克雷白氏肺炎桿菌線毛的分佈與表現

Abstract

克雷白氏肺炎桿菌是一株引起伺機性感染的革蘭式陰性細菌，大多於免疫力不全的病人身上，引起化膿性的潰瘍、菌血症、尿道以及呼吸道的感染。為了探討克雷白氏肺炎桿菌的致病機制，在本論文中，我們針對在感染初期扮演著重要角色的線毛的分佈及表現調控進行研究。 我們藉由分析克雷白氏肺炎桿菌NTUH-K2044的全基因體序列，共發現了九組獨立的線毛基因群。除了過去已知的第一型與第三型線毛基因組外，其餘的七組尚未有文獻報導，我們將其分別命名為kpa、kpb、kpc、kpd、kpe、kpf、kpg基因組。我們進一步分析了克雷白氏肺炎桿菌的臨床分離菌株，發現kpb與kpc基因組在K1莢膜血清型的菌株中有顯著較高的存在率。隨後，我們針對Kpc線毛進行了特性分析。在大腸桿菌中表現Kpc線毛的生合成基因組kpcABCD後，可以致使細菌製造出Kpc線毛並且增強其生物膜的形成能力。我們也發現Kpc線毛的表現受到了相變機制（phase variation）的調控，而此相變機制則是由KpcI專一性DNA重組酶所負責。最後，我們著重於第三型線毛的表現調控進行研究。我們發現位於第三型線毛基因組下游的mrkHIJ操作組可生合成出三個調控蛋白質來影響第三型線毛的表現。mrkI基因被預測可生合成出LuxR類型的轉錄因子，而我們將mrkI在克雷白氏肺炎桿菌中進行剔除後，發現會破壞第三型線毛的基因轉錄。同時我們也證明，具有PilZ模組的MrkH與具有EAL模組的MrkJ，可分別正向與負向調控第三型線毛的表現。此外，我們發現Fur攝鐵調控子會經由調控mrkHIJ操作組來影響第三型線毛的表現。而二次代謝傳導物c-di-GMP也被證明可經由Fur與MrkI來活化第三型線毛的表現。我們還發現缺氧環境可能是影響第三型線毛表現的因子。 在本論文中，我們首度報導了克雷白氏肺炎桿菌線毛的基因體學分析，這個報導將有助於未來對於此細菌線毛黏附作用的研究（第二章）。我們也分析了Kpc線毛的功能性與相變調控表現（第三章），以及探討影響第三型線毛表現的多重調控因子（第四章）。

Klebsiella pneumoniae is a Gram-negative pathogen which causes suppurative lesions, bacteremia and urinary as well as respiratory tract infections mostly in patients with underlying diseases. To investigate the pathogenicity of K. pneumoniae, we focused on the study of prevalence and expression of fimbriae, which are important virulence determinants during the initial infection. By analyzing the whole genome sequence of K. pneumoniae NTUH-K2044, nine distinct fimbrial gene clusters were identified. Besides type 1 and type 3 fimbrial genes, the other seven are novel and designated kpa, kpb, kpc, kpd, kpe, kpf, and kpg. The following prevalence analysis among K. pneumoniae clinical isolates indicated that the kpb and kpc genes were more prevalent in the strains of capsular serotype K1. Subsequently, the Kpc fimbria, encoded by kpcABCD genes, was characterized. Induced expression of the recombinant kpcABCD genes in Escherichia coli resulted in Kpc fimbriation and increased bacterial biofilm formation. The Kpc fimbriae expression was also found to be regulated under phase variation mediated by the site-specific recombinase KpcI. Finally, the expressional control of type 3 fimbriae in K. pneumoniae CG43 was investigated. We described that the type 3 fimbriae expression was mediated by three regulatory proteins encoded by the mrkHIJ operon which located downstream to the type 3 fimbrial genes. Deletion of mrkI, which encoded a LuxR-type response regulator, from K. pneumoniae was found to abolish the expression of type 3 fimbriae at transcriptional level. Moreover, MrkH, a PilZ domain protein, and MrkJ, an EAL domain protein, appeared to act as an activator and a repressor, respectively, for the type 3 fimbriae expression. Besides, we showed that the ferric uptake regulator Fur could activate the expression of type 3 fimbriae through regulation on the mrkHIJ operon. The second messenger c-di-GMP was also found to activate the expression of type 3 fimbriae through Fur and MrkI. Furthermore, we identified that oxygen-limitation was possibly an environmental stimulus for activating the type 3 fimbriae expression. In this dissertation, we reported the first genomic analysis of fimbrial gene sequences in K. pneumoniae, which pave the way for future study of the bacterial adherence (Chapter 2). Subsequently, the functional role and phase-variable expression of Kpc fimbriae were characterized (Chapter 3). Moreover, a multi-factorial regulation of type 3 fimbriae expression was elucidated (Chapter 4).