- 學位論文
產基因毒素尿道致病型大腸桿菌對人類膀胱細胞株的影響性探討
Exploring the effects of genotoxic uropathogenic Escherichia coli on human bladder cells
摘要
中文摘要 許多女性經常會有泌尿道感染的問題，且通常會有反覆感染的情況，而造成泌尿道感染的致病菌中大約有七成是尿道致病型大腸桿菌感染所導致，而尿道致病型大腸桿菌有許多毒力因子可以幫助其感染並生存在泌尿道系統中，本篇研究主要針對大腸桿菌的基因毒素Colibactin對於膀胱上皮癌細胞株的影響，Colibactin是一個基因毒素次級代謝物，是由54 kb的基因簇（從clbA到clbS）所編碼的Non-ribosomal peptide synthetase-polyketide synthase（NRPS-PKS）來合成，首先我利用PCR從泌尿道感染病人尿液中所分離出的臨床分離菌株來篩選擁有clb基因的菌株，我們鑑定出兩株帶有clb基因的大腸桿菌並命名為U013和U030，並確認了其在親緣分型上屬於B2分型即主要為腸外致病型大腸桿菌，接著為了證實Colibactin的基因毒性，我利用lambda red基因重組系統建立了U013及U030的clbA剔除菌株（△Clb A），並利用膀胱上皮癌細胞株T24以及5637來進行感染實驗，當T24以及5637細胞株短暫感染野生型菌株U013時比起△Clb A明顯誘導了較多的細胞呈現Megalocytosis的現象，Megalocytosis即為細胞有細胞核以及細胞本身的增大現象，此結果意味著UPEC中的Colibactin產生的基因毒素促使泌尿道細胞DNA損傷而導致細胞週期停滯，此外當短暫感染U013時會誘導T24細胞株的exosome分泌，我們利用蛋白質組學來分析U013及△Clb A感染T24細胞時所分泌的exosome中之蛋白質差異，結果顯示與△Clb A相比經U013刺激之T24細胞株所分泌的exosome中具有更多的糖解酵素Enolase-1及蛋白質合成因子eEF2，然而其在泌尿道細胞中Colibactin相關性的腫瘤生成所扮演的角色仍然需要更進一步的研究來闡明。
關鍵字
大腸桿菌並列摘要
Abstract Many women have problems with recurrent urinary tract infections (UTIs). About 70% of UTIs are caused by Uropathogenic Escherichia coli (UPEC). UPEC has a lot of virulence factors which can help them infect and survive in the urinary tract system. I aim in this study to investigate the effect of the genotoxin colibactin on bladder epithelial carcinoma cell lines. Colibactin is a genotoxic secondary metabolite, which is biosynthesized by a non-ribosomal peptide synthetase-polyketide synthase (NRPS-PKS) assembly line encoded in a 54 kb gene cluster (from clbA to clbS).First, I performed a PCR-screening of clb genes in clinical isolates which were collected from the urine samples of UTI patients. Two clb-positive E. coli strains were identified and named U013 and U030. Their genotype belongs to the B2 subtype which is the major cluster of extraintestinal pathogenic E. coli (ExPEC). Next, to validate the genotoxicity of colibactin, I used the lambda red gene recombination system to generatean isogenic mutant of clbA(△Clb A) in U013 and U030. Bladder epithelial carcinoma cell line, T24 and 5637, was used for infection experiments. Transient infection of T24 and 5637 cells with the wild type strain U013 induced a higher number of cells undergone megalocytosis than that with △Clb A. Megalocytosis means that the cells have enlargement of the cell body and nucleus. This result suggests that the genotoxicity of UPEC colibactin causes DNA damages in the urinary cells, leading to cell cycle arrest. Moreover, the transient infection with U013 evoked increased release of exosome from T24 cells. A proteomic approach was used to compare the protein content of the exosome released from the U013-infected T24 cells with the △Clb A group. Data show that U013 stimulated T24 cell line to secrete exosome with more glycolytic enzyme Enolase-1 and protein synthesis factor eEF2 when compared to △Clb A. Their role in the colibactin-related tumorigenesis in urinary cells needs a further study to elucidate.