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

摘要:

【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。

Abstract:

[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 the ΔPrtV were hardly changed, but the physicochemical properties of decomposing urea, glycine, coumarinate, ornithine, and lysine were changed. The extracellular product protease activity and cytotoxicity of ΔPrtV 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.