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研究生： 黃智玟
Huang, Chih-Wen
論文名稱： 探討CDK1在出血性大腸桿菌誘發宿主微絨毛肌動蛋白重新排列之現象中所扮演的角色
Characterization of CDK1 in the Enterohemorrhagic E. coli-induced Host Microvillar Actin Rearrangement
指導教授： 陳昌熙
Chen, Chang-Shi
學位類別： 碩士
Master
系所名稱： 醫學院 - 生物化學暨分子生物學研究所
Department of Biochemistry and Molecular Biology
論文出版年： 2017
畢業學年度： 105
語文別： 英文
論文頁數： 71
中文關鍵詞： 出血性大腸桿菌 、線蟲 、人類大腸直腸癌細胞株 、微絨毛肌動蛋白重新排列 、細胞週期蛋白B3 、週期蛋白依賴性激酶1
外文關鍵詞： Enterohemorrhagic E. coli (EHEC), C. elegans, Caco-2 cells, microvillar actin rearrangement, CCNB3, CDK1
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    • 出血性大腸桿菌血清型O157:H7屬於一種人類的致病菌，會導致人類出現嚴重症狀，例如出血性腸炎、溶血性尿毒症等等症狀甚至會導致死亡，而出血性大腸桿菌血清型O157:H7主要是藉由糞口途徑進入人體並且寄殖在人類的腸道。在臨床方面，目前還沒有較有效的抗生素能夠治療出血性大腸桿菌感染所導致的症狀，因此更進一步的了解出血性大腸桿菌的致病機轉是必須的。從先前許多研究指出出血性大腸桿菌感染宿主時會出現A/E lesion的病理特徵，並且使得宿主腸道的微絨毛出現抹除的現象，而在我們的研究中也發現出血性大腸桿菌感染線蟲會出現微絨毛肌動蛋白重新排列的現象。為了去釐清參予出血性大腸桿菌誘發微絨毛肌動蛋白重新排列的宿主因子，我們利用核糖核酸抑制(RNAi)的技術來找出在出血性大腸桿菌誘發微絨毛肌動蛋白重新排列的過程中參予的宿主因子，在找到的宿主基因中，抑制cyb-3基因能夠最顯著阻止出血性大腸桿菌誘發的病理特徵。在G2/M時期，由cyb-3基因轉譯出的細胞週期蛋白B3能夠透過和週期蛋白依賴性激酶1(CDK1)結合形成活化態的複合體進而調控細胞週期。因此我們想去探討細胞週期蛋白B3以及週期蛋白依賴性激酶1(CDK1)在出血性大腸桿菌誘發哺乳動物細胞微絨毛肌動蛋白重新排列的現象中所扮演的角色，在我們的結果中發現當出血性大腸桿菌感染人類大腸直腸癌細胞株(Caco-2 cells)時會導致在G2/M時期的細胞數顯著得增加，接著我們利用也利用掃描式電子顯微鏡觀察腸道細胞表面上微絨毛的結構，根據我們的實驗證實了週期蛋白依賴性激酶1(CDK1)對於出血性大腸桿菌誘發的病理機制是必需的，並且我們也發現週期蛋白依賴性激酶1(CDK1)的酵素受質diaphanous-related formin 1 (DIAPH1)也參予在出血性大腸桿菌誘發的病理機制中。總結我們的所有實驗結果，我們發現週期蛋白依賴性激酶1(CDK1)在出血性大腸桿菌誘發哺乳動物細胞微絨毛肌動蛋白重新排列的過程中扮演重要的角色。

    Enterohemorrhagic E. coli (EHEC) serotype O157:H7 is a human pathogen that can cause serious diseases such as hemorrhagic colitis, hemolytic uremic syndrome (HUS) and even death. EHEC is transmitted to humans primarily through the fecal–oral route and colonizing on human intestine. There are no available antibiotics to improve the course of disease, therefore understanding the pathogenesis of EHEC is required. Previous studies indicated that EHEC can cause specific attaching and effacing (A/E) lesions which lead to microvillar effacement in host intestine. Moreover, in our studies found there is microvillar actin rearrangement in C. elegans during EHEC infection. To identify the host factors required for this EHEC-induced microvillar actin rearrangement, we conducted a genetic suppressor screen to identify the host genes that are required for the EHEC-induced microvillar actin rearrangement. Among the genes identified, knockdown of the cyb-3 gene significantly abolished this EHEC-induced pathology. Cyclin B3, encoded by cyb-3, regulates cell cycle through binding to the cyclin-dependent kinase 1 (CDK1) and then forming an activating complex during G2/M phase. We therefore aimed to study the role of cyclin B3 and CDK1 in mammalian cells during EHEC infection. We found that when the Caco-2 cells, which derived from a human colorectal carcinoma, are infected with EHEC, the G2/M phase population is significantly increased. Next, we monitored the morphology of human intestinal microvilli after EHEC infection by scanning electron microscopy (SEM). In these experiments, we demonstrated that CDK1 is essential for the EHEC-induced microvillar actin rearrangement. Moreover, we found that diaphanous-related formin 1 (DIAPH), a CDK1 kinase substrate, also involves in this EHEC-induced pathology. Taken all together, our current data showed that CDK1 plays an important role in the EHEC-induced microvillar actin rearrangement in mammalian cells.

    摘要 I Abstract III 誌謝 V Introduction 1 Enterohemorrhagic Escherichia coli (EHEC) 1 Attaching and effacing (A/E) lesion of EHEC 1 EHEC virulence factors 2 Animal model: Caenorhabditis elegans 2 EHEC-induced ACT-5 ectopic expression in C. elegans 3 Genetic suppressor screen for host factors required for EHEC-induced ACT-5 ectopic expression 4 CYB-3 (CCNB3) and CDK-1 (CDK1) 4 Cell model: Caco-2 cells 5 The significance of this study 6 Materials and methods 7 Nematode strains 7 Bacterial strains 7 Cell culture 8 EHEC Infection 9 Cells synchronize by thymidine and Flow cytometry 9 Western blotting 10 Plasmid construction and Transfection 15 Quantitative RT-PCR 17 Scanning electron microscopy 19 Immunofluorescence microscopy 20 Specific CDK-1 Inhibitor treatment in vitro 21 Specific CDK-1 Inhibitor treatment in vivo 21 C. elegans ACT-5 Ectopic Expression assay 22 Results 23 Gram negative intestinal pathogens induce significant microvillar actin (ACT-5) ectopic expression in C. elegans during five days infection 23 G2/M population of cell cycle is increased during EHEC infection 24 Microvillar actin rearrangement can be observed during EHEC infection 25 CCNB3 is essential for EHEC-induced microvillar effacement 26 CDK1 plays an important role in EHEC-induced microvillar effacement 27 DIAPH1 may be induced and regulated by CDK1 and CCNB3 during EHEC infection 28 Type III secretion system may not involve in EHEC-induced microvillar effacement 29 CDK1 may involves in the attachment of EHEC-induced A/E lesion 30 Conclusion 32 Discussion 33 Reference 35 Figures 39 Fig. 1 Gram negative intestinal pathogens induce significant microvillar actin (ACT-5) ectopic expression in C. elegans during five days infection. 40 Fig. 2 The cell population of G2/M phase was induced during EHEC infection. 45 Fig. 3 Microvillar actin rearrangement can be observed during EHEC infection. 47 Fig. 4 CCNB3 is essential for EHEC-induced microvillar actin rearrangement. 50 Fig. 5 CDK1 plays an important role in EHEC-induced microvillar effacement. 55 Fig. 6 DIAPH1 may be induced and regulated by CDK1 and CCNB3 during EHEC infection. 59 Fig. 7 Type III secretion system may not involve in EHEC-induced microvillar effacement. 64 Fig. 8 CDK1 may involves in the attachment of EHEC-induced A/E lesion. 68 Fig. 9 The model of EHEC-induced microvillar actin rearrangement in mammalian cells. 69 Appendix 70 Appendix 1 Bacterial strains 70 Appendix 2 Genetic suppressor screen results 71

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