克雷白氏肺炎桿菌NTUH-K2044中纖毛的功能性分析

Abstract

纖毛黏附蛋白是一種重要的致病因子，影響著黏附到宿主細胞的專一性。在克雷白氏肺炎桿菌NTUH-K2044的基因體中，藉由HMMER找到了九套纖毛基因組，其中包含已知的第一型纖毛fim基因組和第三型纖毛mrk基因組，以及七套未曾被報導的纖毛基因組，分別被命名為kpa、kpb、kpc、kpd、kpe、kpf和kpg。為了闡明這些新纖毛的功能，我們以聚合酶連鎖反應由NTUH-K2044染色體中選殖這些纖毛基因組到無纖毛的大腸桿菌BL21(DE3)。藉由SDS-PAGE分析，我們可以偵測到kpa、kpb以及kpe纖毛的主要結構蛋白（pilin），此外kpa纖毛基因組重組質體的表現可以增進大腸桿菌對細胞的黏附和些許生物膜的生成能力。位於纖毛末端的黏附蛋白為決定細菌黏附專一性的主要因子，為了測試黏附蛋白的活性，我們進一步以過去建立的第三型纖毛表現系統為平台，將MrkD黏附蛋白基因分別置換為kpa、kpb、kpc、kpd、kpe、kpf或kpg的黏附蛋白基因，並經西方墨點法確認這些重組第三型纖毛的結構蛋白的表現和組裝正常。而在紅血球凝集測試中，我們發現KpfD具有黏附天竺鼠紅血球以及兔子紅血球的能力，而KpaE、KpbD、KpdD及KpgD對兔子的紅血球也具有不同程度的黏附能力，其中KpfD與天竺鼠紅血球的凝集作用可被0.5 mM的甘露醣抑制，顯示kpf纖毛與第一型纖毛同樣具有對甘露醣敏感型的血球凝集活性。

Fimbrial adhesin is an important bacterial virulence factor which affects the adherence specificity to host tissues. In the genome of Klebsiella pneumoniae NTUH-K2044, we found nine fimbrial gene cluters including fim, mrk, and seven novel fimbrial operons, namely kpa, kpb, kpc, kpd, kpe, kpf, and kpg. These fimbrial gene clusters were cloned into the expression plasmid pET30b and then transformed to an afimbriated E. coli. Expression of the major pilin of the recombinant kpa, kpb or kpe fimbriae could be detected by SDS-PAGE analysis. Moreover, the kpa fimbriae appeared to conferr the bacteria an adherence ability to HCT-8 cell and also biofilm formation capability. The adhesin protein at the tip of fimbriae is a major determinant of the adhesion specificity. To investigate the presence of specific adhesion activity, the mrkD adhesin gene of the established type 3 fimbriae expression system was replaced respectively by each of the fimbrial adhesins. Transmission electron microscopy and western blot analysis revealed that most of the recombinant type 3 fimbriae expressed and assembled properly. The hemagglutination (HA) activity assay indicated that the recombinant KpfD could bind to erythrocytes of guinea pig and rabbit. Different binding activity levels of the recombinant adhesins KpaE, KpbD, KpdD and KpgD to rabbit erythrocytes were also demonstrated. Moreover, the HA activity of KpfD to guinea pig erythrocytes was found to be inhibited by 0.5 mM mannose.