"尿道致病性奇異變形桿菌中肽聚醣水解蛋白之活化劑基因, envC, 抗銅及調控毒力因子之研究"

Abstract

奇異變形桿菌(P. mirabilis)是一種重要的泌尿道病原體，主要是經由尿道或導尿管進入泌尿道，爬行進入膀胱並增生，接著再沿著輸尿管上行至腎臟，造成腎臟感染。而銅是所有生命皆必需的微量元素，但過量也會對生物體造成毒性。以P. mirabilis引發的UTI期間，尿液中銅離子的濃度會顯著上升，其中可使尿液中銅濃度上升，銅在巨噬細胞亦會發揮殺菌作用，因此我們利用跳躍子卡匣突變法構建突變菌株，並以高濃度之銅來篩選出對銅有高感受性的突變株。其中，我們發現envC突變株對銅感受性增加，經基因分析後發現EnvC蛋白在Escherichia coli及Salmonella enterica serovar Typhimurium中作為peptidoglycan的水解酶AmiA和AmiB的activator，對細菌的細胞分裂以及細胞膜的完整性維持有關。為了探討EnvC在P. mirabilis所扮演的角色，本篇分析了野生株以及envC突變株的抗銅及致病因子之表達，結果顯示P. mirabilis中envC突變對不同銅濃度較野生株更有感受性，而細胞有分裂異常的情況，envC突變株對比野生株表現出運動性顯著降低、生物膜生成量顯著上升、細胞黏附率下降、細胞入侵率下降、在氧化壓力下存活率顯著下降、外膜通透性及藥物感受性改變，以及對小鼠的定殖能力顯著下降。綜上所述，EnvC參與了P. mirabilis的抗銅和致病因子之表達。這是首次發現EnvC參與尿道致病菌P. mirabilis的研究，我們的研究結果表明，參與peptidoglycan裂解的EnvC 可能會影響細胞表面特性，從而改變細胞膜相關之致病因子，包括細胞膜結構、運動性、生物膜形成，在壓力環境下的存活、細胞黏附率與入侵率，以及對小鼠的定殖能力。

Proteus mirabilis is an important uropathogen. Copper is an essential trace element for all lives but lethal upon excess. During UTI, the urine copper level rises, and copper exerts bacterial killing in macrophages. Previously we found copper affects P. mirabilis virulence factors such as urease activity and biofilm formation. We then performed transposon mutagenesis to investigate how P. mirabilis deploys copper detoxification system to maintain copper homeostasis. We isolated several mutants exhibited increased copper susceptibility. An envC mutant displaying increased copper susceptibility was identified. The envC gene, encoding a murein hydrolase activator, has been found to be involved in cell division and membrane integrity maintenance in E. coli and Salmonella. We found P. mirabilis envC mutant cells are longer than the wild-type and swarming and swimming motility ability was significantly decreased in the mutant. The mutant also exhibited impaired survival on exposure to oxidative stress and significantly reduced ability to adhere and invade urothelial cells. Accordingly, a defect in mouse colonization of the envC mutant was observed. In summary, EnvC participates in copper tolerance and virulence factor expression of P. mirabilis. This is the first study to investigate the role of envC gene in uropathogenic P. mirabilis. Our results suggest that the P. mirabilis EnvC, participating in cell wall development, may influence the cell surface properties, thereby altering the cell envelope-associated phenotypes including membrane composition, motility, survival in the stressful environment, adhesion and invasion and also colonization in mice.