铜绿假单胞菌pcr2基因功能的研究

Characterization of pcr2 gene of Pseudomonas aerugionosa

摘要:

Ⅲ型分泌系统(type Ⅲ secretion system,TTSS)是铜绿假单胞菌的重要致病因子,pcr2基因位于TTSS基因簇中popN操纵子的第三位,有关该基因的具体功能研究还是空白。首先,本研究采用定点诱变方法构建pcr2-突变体,发现TTSS表达和分泌ExoS和ExoT蛋白的能力显著下降,在HeLa细胞感染实验中,ExoS和ExoT蛋白注入细胞的数量明显低于野生型菌株。其次,我们采用细菌双杂交系统研究了Pcr2蛋白与其它蛋白结合的可能性,发现Pcr2蛋白与PscB蛋白一起能够结合PopN蛋白,同时Western blot实验发现Pcr2蛋白能够调控PopN蛋白的分泌。最后,实验发现Pcr2蛋白本身也能够分泌到细胞外,可能与TTSS分泌器的早期形成过程有关。

Abstract:

Type Ⅲ secretion system (TTSS) is an important virulence factor encoding by pseudomonas aeruginosa. About 40 genes are involved and they function as structure proteins，chaperons，regulators，and effectors proteins，respectively. Although some genes have been studied previously，functions of many genes remained unknown. Pcr2 gene is the third gene of popN operon that is one of the five operons of the TTSS gene clusters. Its functions were investigated in this study. First，by characterization of the phenotypes of pcr2- mutant，we found that the abilities of secreting or translocating effectors proteins were significantly damaged in the absence of Pcr2 protein，suggesting that Pcr2 protein involved in both the secretion and translocation processes of TTSS. Second，evidences were provided that no PopN protein was detectable in supernatant of pcr2- mutant culture. Combined with the data from the bacterial two-hybrid system，we can conclude that Pcr2 protein might function as part of a chaperone complex for the PopN protein. Third，Pcr2 protein was found secreted in a TTSS-dependent manner，suggesting that secreted Pcr2 may play a role in the TTSS needle biogenesis.