利用哺乳動物細胞探討克雷白氏肺炎桿菌的致病因子

克雷白氏肺炎桿菌 ( Klebsiella pneumoniae )是革蘭氏陰性的腸內菌。可引起社區型及院內感染，包括肺炎、菌血症、肝膿瘍、腦膜炎、尿路感染以及傷口感染等。儘管如此，對於克雷白氏肺炎桿菌致病機轉的研究仍有許多空間。在本篇研究中，首先，建立了肝細胞 (BNL)模型對克雷白氏肺炎桿菌致病機制進行探討。在本篇研究的第一部分確定了一株帶有colibactin 基因簇( clbA- clbQ ) 的K1 血清型菌株 K. pneumoniae 1084。在感染BNL後從phospho-histone 2AX的結果發現會造成DNA的損傷其結果與colibactin –positive大腸桿菌相同。且DNA damage、細胞聚集和megalocytosis隨感染時間改變。本篇研究第二部分，建構了紅血球細胞模型探討了第六型蛋白質分泌系統在K. pneumoniae CG43毒力中扮演的角色。實驗結果發現，icmF( I )在溶血反應中似乎扮演了重要的角色。而T6SS 導致紅血球溶血取決於CPS的多寡。雖然，在動物模型及紅血球溶血上我們確定了T6SS的功能，但對於其分子機制是我們值得更進一步探討的。

Klebsiella pneumoniae is a Gram-negative intestinal bacterium, responsible for community and hospital-acquired infections,including pneumonia, bacteremia, liver abscess, meningitis, urinary tract infections, and wound infections. Nevertheless, our knowledge regarding the pathogenesis mechanism of K. pneumoniae is still restricted. In this study, I established a hepatic cell model with BNL cells, by which K. pneumoniae virulence mechanism was investigated. In the first part of my study, I determined the DNA damage inducing capacity of K. pneumoniae 1084, which is a K1 strain carrying the colibactin gene cluster (clbA-clbQ). Similar to colibactin-positive Escherichia coli, K. pneumoniae 1084 induced DNA damages in the transiently infected BNL cells, as revealed by significantly increased levels of phospho-histone 2AX, megalocytosis, and colony formation at different time intervals post-infection. In the second part of my study, I determined the role of type VI secretion system (T6SS) in K. pneumoniae CG43 virulence. Genome sequence analysis identified two genetic loci carrying genes of T6SS. Previous study in our lab revealed that icmF gene of T6SS-I (the first genetic loci of T6SS) was required for K. pneumoniae virulence in murine models. By using a red blood cell model, I found that the loss of icmF significantly attenuated hemolytic capacity of K. pneumoniae. The T6SS-dependent hemolysis of K. pneumoniae was significantly enhanced in a nonmucoid mutant, which had deletion in galU gene. As this mutant lost the integrity of capsular saccharides, this result suggested a possible interference of capsule on the display of T6SS in K. pneumoniae. Although we determined that T6SS-I was required for Klebsiella pneumoniae virulence and in vitro hemolytic capacity. The detailed molecular mechanism of T6SS is largely unknown. It deserves further studies to explore.