系統識別號 U0026-3108201514300100
論文名稱(中文) 探討困難梭狀桿菌新穎膠原蛋白質 Csp1 之調控關係
論文名稱(英文) Regulation of a novel adhesin Csp1 in Clostridium difficile
校院名稱 成功大學
系所名稱(中) 微生物及免疫學研究所
系所名稱(英) Department of Microbiology & Immunology
學年度 103
學期 2
出版年 104
研究生(中文) 林東昇
研究生(英文) Tung-Sheng Lin
學號 S46011126
學位類別 碩士
語文別 英文
論文頁數 53頁
口試委員 指導教授-黃一修
中文關鍵字 困難梭狀桿菌  膠原蛋白結合蛋白質  金屬蛋白酶  c-di-GMP 
英文關鍵字 Clostridium difficile  collagen-binding protein  metalloprotease  c-di-GMP 
中文摘要 困難梭狀桿菌是一株革蘭氏陽性產孢的人類致病菌,同時也是使用抗生素而導致腹瀉的主因之一。困難梭狀桿菌感染的病徵從輕微的腹瀉到致死率極高的惡性結腸炎。自從2000年高致病菌株出現在北美與歐洲地區,困難梭狀桿菌感染的疫情被已擴散到全世界各地,並且在過去十年當中不斷攀升。困難梭狀桿菌的毒性主要取決於分泌兩種醣基化毒素,毒素A 與毒素 B。然而,其他因子,例如困難梭狀桿菌附著到宿主細胞的能力以及困難梭狀桿菌如何導致反覆復發的主因仍尚未釐清。在細菌造成感染前,細菌必須先與宿主細胞進行附著,而困難梭狀桿菌具有許多的表面蛋白,對於細菌在附著上扮演重要的角色。本研究主要聚焦在由分選酶錨定的表面蛋白質裏,其中一種能與膠原蛋白結合的蛋白質Csp1(CD2831),在前人的研究中,發現Csp1 大部分為外泌型蛋白質,而非附著於細菌表面蛋白。另一方面,Zmp1(CD2830),一種外泌型的金屬蛋白酶,會去對Csp1 進行剪切,同時,這兩分子又受到一個二級訊息分子c-di-GMP 所調控,因此,本篇研究主要探討Csp1, Zmp1 與c-di-GMP之間的調控關係。結果顯示,我們證明 Csp1 的表現是由二級訊息因子 c-di-GMP 所調控。c-di-GMP 會正相調控Csp1,同時也會反相調控金屬蛋白酶 Zmp1。 c-di-GMP會去控制 Csp1 是附著於細菌細胞壁上或是分泌於細菌體外。未來的研究將聚焦在感染過程中 c-di-GMP 如何去調節 Csp1 的定位。
英文摘要 Clostridium difficile is a Gram positive, spore forming obligate anaerobic bacteria, causive agent of the antibiotic associated diarrhea. C. difficile infection (CDI) is caused by host microflora disruption through broad-spectrum antibiotics. The emergence of hypervirulent C. difficile strains resulting in high morbidity and mortality has occurred in many countries. The C. difficile secrets toxins TcdA, TcdB and binary toxin CDT. TcdA and TcdB are responsible for gastrointestinal inflammation, epithelial cell tight junction lose and apoptosis. Before infection, the pathogens need to attach to the host cell first. In C. difficile, surface protein are responsible for interaction with host cell and extracellular matrix of vertebrates. Csp1, a potential cell wall protein anchored on cell wall by sortase, and a putative collagen binding adhesin. Zmp1, a metalloprotease which can cleave Csp1 in vitro. c-di-GMP, a second messenger molecules modulate C. difficile motility, biofilm and toxin. In our study, we focus on Csp1 regulation between Zmp1 and c-di-GMP. Our results showed that in the absence of metalloprotease, Csp1 localization is increased on the cell wall, c-di-GMP enhance the quantity of Csp1 , and the PPKTG motif is needed for recognization by sortase. In summary, our results demonstrated the localization and regulation of a novel adhesin Csp1 by Zmp1 and c-di-GMP.
論文目次 中文摘要 I
Abstract II
Acknowledgements III
Table of Contents V
List of tables VII
List of figures VIII
Abbreviations IX
Chapter 1 1
Introduction 1
1.1 Clostridium difficile infectious disease 1
1.2 Clostridium difficile infection (CDI) epidemiology 1
1.3 Clostridium difficile 630 2
1.4 Clostridium difficile virulence factors 2
1.5 Clostridium difficile non-toxin virulence factors 3
1.6 Sortase 3
1.7 Csp1 (CD2831) (cell surface protein 1) 5
1.8 Zmp1 (CD2830) 5
1.9 c-di-GMP 6
1.10 Rationale and aim of this study 7
Chapter 2 8
Material and methods 8
2.1 Bacterial strains and growth conditions 8
2.2 Cell strain 8
2.3 Bacterial growth curve 8
2.4 DNA isolation 9
2.5 Plasmid isolation 9
2.6 Polymerase chain reaction, PCR 10
2.7 Enzyme digestion 11
2.8 Ligation 11
2.9 Transformation 12
2.10 RNA preparation, cDNA synthesis and qRT-PCR 12
2.11 Mutagenesis and genetic complementation studies. 14
2.12 Conjugation 15
2.13 Cell fractionation 15
2.14 SDS-PAGE, and immunoblotting. 16
2.15 Immunofluorescence microscopy 17
2.16 Cell adhesion assay 17
Chapter 3 19
Results 19
3.1 Insertional inactivation of C. difficile 630DPS zmp1 19
3.2 Zmp1 is involved in cleaving Csp1 in C. difficile 20
3.3 Inverse regulation of zmp1 and csp1 by c-di-GMP 20
3.4 High level of c-di-GMP results in increasing Csp1 expression in C. difficile 21
3.5 Csp1 is located on the surface of C. difficile under high level of c-di-GMP 22
3.6 The Csp1 cell wall anchoring motif PPKTG is implicated in sortase-associated transpepidation 23
3.7 Csp1 is involved in adhesion to fibroblast cell 24
Chapter 4 25
Discussion 25
References 28
Tables 34
Figures 38
Appendix 50

List of tables
Table 1. Strains and Plasmids Used in this Study 34
Table 2. Sequences of oligonucleotide primers used in this study 36

List of figures
Fig. 1 Schematic representation of mutant generation using the ClosTron and genetic organization of the C. difficile CD2830 and CD2831 locus. . 38
Fig. 2 Inactivation of genes encoding Zmp1 in C. difficile using the Clos Tron system.. 40
Fig. 3 Without Zmp1, localization of Csp1 shifts from supernatant to cell wall. 41
Fig. 4 Transcriptional expression level of zmp1 or csp1 in different C. difficile strains by quantitative real-time PCR analysis. 42
Fig.5 Csp1 increase under high level of c-di-GMP in different strains by western-blot analysis. 44
Fig. 6 Immuno-fluorescent detection for Csp1 on C. difficile. 47
Fig. 7 PPKTG motif on Csp1 is implicated in cell wall sorting signal which can be recognized by sortase. 48
Fig. 8 Binding of DPS, ∆csp1, and ∆zmp1 mutant strain to collagen-productive fibroblast cell line NIH3T3 49

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