克雷白氏肺炎桿菌第三型纖毛黏附蛋白特性

Abstract

曾經有報導指出第三型纖毛頂端的黏附蛋白質 (MrkD)的表現與克雷白氏肺炎桿菌感染的部位有很密切的相關性。MrkD黏附蛋白可與單寧酸處理的紅血球凝集，並不像第一型纖毛的黏附蛋白會因為添加甘露糖而影響凝集紅血球的現象。在本論文裡，我們探討此黏附蛋白質MrkD與引起腦膜炎的克雷白氏肺炎桿菌的相關性：首先，我們將收集的19株腦膜炎菌株作質體分型，結果發現大多數的菌株都帶有較大質體 (plasmids≧100 kb)，而以聚合酶連鎖反應 (Polymerase chain reaction, PCR)及南方墨點法分析確認帶有mrkD基因的菌有18株；我們進一步以Sau3AI 截切分析把這些PCR產物分成四種Retriction fragment length polymorphisms (RFLPs)型，經過核酸定序結果發現與已發表的mrkDC1，mrkDC2，mrkDp序列有部分差異，所以我們將這四種mrkD alleles分別命名為mrkDv1、v2、v3、v4。為了探討這四種mrkD基因型與其黏附活性的相關性，我們構築了可以表現第三型纖毛且分別帶有不同mrkD基因型的重組質體，以大腸桿菌JM109為表現載體，我們以西方墨點法與穿透式電子顯微鏡來證實在大腸桿菌上第三型纖毛的表現，同時分別以已建立的紅血球凝集測試、膠原蛋白的黏附分析、細胞黏附活性與生物膜的形成測試來測量其活性差異。其中我們發現黏附蛋白MrkDv3對膠原蛋白與細胞的黏附活性和生物膜的形成皆高於其他三種黏附蛋白。而由電顯圖也證實纖毛的表現量也以E. coli JM109 [pMrkABCDv3]最高。

It has been demonstrated that the deployment of type 3 fimbrial adhesins (MrkD) is strongly associated with their host specificity of Klebsiella pneumoniae. The fimbrial adhesins mediate the agglutination reaction of tannic acid-treated erythrocytes in vitro. In this study whether MrkD was correlated with meningitis-associated K. pneumoniae was investigated. In the beginning, 19 meningitis-associated K. pneumoniae isolates from patients were identified for plasmid profile analysis. Almost all isolates were found to carry a plasmid(s) larger than 100 kb. Polymerase chain reaction (PCR) and Southern blotting analysis were also used to demonstrate that eighteen of the isolates carry mrkD gene. The PCR products were further classified into 4 Retriction fragment length polymorphisms (RFLPs) types by Sau3AI. The 4 polymorphic mrkD alleles were classified as mrkDv1, v2, v3, and v4, respectively. In order to investigate the structural and functional relationship of these variants, DNA fragments encoding the major fimbrial proteins MrkA, MrkB, MrkC were cloned and expressed together with either one of the four MrkD adhesins in Escherichia coli JM109. Expression of the type 3 fimbriae in E. coli JM109 was confirmed by Western blotting analysis and transmission electron microscopic observation. MrkDv3 was found to be the best adhesin while these adhesins analyzed on the basis of their capabilities of hemagglutination, cell adhesion, collagen binding, and biofilm formation. Transmission electron microscopy (TEM) study also demonstrated that the E. coli JM109 [pMrkABCDv3]showed the highest expression level of fimbriae.