铜绿假单胞菌popN^-突变子Ⅲ型分泌水平及与蛋白酶关系的研究

Type Ⅲ Secretion Study of popN^- Mutant of Pseudomonas aeruginosa and Proteases Degradation

铜绿假单胞菌(Pseudomonas aeruginosa)Ⅲ型分泌系统(typeⅢsecretion system,TTSS)是重要的细菌致病因子之一,能够将酶蛋白直接注入到宿主细胞内,导致细胞损害的发生。重点研究了TTSS中的popN基因的功能,通过构建popN-突变子,发现该突变子在非诱导条件下,能够分泌酶蛋白,显示popN基因编码的蛋白对TTSS蛋白的分泌具有负调控作用。进一步研究发现,popN-突变子在不同培养基中TTSS的分泌水平存在着显著差异,影响对popN基因的功能的判断。为了解决这一矛盾,从几个方面分析了造成表型差异的可能因素,确定蛋白酶对TTSS分泌蛋白的降解作用,是表型差异存在的主要原因,从而首次系统地阐明popN-突变子在不同培养基中都具有TTSS组成型表达的表型,对于深入研究TTSS的调控机制具有重要意义。

Pseudomonas aeruginosa is an important opportunistic human pathogen.It encodes many virulence factors and one of them is type Ⅲ secretion system（TTSS）.Effectors proteins can be delivered into host cells directly by this system,causing necrosis or apoptosis.popN gene is the first gene in the popN operon of TTSS gene cluster.To investigate its function,popN gene deletion mutant was generated in this study,and we found this mutant can secrete effectors proteins constitutively under non-inducting condition in DMEM medium containing serum.The results indicated that PopN is a negative regulator of the TTSS expression.However,no secreted effector proteins were detectable when the popN-mutant was grown in LB medium under non-inducting condition.To investigate the possible reasons,effects of growth status and protease（s）inhibitors on the TTSS were investigated.We present evidences that indicate protease mediated degradation of secreted effector proteins played a key role in the phenotypic inconsistency of popN-mutant.