV基因编码含多囊肾病(polycystic kidney disease)结构域的金属蛋白酶,其在多种细菌的致病过程中具有重要作用。拟态弧菌是一种感染多种水生动物的重要病原菌,PrtV基因在拟态弧菌致病中的作用尚不清楚。【目的】探究PrtV基因对拟态弧菌致病相关生物学特性的影响。【方法】采用自然转化的方法构建拟态弧菌PrtV基因缺失株(ΔPrtV),同时通过基因与质粒重组后电转化导入缺失株构建回补株(ΔPrtV/pPrtV),对突变株的生长特性、生化特征、生物被膜形成、自聚集能力、胞外产物卵磷脂酶和蛋白酶活性,以及致病性和细胞毒性等进行分析。【结果】与野生株相比,缺失株的生长特性、生物被膜形成、自聚集能力和卵磷脂酶活性无变化,但分解尿素、甘氨酸、香豆酸盐、鸟氨酸和赖氨酸的理化特性改变;胞外产物蛋白酶活性显著降低(P<0.05),细胞毒性显著下降(P<0.05),对杂交鲇的致病力下降10倍。【结论】PrtV基因与拟态弧菌的细胞毒性及致病性等多种生物学特性有关。该结果为进一步解析拟态弧菌PrtV基因功能及其致病机制提供了依据。

[Background]Studies have found that PrtV gene encodes a metalloprotease containing polycystic kidney disease(PKD)domains,which plays an important role in the pathogenic process of various bacteria.Vibrio mimicus is one of the important pathogenic bacteria that infects a variety of aquatic animals,but the role of PrtV gene in the pathogenicity of V.mimicus is not clear.[Objective]To explore the effect of PrtV gene on the pathogenicity-related biological characteristics of V.mimicus.[Methods]The natural transformation method was used to construct the PrtV gene-deletion strain(ΔPrtV)of V.mimicus,and the complement strain(ΔPrtV/pPrtV)was constructed by electrotransformation after gene combined with plasmid.The mutant’s biochemical characteristics,biofilm formation,self-aggregation ability,extracellular product protease activity,pathogenicity,and cytotoxicity were analyzed.[Results]As compared with wild stains,the growth,biofilm formation,self-aggregation ability,and lecithinase activity of theΔPrtV were hardly changed,but the physicochemical properties of decomposing urea,glycine,coumarinate,ornithine,and lysine were changed.The extracellular product protease activity and cytotoxicity ofΔPrtV were significantly decreased(P<0.05),and the pathogenicity to hybrid catfish was decreased by 10 times.[Conclusion]PrtV gene is related to various biological functions such as cytotoxicity and pathogenicity of V.mimicus.The results provide a basis for further analysis of the features of PrtV gene and its pathogenic mechanism in V.mimicus.