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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
口試委員-陳振暐
口試委員-鄧景浩
口試委員-許鴻猷
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 
學科別分類
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


參考文獻 1. Bartlett JG. 2006. Narrative review: the new epidemic of Clostridium difficile-associated enteric disease. Annals of internal medicine 145:758-764.
2. Hall IC, O'Toole E. 1935. Intestinal flora in new-born infants: With a description of a new pathogenic anaerobe, bacillus difficilis. American Journal of Diseases of Children 49:390-402.
3. Pepin J, Valiquette L, Alary ME, Villemure P, Pelletier A, Forget K, Pepin K, Chouinard D. 2004. Clostridium difficile-associated diarrhea in a region of Quebec from 1991 to 2003: a changing pattern of disease severity. CMAJ : Canadian Medical Association journal = journal de l'Association medicale canadienne 171:466-472.
4. Prevention. CfDCa. 2013. Antibiotic Resistance Threats in the United States, 2013.
5. Zimlichman E, Henderson D, Tamir O, Franz C, Song P, Yamin CK, Keohane C, Denham CR, Bates DW. 2013. Health care-associated infections: a meta-analysis of costs and financial impact on the US health care system. JAMA internal medicine 173:2039-2046.
6. Rupnik M, Wilcox MH, Gerding DN. 2009. Clostridium difficile infection: new developments in epidemiology and pathogenesis. Nature reviews. Microbiology 7:526-536.
7. Kyne L, Sougioultzis S, McFarland LV, Kelly CP. 2002. Underlying disease severity as a major risk factor for nosocomial Clostridium difficile diarrhea. Infection control and hospital epidemiology 23:653-659.
8. Navaneethan U, Mukewar S, Venkatesh PG, Lopez R, Shen B. 2012. Clostridium difficile infection is associated with worse long term outcome in patients with ulcerative colitis. Journal of Crohn's & colitis 6:330-336.
9. Redelings MD, Sorvillo F, Mascola L. 2007. Increase in Clostridium difficile-related mortality rates, United States, 1999-2004. Emerging infectious diseases 13:1417-1419.
10. Kelly CP. 2012. Current strategies for management of initial Clostridium difficile infection. Journal of hospital medicine 7 Suppl 3:S5-10.
11. McDonald LC, Killgore GE, Thompson A, Owens RC, Jr., Kazakova SV, Sambol SP, Johnson S, Gerding DN. 2005. An epidemic, toxin gene-variant strain of Clostridium difficile. The New England journal of medicine 353:2433-2441.
12. Lessa FC, Gould CV, McDonald LC. 2012. Current status of Clostridium difficile infection epidemiology. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America 55 Suppl 2:S65-70.
13. Pepin J, Valiquette L, Cossette B. 2005. Mortality attributable to nosocomial Clostridium difficile-associated disease during an epidemic caused by a hypervirulent strain in Quebec. CMAJ : Canadian Medical Association journal = journal de l'Association medicale canadienne 173:1037-1042.
14. Kuijper EJ, Coignard B, Tull P. 2006. Emergence of Clostridium difficile-associated disease in North America and Europe. Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases 12 Suppl 6:2-18.
15. Clements AC, Magalhaes RJ, Tatem AJ, Paterson DL, Riley TV. 2010. Clostridium difficile PCR ribotype 027: assessing the risks of further worldwide spread. The Lancet. Infectious diseases 10:395-404.
16. Sebaihia M, Wren BW, Mullany P, Fairweather NF, Minton N, Stabler R, Thomson NR, Roberts AP, Cerdeno-Tarraga AM, Wang H, Holden MT, Wright A, Churcher C, Quail MA, Baker S, Bason N, Brooks K, Chillingworth T, Cronin A, Davis P, Dowd L, Fraser A, Feltwell T, Hance Z, Holroyd S, Jagels K, Moule S, Mungall K, Price C, Rabbinowitsch E, Sharp S, Simmonds M, Stevens K, Unwin L, Whithead S, Dupuy B, Dougan G, Barrell B, Parkhill J. 2006. The multidrug-resistant human pathogen Clostridium difficile has a highly mobile, mosaic genome. Nature genetics 38:779-786.
17. Voth DE, Ballard JD. 2005. Clostridium difficile toxins: mechanism of action and role in disease. Clinical microbiology reviews 18:247-263.
18. Hennequin C, Janoir C, Barc MC, Collignon A, Karjalainen T. 2003. Identification and characterization of a fibronectin-binding protein from Clostridium difficile. Microbiology 149:2779-2787.
19. Waligora AJ, Hennequin C, Mullany P, Bourlioux P, Collignon A, Karjalainen T. 2001. Characterization of a cell surface protein of Clostridium difficile with adhesive properties. Infection and immunity 69:2144-2153.
20. Barroso LA, Wang SZ, Phelps CJ, Johnson JL, Wilkins TD. 1990. Nucleotide sequence of Clostridium difficile toxin B gene. Nucleic acids research 18:4004.
21. Dove CH, Wang SZ, Price SB, Phelps CJ, Lyerly DM, Wilkins TD, Johnson JL. 1990. Molecular characterization of the Clostridium difficile toxin A gene. Infection and immunity 58:480-488.
22. Ng J, Hirota SA, Gross O, Li Y, Ulke-Lemee A, Potentier MS, Schenck LP, Vilaysane A, Seamone ME, Feng H, Armstrong GD, Tschopp J, Macdonald JA, Muruve DA, Beck PL. 2010. Clostridium difficile toxin-induced inflammation and intestinal injury are mediated by the inflammasome. Gastroenterology 139:542-552, 552 e541-543.
23. Bacci S, Molbak K, Kjeldsen MK, Olsen KE. 2011. Binary toxin and death after Clostridium difficile infection. Emerging infectious diseases 17:976-982.
24. Perelle S, Gibert M, Bourlioux P, Corthier G, Popoff MR. 1997. Production of a complete binary toxin (actin-specific ADP-ribosyltransferase) by Clostridium difficile CD196. Infection and immunity 65:1402-1407.
25. Calabi E, Calabi F, Phillips AD, Fairweather NF. 2002. Binding of Clostridium difficile surface layer proteins to gastrointestinal tissues. Infection and immunity 70:5770-5778.
26. Barketi-Klai A, Hoys S, Lambert-Bordes S, Collignon A, Kansau I. 2011. Role of fibronectin-binding protein A in Clostridium difficile intestinal colonization. Journal of medical microbiology 60:1155-1161.
27. Lin YP, Kuo CJ, Koleci X, McDonough SP, Chang YF. 2011. Manganese binds to Clostridium difficile Fbp68 and is essential for fibronectin binding. The Journal of biological chemistry 286:3957-3969.
28. Twine SM, Reid CW, Aubry A, McMullin DR, Fulton KM, Austin J, Logan SM. 2009. Motility and flagellar glycosylation in Clostridium difficile. Journal of bacteriology 191:7050-7062.
29. Stabler RA, He M, Dawson L, Martin M, Valiente E, Corton C, Lawley TD, Sebaihia M, Quail MA, Rose G, Gerding DN, Gibert M, Popoff MR, Parkhill J, Dougan G, Wren BW. 2009. Comparative genome and phenotypic analysis of Clostridium difficile 027 strains provides insight into the evolution of a hypervirulent bacterium. Genome biology 10:R102.
30. Varga JJ, Nguyen V, O'Brien DK, Rodgers K, Walker RA, Melville SB. 2006. Type IV pili-dependent gliding motility in the Gram-positive pathogen Clostridium perfringens and other Clostridia. Molecular microbiology 62:680-694.
31. Maresso AW, Schneewind O. 2008. Sortase as a target of anti-infective therapy. Pharmacological reviews 60:128-141.
32. Hendrickx AP, Budzik JM, Oh SY, Schneewind O. 2011. Architects at the bacterial surface - sortases and the assembly of pili with isopeptide bonds. Nature reviews. Microbiology 9:166-176.
33. Schneewind O, Mihaylova-Petkov D, Model P. 1993. Cell wall sorting signals in surface proteins of gram-positive bacteria. The EMBO journal 12:4803-4811.
34. Speziale P, Pietrocola G, Rindi S, Provenzano M, Provenza G, Di Poto A, Visai L, Arciola CR. 2009. Structural and functional role of Staphylococcus aureus surface components recognizing adhesive matrix molecules of the host. Future microbiology 4:1337-1352.
35. Patti JM, Allen BL, McGavin MJ, Hook M. 1994. MSCRAMM-mediated adherence of microorganisms to host tissues. Annual review of microbiology 48:585-617.
36. Budzik JM, Marraffini LA, Souda P, Whitelegge JP, Faull KF, Schneewind O. 2008. Amide bonds assemble pili on the surface of bacilli. Proceedings of the National Academy of Sciences of the United States of America 105:10215-10220.
37. Marraffini LA, Schneewind O. 2006. Targeting proteins to the cell wall of sporulating Bacillus anthracis. Molecular microbiology 62:1402-1417.
38. Spirig T, Weiner EM, Clubb RT. 2011. Sortase enzymes in Gram-positive bacteria. Molecular microbiology 82:1044-1059.
39. Comfort D, Clubb RT. 2004. A comparative genome analysis identifies distinct sorting pathways in gram-positive bacteria. Infection and immunity 72:2710-2722.
40. Deivanayagam CC, Rich RL, Carson M, Owens RT, Danthuluri S, Bice T, Hook M, Narayana SV. 2000. Novel fold and assembly of the repetitive B region of the Staphylococcus aureus collagen-binding surface protein. Structure 8:67-78.
41. Cafardi V, Biagini M, Martinelli M, Leuzzi R, Rubino JT, Cantini F, Norais N, Scarselli M, Serruto D, Unnikrishnan M. 2013. Identification of a novel zinc metalloprotease through a global analysis of Clostridium difficile extracellular proteins. PloS one 8:e81306.
42. Hensbergen PJ, Klychnikov OI, Bakker D, van Winden VJ, Ras N, Kemp AC, Cordfunke RA, Dragan I, Deelder AM, Kuijper EJ, Corver J, Drijfhout JW, van Leeuwen HC. 2014. A novel secreted metalloprotease (CD2830) from Clostridium difficile cleaves specific proline sequences in LPXTG cell surface proteins. Molecular & cellular proteomics : MCP 13:1231-1244.
43. Jenal U, Malone J. 2006. Mechanisms of cyclic-di-GMP signaling in bacteria. Annual review of genetics 40:385-407.
44. Hengge R. 2009. Principles of c-di-GMP signalling in bacteria. Nature reviews. Microbiology 7:263-273.
45. Ryan RP, Fouhy Y, Lucey JF, Dow JM. 2006. Cyclic di-GMP signaling in bacteria: recent advances and new puzzles. Journal of bacteriology 188:8327-8334.
46. McDougald D, Rice SA, Barraud N, Steinberg PD, Kjelleberg S. 2012. Should we stay or should we go: mechanisms and ecological consequences for biofilm dispersal. Nature reviews. Microbiology 10:39-50.
47. Soutourina OA, Monot M, Boudry P, Saujet L, Pichon C, Sismeiro O, Semenova E, Severinov K, Le Bouguenec C, Coppee JY, Dupuy B, Martin-Verstraete I. 2013. Genome-wide identification of regulatory RNAs in the human pathogen Clostridium difficile. PLoS genetics 9:e1003493.
48. Wren BW, Tabaqchali S. 1987. Restriction endonuclease DNA analysis of Clostridium difficile. Journal of clinical microbiology 25:2402-2404.
49. Heap JT, Kuehne SA, Ehsaan M, Cartman ST, Cooksley CM, Scott JC, Minton NP. 2010. The ClosTron: Mutagenesis in Clostridium refined and streamlined. Journal of microbiological methods 80:49-55.
50. Heap JT, Pennington OJ, Cartman ST, Carter GP, Minton NP. 2007. The ClosTron: a universal gene knock-out system for the genus Clostridium. Journal of microbiological methods 70:452-464.
51. Perutka J, Wang W, Goerlitz D, Lambowitz AM. 2004. Use of computer-designed group II introns to disrupt Escherichia coli DExH/D-box protein and DNA helicase genes. Journal of molecular biology 336:421-439.
52. Purdy D, O'Keeffe TA, Elmore M, Herbert M, McLeod A, Bokori-Brown M, Ostrowski A, Minton NP. 2002. Conjugative transfer of clostridial shuttle vectors from Escherichia coli to Clostridium difficile through circumvention of the restriction barrier. Molecular microbiology 46:439-452.
53. Williams DR, Young DI, Young M. 1990. Conjugative plasmid transfer from Escherichia coli to Clostridium acetobutylicum. Journal of general microbiology 136:819-826.
54. Heap JT, Pennington OJ, Cartman ST, Minton NP. 2009. A modular system for Clostridium shuttle plasmids. Journal of microbiological methods 78:79-85.
55. Hennequin C, Collignon A, Karjalainen T. 2001. Analysis of expression of GroEL (Hsp60) of Clostridium difficile in response to stress. Microbial pathogenesis 31:255-260.
56. van Leeuwen HC, Klychnikov OI, Menks MA, Kuijper EJ, Drijfhout JW, Hensbergen PJ. 2014. Clostridium difficile sortase recognizes a (S/P)PXTG sequence motif and can accommodate diaminopimelic acid as a substrate for transpeptidation. FEBS letters 588:4325-4333.
57. Donahue EH, Dawson LF, Valiente E, Firth-Clark S, Major MR, Littler E, Perrior TR, Wren BW. 2014. Clostridium difficile has a single sortase, SrtB, that can be inhibited by small-molecule inhibitors. BMC microbiology 14:219.
58. Kang M, Ko YP, Liang X, Ross CL, Liu Q, Murray BE, Hook M. 2013. Collagen-binding microbial surface components recognizing adhesive matrix molecule (MSCRAMM) of Gram-positive bacteria inhibit complement activation via the classical pathway. The Journal of biological chemistry 288:20520-20531.
59. Sinha B, Francois P, Que YA, Hussain M, Heilmann C, Moreillon P, Lew D, Krause KH, Peters G, Herrmann M. 2000. Heterologously expressed Staphylococcus aureus fibronectin-binding proteins are sufficient for invasion of host cells. Infection and immunity 68:6871-6878.
60. Paredes-Sabja D, Cofre-Araneda G, Brito-Silva C, Pizarro-Guajardo M, Sarker MR. 2012. Clostridium difficile spore-macrophage interactions: spore survival. PloS one 7:e43635.
61. Purcell EB, McKee RW, McBride SM, Waters CM, Tamayo R. 2012. Cyclic diguanylate inversely regulates motility and aggregation in Clostridium difficile. Journal of bacteriology 194:3307-3316.
62. Kuehne SA, Minton NP. 2012. ClosTron-mediated engineering of Clostridium. Bioengineered 3:247-254.
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