zapD對奇異變形桿菌生物膜合成能力的影響及其機制探討

Abstract

奇異變形桿菌(Proteus mirabilis)為格蘭氏陰性腸內菌，在健康人體腸道屬於正常菌叢，但在長期使用導尿管病患身上，會造成伺機性感染，嚴重甚至可能導致腎臟病、肺炎等併發症，目前研究發現P. mirabilis在導尿管形成生物膜後具有顯著的感染能力，造成導尿管病患嚴重感染，然而生物膜合成的機制至今還是不清楚。本篇利用transposon mutagenesis的方式建構zapD突變株，實驗結果發現zapD突變株生物膜合成能力大幅下降，探究其可能原因，我們發現zapD突變株的cpxAR基因表現量有顯著的降低，先前實驗室的研究發現，cpxAR的突變株，會影響細菌MR/P fimbriae的表現，進而導致生物膜合成能力下降，所以，我們觀察zapD突變株 MR/P fimbriae的蛋白表現量，我們也發現zapD突變株其MR/P fimbriae蛋白表現量有明顯的下降；此外，我們也觀察zapD上游的zapA基因，我們發現細菌的potease活性下降且zapA基因的表現明顯的降低，我們利用reporter assay及EMSA發現，zapA基因會直接受到Cpx pathway的調控；另外，我們探討其他毒力因子發現zapD突變株的細胞，FlaA蛋白表現量也有明顯的上升。總結而論，我們發現zapD突變會影響P. mirabilis生物膜合成能力，其機制是因為zapD突變導致cpxAR基因表現下降，影響MR/P fimbriae蛋白的表現使生物膜合成能力降低，同時，Cpx pathway會直接調控zapD的上游基因zapA的表現。

Proteus mirabilis, a facultative Gram-negative bacterium, is a common cause of catheter-associated urinary tract infections. Recently studies have showed that P. mirabilis can form biofilm on catheter leading to blockage of catheter and cause severe infections. There has been a report that mannose-resistant proteus like (MR/P) fimbriae contributes P. mirabilisbiofilm formation. However, the genes and its mechanisms in P. mirabilisbiofilm formation remain unclear. In previous study, we have showed that mrp operon was regulated by Cpx pathway. In this study, we constructedzapD- mutant by transposon mutagenesis. Data from biofilm formation test demonstrated that, in the absence of zapD, P. mirabilisbiofilm formation dramatically decreased. Further, we also found thatthe expression of MR/P fimbriae and cpxAR gene expression significantly decreased in zapD mutant. Besides, we revealed that zapA, an upstream gene of zapD, was directly regulated by CpxR by use of reporter assay and EMSA. In sum, this study shows that zapD plays an important role in P. mirabilisbiofilm formation involving in Cpx pathway which can impair mrp operon expression. Moreover, we also show that zapD has an influence on its upstream gene, zapA, which contains CpxR-binding site on its promoter and directly regulated by Cpx pathway.