克雷白氏肺炎桿菌外膜蛋白質致病機轉之研究：探討RNA chaperone Hfq扮演之角色

克雷白氏肺炎桿菌（Klebsiella pneumoniae）為一種經常引起院內及社群感染的重要病原菌，主要造成病人尿道感染（urinary tract infections）、肺炎（nosocomial pneumonia）、腦膜炎（meningitis）以及化膿性肝膿瘍（pyogenic liver abscess）。K. pneumoniae引起的感染症具有高致病性與致死率，好發於免疫不全的患者，尤其是糖尿病病人。在台灣地區K. pneumoniae是引起化膿性肝膿瘍中最重要的致病菌。然而，關於該菌致病機轉的研究現今卻仍停留在起步階段。由於外膜蛋白質對革蘭氏陰性細菌的生理特性和致病能力相當重要，因此本研究中我們探討外膜蛋白質在K. pneumoniae致病機轉上所扮演的角色，以及影響其表現的可能調控機轉。以K. pneumoniae外膜蛋白萃取物針對BALB/c小鼠進行接種，六週後發現預先接種外膜蛋白的小鼠可對抗野生型K. pneumoniae感染，這個結果說明外膜蛋白在K. pneumoniae 進行感染的過程中扮演重要的角色。 Guillier, et. al 在 2006 的報告中指出大腸桿菌外膜蛋白質的表現受到 small regulatory RNAs 的調控，而這些 srRNAs 的表現需仰賴 RNA chaperone Hfq。為進一步探討Hfq對於外膜蛋白質的調控機轉，我們以同源重組技術建構hfq基因缺失突變菌株。在K. pneumoniae感染小鼠的模式中可發現，hfq基因缺失造成K. pneumoniae的致病力增加。此外，相較於野生型菌株，hfq基因缺失會影響K. pneumoniae多方面的生理特性，包括在不同培養條件下的細菌生長曲線、莢膜多醣體的產量、細菌的黏性和外膜蛋白質的表現圖譜等。而hfq基因缺失亦會使得外膜蛋白OmpA的表現有增加的趨勢。在蛋白質體學的比較分析中發現，有多個外膜蛋白質在野生型與hfq基因缺失的突變菌株間有表現量的差異，然而，這些外膜蛋白質究竟如何受到Hfq的調控以及在K. pneumoniae致病上扮演何種角色，仍需後續的實驗加以驗證。

Klebsiella pneumoniae is a common pathogen of hospital- and community-acquired infections, causing urinary tract infections, nosocomial pneumonia, meningitis and pyogenic liver abscess. K. pneumoniae infections tend to occur in immunocompromised individuals, especially diabetic patients with high morbidity and mortality rates. In Taiwan, K. pneumoniae is the primary pathogen for pyogenic liver abscesses; however, its pathogenesis mechanism still remains unclear. Since outer membrane proteins are important for the pathogenesis and physiology of Gram-negative bacteria, in this study, we determined the role of outer membrane proteins in the pathogenesis of K. pneumoniae as well as the possible regulatory mechanism controlling the expression of these proteins. Inoculation with BALB/c mice with outer membrane fractions of K. pneumoniae was found to provide mice immunity against the wild type infection after six weeks, indicating that outer membrane proteins played a crucial role during K. pneumoniae infections. Given Guillier, et. al in 2006 reported that expression of outer membrane proteins is controlled by small regulatory RNAs via mediation with the RNA chaperone Hfq, in order to examine the possible role of Hfq in the outer membrane expression, a hfq deletion mutant was initially constructed with allelic exchange technique. In our BALB/c infectious model via the GI route, the deletion of hfq gene slightly increased bacterial virulence of K. pneumoniae. Besides, the deletion of hfq gene also affected the physiology of K. pneumoniae in many aspects including bacterial growth curves upon different conditions, production of capsular polysaccharides mucoviscosity of colony, and the expression profiles of outer membrane proteins. Moreover, this deletion slightly increased the protein level of outer membrane protein OmpA. Based on a proteomics analysis, several outer membrane proteins were found to express differentially in the hfq deletion mutant when compared with the wild type strain. Characterization of these Hfq-dependent outer membrane proteins and determination of the underlying regulatory circuits require further studies.