奇異變形桿菌中rpoE基因功能之研究

Abstract

奇異變形桿菌(Proteus mirabilis)屬於伺機性感染人類泌尿道的病原菌，感染的對象主要為尿道不健全或長期插尿導管的病人，膀胱炎、腎結石，腎臟病。先天免疫系統的活化、抗菌胜肽以及中性球的聚集可以清除泌尿道中的細菌感染，而細菌的毒力及適應因子可以幫助其逃避免疫機制。本篇研究的目的在於找出奇異變形桿菌如何感受宿主環境及啟動免疫逃避的機制，我們發現sigma factor E (RpoE)可以調控奇異變形桿菌困境生存的基因及在逃避宿主免疫中扮演重要角色。 為了觀察奇異變形桿菌RpoE在逃避宿主免疫機制中的角色，我們創造了rpoE及rseA突變株 (RseA蛋白的功能在於抑制RpoE，若被去除將造成RpoE過度表現)。本研究利用qPCR發現RpoE正向調控MR/P纖毛基因及抑制鞭毛合成基因flhDC及fliC1的表現。MrpI是一種重組蛋白，其功能為轉換mrp operon的啟動子，進而調控MR/P纖毛的表現，本研究發現RpoE正向調控mrpI並使得mrp operon轉換成開啟模式。我們又發現了RpoE抑制奇異變形桿菌爬行的能力，進而抑制其爬行時溶血酶的生成及對細胞產生的毒性。人類的表皮細胞可分泌細胞激素以吸引中性球來殺死致病菌，而在泌尿道感染中，膀胱細胞可分泌IL-8達到此目的。本研究發現rpoE 突變株可刺激人類膀胱癌細胞NTUB1產生大量的IL-8而rseA突變株卻相反，造成此現象的原因可能是LPS修飾發生改變。動物實驗中發現rpoE突變株幾乎無法在小鼠泌尿道中生存，且RpoE可被尿液中的成分尿素所活化。為探討為何rpoE突變株無法小鼠泌尿道中生存的原因，我們利用組織免疫染色觀察三種菌株感染小鼠後的膀胱及腎臟，我們發現在第一天時rpoE mutant刺激大量的免疫細胞聚集而使其容易被清除，因此RpoE對於奇異變形桿菌在泌尿道的感染中扮演著重要的角色。

Proteus mirabilis (P. mirabilis) is an opportunistic human pathogen causing urinary tract infection (UTI) in individuals with abnormalities or long-term catheterization. Innate immune responses induced or mediated by pattern recognition receptor signalling, antimicrobial peptides, and neutrophils are vital to clear bacteria during UTIs. Bacterial virulence and fitness factors include fimbriae and flagella that may function in immune evasion. The mechanism by which P. mirabilis sense the host environment and then trigger immune evasion is still unclear. In this study, we showed that sigma factor E (RpoE), which regulates expression of genes for survival in stresses, is important for host immune evasion. To investigate the role of P. mirabilis RpoE in immune evasion, we constructed rpoE mutant and rseA mutant (RseA, an anti-sigma E factor, rseA mutation causing RpoE overexpression). We found that the expression of mannose-resistant Proteus-like (MR/P) fimbriae of rseA mutant was higher than others, while the expression of flhDC and fliC1 of rseA mutant was the lowest in the three strains by qPCR assay. MrpI is a recombinase that control orientation of mrp operon promoter and we observe RpoE positively regulates mrpI by real-time PCR. The promoter invertible element (IE) assay also showed IE of wild-type and rseA mutant are largely on compared to rpoE mutant. Consistent with the results, rseA mutant had the best invasion ability and swarmed the least of all. Human urothelial cells have been known to secrete IL-8 cytokines to attract neutrophils to eliminate the pathogens. In this regard, we found NTUB1 produced more IL-8 and had the highest level of IL-8 mRNA after infecting with rpoE mutant by cytokine array and qPCR. The mouse UTI model further indicated almost no colonization of rpoE mutant in the bladder and kidney. Finally, we found that urea (the major component in urine) and polymyxin B (a kind of antimicrobial peptides) can induce high expression of rpoE in the wild-type. Altogether, our findings support that rseA mutant and rpoE mutant were phenotypically distinct and for the first time, we demonstrate that RpoE is important in sensing environmental cues and subsequently triggers expression of genes associated with virulence and fitness factors to evade host immunity and build up UTIs.