臺灣博碩士論文加值系統

血鏈球菌(Streptococcus sanguinis) 是形成牙菌斑生物膜菌種之一且可入侵血液並引發伺機性亞急性心內膜炎。依已知口腔鏈球菌基因體得知血鏈球菌是唯一含第四型纖毛 (Type IV pili) 基因群的口腔鏈球菌。先前本實驗室的研究在第四型纖毛基因群的上游發現三個轉錄起始位置。利用電子顯微鏡觀察野生株時可在菌體表面發現由PilA 構成之短纖毛結構。然而，血鏈球菌的第四型纖毛之功能仍然不詳。本研究目的在分析此基因群的表現與功能。由纖毛啟動子結合報導基因分析結果得知，預測的三個啟動子均具活性，且CcpA蛋白質可負向調控纖毛啟動子的表現。經由西方墨點法分析得知生長時期影響菌體表面PilA的表現量，而以初期靜止期的表現量最高。利用第四型纖毛PilA的突變株得知第四型纖毛參與血鏈球菌與HeLa細胞及口腔鱗狀上皮細胞的沾附。綜合以上，第四型纖毛基因群的表現是由一複雜系統所調控，且經由這系統產生的纖毛有沾附宿主細胞的能力，顯示第四型纖毛對血鏈球菌致病機轉的重要性。

Streptococcus sanguinis is a member of the dental plaque and occasionally causes infective endocarditis. Thus far the gene cluster (pil) encoding type IV pili (Tfp) was found only in the genome of Streptococcus sanguinis SK36. Previous studies by using 5’ RACE analysis revealed 3 putative transcription initiation sites 5’ to the pil cluster. Short hair-like structures were observed on the surface of SK36 by using anti-SSA_2315 (PilA) antiserum under transmission electron microscopy. However, the biological functions of the Tfp in S. sanguinis SK36 remains unknown. This study aims to analyze the expression and function of the pil cluster. By using various pil promoter-reporter fusion strains, it was found that all 3 promoters were functional. The activity of a transcriptional fusion containing all 3 promoters was higher in the ccpA-deficient host than that in the wild-type background, indicating that the expression of the pil operon is subject to the regulation of CcpA. Western analysis of the PilA protein indicated that the biogenesis of Tfp was regulated by growth phases, with the highest expression at the early stationary phase. Inactivation of SSA_2313-2315 led to a 40% reduction in adherence to HeLa cells and squamous cell carcinoma (SCC-4) compared to the wild-type strain. Taken together, the expression of the pil cluster was regulated by a complex system and the biosynthesis of Tfp was closely associated with the development of growth phase. The binding of S. sanguinis SK36 Tfp to host cells supports the role of Tfp in the pathogenesis of S. sanguinis SK36.

TABLE OF CONTENTS
論文指導教授推薦書.........................................i
論文口試委員審定書......................................... ii
長庚大學博碩士紙本論文著作授權書.............................iii
誌謝.................................................... iv
中文摘要................................................. vi
ABSTRACT................................................vii
TABLE OF CONTENTS.......................................viii
LIST OF TABLES...........................................ix
LIST OF FIGURES...........................................x
LIST OF APPENDIX.........................................xi
INTRODUCTION..............................................1
MATERIALS AND METHODS.....................................10
RESULTS...................................................17
DISCUSSION................................................23
TABLES....................................................27
FIGURES...................................................30
APPENDIX..................................................40
REFERRENCE................................................42


LIST OF TABLES
TABLE 1. Bacteria strains constructed in this study.....................................27

 
LIST OF FIGURES
FIG. 1. S. sanguinis SK36 pil cluster and the construction of pil-
deletion derivatives....................................................................30
FIG. 2. The promoter activity of the type IV pili gene cluster of S. sanguinis SK36....31
FIG. 4. The regulation of pil promoters of the type IV pili gene cluster of S. sanguinis SK36 by CcpA............................................................................34
FIG. 5. The regulation of pil promoters of the type IV pili gene cluster of S. sanguinis SK36 by GlnR............................................................................35
FIG. 6. The expression of Tfp of wild-type S. sanguinis SK36 at different growth phases ................................................................................36
FIG. 7. Piliation of the wild-type and SSA_2313-2315 mutant strain examined by TEM.....37
FIG. 8. The adherence efficiency of wild-type S. sanguinis SK36 and
its derivatives to HeLa cells.......................................................... 38
FIG. 9. The adherence efficiency of wild-type S. sanguinis SK36 and
its derivatives to squamous cell carcinoma (SCC-4)......................................39

 
LIST OF APPENDIX
Appendix 1. Properties and BlastP search results of S. sanguinis SK36
pil cluster............................................................................40

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