vcrV基因对副溶血弧菌生物学特性及Ⅲ型分泌系统1效应蛋白易位的影响

vcrV gene affects translocation of T3SS1 effector protein and biological characteristics of Vibrio parahaemolyticus

【背景】副溶血弧菌(Vibrio parahaemolyticus)具有两套Ⅲ型分泌系统(type Ⅲ secretion system,T3SS)。T3SS1通过向宿主细胞分泌效应蛋白发挥致病作用,vcrV基因位于T3SS1编码基因簇。【目的】以vcrV基因为研究对象,探索其对副溶血弧菌生物学特性及T3SS1致病机制的影响。【方法】以副溶血弧菌POR-1株为参考菌株,利用同源重组技术构建vcrV基因缺失株ΔvcrV和回补株CΔvcrV,比较各菌株在生长性能、生物被膜形成能力、细胞黏附及细胞毒性等生物学特性的差异,应用Western Blot检测T3SS1诱导条件下POR-1、ΔvcrV和CΔvcrV等菌株效应蛋白分泌量,进一步利用具有过表达载体pMMB207-vp1683-CyaA的各菌株侵染HeLa细胞,通过Western Blot检测细胞内效应蛋白VopR(Vp1683)的易位量。【结果】与基础菌株POR-1相比,缺失vcrV基因不影响副溶血弧菌的生长性能、生物被膜形成能力及细胞黏附等生物学特性,显著降低了对HeLa细胞的毒性作用,具有过表达载体的POR-1、ΔvcrV和CΔvcrV等菌株效应蛋白VopR的分泌差异不显著,ΔvcrV菌株侵染HeLa细胞的过程中,VopR的易位量显著下降。【结论】vcrV基因参与T3SS1介导的细胞毒性过程,对于副溶血弧菌T3SS1效应蛋白易位是必需的,具有过表达载体的ΔvcrV仍能分泌效应蛋白VopR。

[Background]vcrV is an important gene located in the T3SS1 gene cluster,which is one of the two sets of type Ⅲ secretion systems of Vibrio parahaemolyticus and plays a pathogenic role by secreting effector proteins to host cells.[Objective]In this study,the effect of vcrV was explored on the T3SS1 pathogenesis and the biological characteristics of V.parahaemolyticus.[Methods]The vcrV-deleted strainΔvcrV and the complemented strain CΔvcrV were constructed by homologous recombination technology,using V.parahaemolyticus POR-1 as the reference strain.The growth performance,biofilm formation ability,cell adhesion,and cytotoxicity were compared among different strains.Western Blot was employed to detect the effector proteins secreted by POR-1,ΔvcrV,and CΔvcrV under T3SS1 induction.The translocation of the effector protein VopR(Vp1683)was detected by Western Blot after each strain with the pMMB207-vp1683-CyaA overexpression vector was used to infect HeLa cells.[Results]The deletion of vcrV did not affect the growth performance,biofilm formation ability,or cell adhesion while significantly reduced the strain toxicity to HeLa cells.The effector protein VopR secretion had no significant difference among POR-1,ΔvcrV,and CΔvcrV with the overexpression vector.The translocation of VopR decreased significantly whenΔvcrV infected HeLa cells.[Conclusion]The T3SS1-mediated cytotoxicity involving with vcrV is critical for the translocation of T3SS1 effector protein in V.parahaemolyticus,while the effector protein VopR could still be secreted byΔvcrV with overexpression vector.