困難梭狀芽孢桿菌是革蘭氏陽性會產生孢子的厭氧菌，是造成健康護理相關感染的主要致病源，包括抗生素治療後誘發的相關性腹瀉和偽膜性腸炎。近年來，特定核糖型困難梭狀芽孢桿菌的出現與流行，造成嚴重困難梭狀芽孢桿菌感染(CDI)的病例增加。孢子、TcdA和/或TcdB毒素都是CDI的主要的毒力因子。困難梭狀芽孢桿菌的孢子是休眠細胞，對胃的酸性環境和許多抗微生素具有高度的抗性。一旦孢子進入腸胃道，會黏附在結腸上皮細胞上，並萌發成為營養細胞。此時期的困難梭狀芽孢桿菌會分泌TcdA和/或TcdB毒素，誘導細胞發炎性趨化因子和細胞因子的產生，並造成嗜中性白血球的滲入，最終導致結腸上皮組織細胞的破壞。雖然困難梭狀芽孢桿菌的生理特性和發病機制已經廣泛地被研究，但困難梭狀芽孢桿菌本身的DNA複製合成機制尚未被探索，直至今日我們對困難梭狀芽孢桿菌的DNA聚合酶的生化功能和特性仍然一無所知。在本論文研究中，透過對蛋白質序列的比對，我們發現困難梭狀芽孢桿菌的DNA聚合酶I (CdPol I)與大腸桿菌DNA聚合酶I (EcPol I)具有高度的同源性，為了解CdPol I的生化特性，我們純化了CdPol I蛋白，並利用生物化學方法測定其活性與功能。結果顯示CdPol I含有5'→3'核酸外切酶 (5-Exo) 和5'→3'聚合酶 (DNA Pol) 酵素活性，但缺乏3'→5' 核酸外切酶或複製錯誤校對的功能。另外，我們也確認了CdPol I的5-Exo和DNA Pol活性所需的關鍵殘基。從生化測定的結果顯示，CdPol I含有DNA鏈置換、5'-flap核酸內切酶與相似於核糖核酸酶H的活性。為了瞭解CdPol I在菌內的功能，我們進一步採用ClosTron技術破壞菌中的polA基因。結果顯示，CdPol I的DNA Pol功能的破壞，造成困難梭狀芽孢桿菌的生長速率明顯地降低。然而，DNA Pol缺陷的困難梭狀芽孢桿菌的生長，在氧化壓力的條件下則呈現正常。綜合以上實驗結果，我們推斷CdPol I在困難梭狀芽孢桿菌中在DNA複製和修復中可能扮演重要的作用。
關鍵字 : 困難梭狀芽孢桿菌、DNA聚合酶I

SUMMARY
Clostridium difficile (C. difficile) has become a leading cause of health care-associated infections, including antibiotic-associated diarrheal disease and pseudomembranous colitis. The emergence of epidemic ribotypes of C. difficile attributes to the surge of severe cases of C. difficile infection (CDI). Although the physiology and pathogenesis of C. difficile have been extensively studied, the fundamental mechanism of DNA replication in C. difficile has not been explored. The function and requirement of DNA polymerase I in C. difficile are still unknown. To understand the biochemical features of C. difficile DNA polymerase I, designated as CdPol I in this study, we expressed, purified CdPol I, and characterized the intrinsic properties by in vitro biochemical assays. The results show that CdPol I contains a 5'→3' exonuclease (5'-Exo) and 5'→3' DNA polymerase (DNA Pol) activities, but naturally lacks a 3'→5' exonuclease, or error-proofreading, activity. Additionally, the DNA Pol domain of CdPol I has DNA strand displacement, 5'-flap endonuclease, and RNase H-like activitie. To investigate the requirement of CdPol I in vivo, the ClosTron technology was used to disrupt the polA gene in C. difficile. The disruption of CdPol I DNA Pol domain significantly reduces the growth rate of C. difficile. Taken together, our results suggest that CdPol I may play an important role in DNA replication and repair in C. difficile.

Key words: Clostridium difficile, DNA polymerase I

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