克雷白氏肺炎桿菌致病機轉之研究 (I) 建立克雷白氏肺炎桿菌感染的小鼠與細胞模式 (II) 運用PP-STM突變系統搜尋在小鼠感染模式中之致病相關基因

A study on the pathogenesis of Klebsiella pneumoniae (i) Establishment of an infection model of Klebsiella liver abscess (ii) Identification of virulence-associated gene in Klebsiella pneumoniae CG43 with a PP-STM screening system

涂雅純(Ya-Chun Tu)

指導教授 ： 賴怡琪

中山醫學大學/醫學院/醫學研究所/碩士(2006年)

https://doi.org/10.6834/CSMU.2006.00059

摘要

克雷白氏肺炎桿菌 (Klebsiella pneumoniae) 屬於格蘭氏陰性腸內桿菌科 (Enterobacteriacea) 是經常引起社群及院內感染的重要致病菌。在台灣K. pneumoniae的感染好發於糖尿病患者，易併發多種臨床症狀，如原發性肺炎，化膿性肝膿瘍 (pyogenic liver abscess) ，腦膜炎 (meningitis) ，及轉移性眼內炎 (endocephthalmitis) 。 近二十年來，無論國內外由K. pneumoniae引起之化膿性肝膿瘍的病例都有逐漸增加的趨勢。 在台灣地區有高達 82.1% 的化膿性肝膿瘍是由K. pneumoniae的感染所引起，此病症的發生率約為西方國家的1700倍。 由於K. pneumoniae的感染具有如此的地緣差異與高致死率，近年來有許多國內感染專家學者們致力於該菌分子致病機轉的研究。
目前已知的K. pneumoniae致病因子，包括莢膜 (capsule) ，脂多醣體 (lipopolysaccharides; LPS) ，黏附因子 （adhesion factors） ，抓鐵系統 （siderophores） 與血清抵抗能力 （serum resistance） 等，除此之外，該菌的致病機轉仍不清楚。 為搜尋其他K. pneumoniae感染所需的致病基因以及瞭解這些基因的作用機轉，本研究的第一部份建立K. pneumoniae感染的小鼠肝膿瘍及細胞模式；第二部份運用本實驗室建構的STM突變系統在小鼠感染模式中搜尋K. pneumoniae的致病相關基因。
以餵食方式使6-8週的BALB/c小鼠感染數個GFP-expressing 的K. pneumoniae 臨床分離菌株，包括：K1 血清型的1112 與1084，以及K2血清型的CG43與1015。 小鼠感染後48小時，肝組織出現明顯的膿瘍病徵，以螢光顯微鏡觀察肝組織的冷凍切片可清楚看到病灶周圍有大量的綠色螢光細菌。 在肝癌細胞株Hep 3B與Hep G2的感染模式中，以在小鼠感染中最具侵襲性的K. pneumoniae 1112 所造成的細胞凋亡現象最為明顯。 藉由本研究中第一部份成功建立的小鼠肝膿瘍模式，在第二部分的研究中，將已建構在K. pneumoniae CG43的60個各含48個貼有不同標籤序列的STM突變菌株庫（共有2880個轉位子突變株）以餵食方式感染BALB/c小鼠，48小時後分別取出肝臟脾臟與血液等組織，將仍存在在小鼠組織中的突變菌株的標籤序列以PCR增幅方式放大後藉由雜交反應偵測在感染過程中喪失致病力的突變株。 經過兩次篩選，共有15個突變株在小鼠的感染模式中喪失致病力。 將轉位子插入點的染色體片段進行序列分析結果顯示：有3個在NCBI上無法搜尋到序列相似的相關資料，有4個屬於Lactose operon的基因，有1個是Cellobiose permease IIC component；有2個為hypothetical proteins；有5個和醣的代謝有關， 包括Inner membrane protein yfgF，PGA synthesis protein pgaA precursor，Uracil permease，phosphatidylinositol 3-kinase和dynamin。 這些致病相關基因在 K. pneumoniae CG43致病過程中扮演的生理角色仍需進一步的實驗證明。

關鍵字

克雷白氏肺炎桿菌

並列摘要

Klebsiella pneumoniae, an important pathogen belonging to Gram-negative Enterobacteriaceae, usually causes community- and hospital-acquired infections. In Taiwan, diabetic patients have an increased susceptibility to K. pneumoniae infections combined with several clinical syndromes, including primary pneumonia, pyogenic liver abscess, meningitis and metastatic endocephthalmitis. In the past two decades, the reported incidence of pyogenic liver abscess caused by K. pneumoniae has dramatically increased world wide. In Taiwan, as high as 82.1% of pyogenic liver abscess cases were caused by the K. pneumoniae infection, which was 1700 times more than that in Western countries. Due to the geographic preponderance and high mortality rate of K. pneumoniae infections, groups of researchers endeavor to figure out the underlying mechanism of its pathogenesis and unusual prevalence in Taiwan.
The virulence factors of K. pneumoniae identified so far include capsule, lipopolysaccharides (LPS), adhesion factors, siderophores, and resistance to host serum-killing. Besides these factors, very little is known about its pathogenesis. In order to identify other virulence-associated genes that may participate in bacterial pathogenesis and to further characterize their physiological roles in the K. pneumoniae infection, the first part of this study was to establish a murine infection model with K. pneumoniae-induced pyogenic liver abscess as well as cell models. The second part of this study was to identify virulence-attenuated mutants upon murine infections by using an established STM system in our lab.
GFP-expressing K. pneumoniae isolates, including 1112 and 1084 with K1 serotype and CG43 and 1015 with K2 serotype, were administered by a force-feeding method into 6-8 weeks old BALB/c mice. Forty-eight hours post-infection, several abscess foci were observed in murine liver and numerous green fluorescent K. pneumoniae were also detected in the frozen sections of infected liver. When compared to other K. pneumoniae clinical isolates included in this study, K. pneumoniae 1112 had the highest invasive capability in murine liver abscess model and in consistent with the result, an infection of K. pneumoniae 1112 also resulted in a significant apoptosis in the liver carcinoma cell Hep 3B and Hep G2. Take advantage of the murine model of K. pneumoniae liver abscess established in the first part, a total of 60 STM mutant pools each containing 48 uniquely tagged mutants was screened upon murine infections. Overnight culture from each mutant pool with the dosage of 107 cfu was administered by a force-feeding method into 6-8 weeks old BALB/c mice. Two days post-infection, the infected mice were sacrificed and the homogenates of their spleens, liver and blood were plated onto LB agar supplemented with kanamycin. Genomic DNA was extracted from each output bacterial culture of each mutant pool and used as a template for PCR-amplification of STM tag. The amplified PCR products were then spotted onto 48 membranes and followed by hybridization separately with 48 fluorescein-labeled ST-tag probes. The potentially virulence-attenuated mutants were reassembled into new pools for another round of selection. Finally, fifteen mutants were found to be virulence-attenuated. Sequence determination of the DNA flanking site for each transposon insertion of these mutants revealed: three mutants with disruption in genes with novel sequence; four mutants with disruption in genes belonging to lactose operon, one mutant with disruption in gene encoding cellobiose premease IIC component; two disrupted in genes for hypothetical proteins; and five mutants carrying transposon insertions in genes responsible for carbohydrates metabolisms, including genes encoding inner membrane protein YfgF, PGA synthesis protein PgaA precursor, uracil premease, phosphatidylinositoal 3 kinase and dynamin. However, the physiological roles of these virulence associated genes identified herein remain to be characterized with further experiments.

並列關鍵字

Klebsiella pneumoniae

參考文獻

2. Alcantar-Curiel, D., J. C. Tinoco, C. Gayosso, A. Carlos, C. Daza, M. C. Perez-Prado, L. Salcido, J. I. Santos, and C. M. Alpuche-Aranda. 2004. Nosocomial bacteremia and urinary tract infections caused by extended-spectrum beta -lactamase-producing Klebsiella pneumoniae with plasmids carrying both SHV-5 and TLA-1 genes. Clin Infect Dis 38:1067-1074.

3. Bertrand, X., D. Hocquet, K. Boisson, E. Siebor, P. Plesiat, and D. Talon. 2003. Molecular epidemiology of Enterobacteriaceae producing extended-spectrum beta-lactamase in a French university-affiliated hospital. Int J Antimicrob Agents 22:128-133.

4. Branger, J., S. Florquin, S. Knapp, J. C. Leemans, J. M. Pater, P. Speelman, D. T. Golenbock, and T. van der Poll. 2004. LPS-binding protein-deficient mice have an impaired defense against Gram-negative but not Gram-positive pneumonia. Int Immunol 16:1605-1611.

6. Brook, I., and E. H. Frazier. 1998. Microbiology of liver and spleen abscesses. J Med Microbiol 47:1075-1080.

7. Chang, F. Y., and M. Y. Chou. 1995. Comparison of pyogenic liver abscesses caused by Klebsiella pneumoniae and non-K. pneumoniae pathogens. J Formos Med Assoc 94:232-237.