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研究生：	謝可盈 Hsieh, Ke-Ying
論文名稱：	探討第四型線毛在困難梭狀芽孢桿菌致病過程所扮演角色 The Contribution of Type IV Pili in Clostridium difficile pathogenesis
指導教授：	黃一修 Huang, I-Hsiu
學位類別：	碩士 Master
系所名稱：	醫學院 - 微生物及免疫學研究所 Department of Microbiology & Immunology
論文出版年：	2018
畢業學年度：	106
語文別：	英文
論文頁數：	72
中文關鍵詞：	困難梭狀芽孢桿菌、抗生素相關腹瀉、第四型線毛
外文關鍵詞：	Clostridium difficile, antibiotic-associated diarrhea, type Ⅳ pili
相關次數：	點閱：99 下載：0
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困難梭狀芽孢桿菌(Clostridium difficile)是革蘭氏陽性且會產生孢子的厭氧菌，也是院內感染中抗生素相關腹瀉的重要致病菌，能藉著孢子型態散播在環境，經由糞口途徑進入宿主腸道當中。近年研究發現在此種細菌的細胞壁上有第四型線毛(Type IV pili)的蛋白結構，已知它的功能跟細菌的自我聚集 (Auto-aggregation)、滑行運動 (Gliding motility)及生物膜(Biofilm formation)形成有關，然而，第四型線毛在困難梭狀芽孢桿菌的致病過程中扮演的角色還不清楚。在我們的研究中，我們利用修改後的 Clostron 方法將第四型線毛的線毛蛋白基因 pilA1 (major pilin)及收縮 ATP 蛋白酶基因 pilT(putative retraction ATPase) 進行插入性突變，藉此比較野生株以及突變株之間的差異，我們發現在小鼠感染模式中，菌株R20291第四型線毛的突變株會造成小鼠比較嚴重的發炎反應，為了找出突變型毒力上升的可能原因，我們利用 Caco-2 細胞的感染實驗，也發現R20291的第四型線毛收縮ATP蛋白酶基因pilT突變株呈現較高的細胞貼附能力，我們推測可能是因為細菌失去了收縮的能量來源而導致線毛無法收縮，因此有較高的機會能貼附到細胞上，因此R20291第四型線毛在細菌定殖腸道的過程扮演了一個角色且能促進細菌貼附的能力。另外，我們同時分析R20291以及630這兩種不同的菌株，第四型線毛野生株和突變株在細菌自我聚集、滑行運動以及生物膜形成的這些功能差異。在我們的結果中，我們發現R20291 的第四型線毛突變型會有較高的自我聚集能力，但是630第四型線毛突變型菌株則相反，而在滑行運動中，R20291的第四型線毛突變型是呈現較低的移動能力，因此我們認為第四型線毛在困難梭狀芽孢桿菌R20291中能促使細菌進行滑行運動。綜合以上結果，我們發現第四型線毛的表達在R20291及630當中可能不盡相同，但我們仍證實了R20291第四型線毛在困難梭狀芽孢桿菌的致病過程中，能透過使細菌貼附在宿主細胞上而讓細菌定殖在腸道中，並且也能透過線毛的收縮來達成滑行運動的功能。

Clostridium diffcile is a Gram-positive spore-forming anaerobic bacterium. It is the leading cause of antibiotic-associated diarrhea in nosocomial infection and the transmission is through fecal-oral route. Recently type IV pili (TFP), a proteinaceous polymer widely studied in many gram-negative pathogens was discovered to be produced by C. difficile and has been reported to promote bacterial auto-aggregation, gliding motility, and biofilm formation. However, the role that TFP plays in C. difficile pathogenesis is still unclear. In our study, TFP structural genes pilA1 (major pilin) and pilT (putative retraction ATPase) were inactivated using a modified ClosTron targeting system. To determine the role of TFP in pathogenesis in vivo, a mouse model of infection was established. We were surprised to find that in strain R20291, TFP mutants were more virulent in mouse model of infection. Furthermore, in vivo and in vitro results demonstrated that pilT mutant had higher adhesion efficiency than wild-type. We surmised that in strain R20291, inactive PilT might lead to longer pili formation because pili lost its energy to retract. Therefore, we speculated that TFP might promote adherence in C. difficile R20291 to help colonize in host intestine. Further investigation of the function of TFP in aggregation, biofilm formation, gliding motility, we used both strain R20291 and 630 to demonstrate it. In strain R20291, TFP mutant showed higher aggregation efficiency and a decreased gliding motility phenotype. However, in strain 630, aggregation efficiency of TFP mutants was lower. Together, our results showed that TFP mediated phenotypes might be different in strain R20291 and 630 and we demonstrated that type　IV　pili is important in CDI and may promote adherence in the process of pathogenesis in strain R20291.

中文摘要	I
Abstract	Ⅱ
致謝	Ⅲ
Abbreviations 	Ⅸ
Chapter 1	1
Introduction	1
1.1 Clostridium difficile infection 	1
1.2 Diagnosis and treatment of C. difficile infection	2
1.3 C. difficile surface proteins and toxins	3
1.4 Type Ⅳ pili 	4
1.5 Rationale	5
Chapter 2	7
Materials and methods	7
2.1 Bacteria stains and growth conditions	7
2.2 Heat-shock transformation of E. coli	7
2.3 E. coli- C. difficile conjugation	7
2.4 Extraction of genomic DNA from C. difficile	8
2.5 Polymerase chain reaction (PCR) primers	9
2.6 Diagnostic PCR	9
2.7 High fidelity PCR	9
2.8 PCR product purification	10
2.9 Agarose gel electrophoresis	10
2.10 Restriction enzyme digestion	10
2.11 Purification and quantification of digested DNA	10
2.12 DNA ligation	11
2.13 ClosTron mutagnenesis of pilT and pilA1	11
2.14 Markerless ClosTron mutagenesis of pilT and pilA1	12
2.15 Generation of strains carrying a plasmid-based inducible pilT and pilA1	12
2.16 Recombinat PilA1 protein expression and purification	13
2.17 Antibody generation	13
2.18 Minimum bactericidal concentration of clindamycin	14
2.19 Preparation of C. difficile spores	14
2.20 Mouse model of C. difficile infection	15
2.21 Survival assay	16
2.22 In vivo adherence assay	16
2.23 Ex vivo adherence assay	16
2.24 Competition assay 	17
2.25 Cytotoxicity assay	17
2.26 Detection of toxin A and B by ELISA	18
2.27 Cell adherence assay	18
2.28 Aggregation assay	19
2.29 Biofilm formation assay	19
2.30 Gliding motility assay	19
2.31 Cellular fractionation assay	20
2.32 Western blot	21
Chapter 3	22
Results	22
3.1 Type Ⅳ pilus operon exists widely in the genome of different C. difficile strains  
and clinical isolates	22
3.2 C. difficile express TFP in vivo during infection and the major pilin PilA1 is 
immunogenic	22
3.3 Construction of modified ClosTron mutant	23
3.4 Markerless ClosTron mutant showed similar susceptibility to clindamycin as  
wild-type in strain R202091	24
3.5 Type Ⅳ pili mutants were more virulent in mouse model of infection	25
3.6 The supernatant of wild-type and type Ⅳ pili mutants showed the same cytotoxicity 
to Caco-2 cell	26
3.7 The pilT mutant showed increased adherence to Caco-2 and MDCK cells	26
3.8 The pilT mutant showed increased adherence to mice intestine in vivo and ex vivo	27
3.9 TFP mutant did not outgrow wild-type in mouse model of infection	28
3.10 Type Ⅳ pili mutants increased bacterial auto-aggregation compared to wild-type 
in strain R20291, but decreased in strain 630	28
3.11 No significant difference in biofilm formation between wild-type and type Ⅳ pili 
mutant	30
3.12 Type Ⅳ pili mutants showed decreased gliding motility in strain R20291	30
3.13 PilA1 expressed on the surface of C. difficile pilT mutant in two forms	31
Chapter 4	32
Discussion	32
References	38
Tables	47
Figures	53
Supplementary materials	71

                                    

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