臺灣博碩士論文加值系統

血鏈球菌 (Streptococcus sanguinis) 是最初沾附在牙齒形成牙菌斑生物膜及可入侵血液進而引發伺機性亞急性心內膜炎的菌種之一。血鏈球菌的染色體含一第四型纖毛 (Type IV pili)基因群，其功能至今不詳。本研究目標在分析此基因群的表現與功能。第四型纖毛基因群共有16個基因，其中包括基因轉譯兩個ATPase (pilB 與 pilT)、細胞膜上的蛋白 (pilC)、3 個結構蛋白 (pilA)、一氧化氮還原酵素 (norD)、5 個未知功能的蛋白(gene 2312、2310與 2305-2303)、組裝相關蛋白 (pilMOP) 與將結構蛋白切割的分解酵素 (pilD)。反轉錄-連鎖聚合酶反應分析結果顯示pilD及下游lytB (轉譯乙醯胺基葡糖甘酶)在轉錄中是相連的，代表lytB也屬第四型纖毛基因群。由快速擴增cDNA法分析結果得知pilB ATG上游有三個轉錄起始位置，分別位於在pilB ATG上游的153 (P1)、536 (P2) 與837 (P3) 鹼基，P2與P3是σ70啟動子 (5’-TTGACA-N17-TATACT)，而P1只具extended -10。利用電子顯微鏡觀察野生株時可在細菌表面發現由PilA構成之纖毛狀結構，而具ATPase與結構蛋白等七個基因缺失的突變株則無此結構。此外第四型纖毛缺失的突變株，其形成生物膜的能力較野生株低，但此突變不影響血鏈球菌與Hela細胞黏附的能力和細菌運動性 (twitching motility)。綜合以上得知，第四型纖毛基因群可合生位於菌表的纖毛，且此纖毛與生物膜的形成有關。此基因群的表現是由三個啟動子共同完成，而其中兩個啟動子具長段的非轉譯區域，暗示出可此基因群的表現是由一複雜機制所調控。

Streptococcus sanguinis is a primary colonizer of human tooth and an opportunistic pathogen for subacute endocarditis. A Type IV pili (Tfp) gene cluster was reported in the complete genome of Streptococcus sanguinis SK36 recently. The goal of this research aimed to analyze the expression and function of this gene cluster. The Tfp gene cluster is composed of total 16 genes, from pilB to pilD. A contiguous transcript was detected between pilD and the downstream lytB by RT-PCR, suggesting that lytB is also part of the operon. Three transcription initiation sites, 153- (P1), 536- (P2) and 837-base (P3) 5’ to the translation start site of pilB, respectively, were detected by rapid amplification of cDNA ends (5’ RACE) analysis. Both the P2 and P3 mapped to a σ70-like promoters (5’-TTGACA-N17-TATACT), whereas only an extended -10 sequence was observed with the P1. An anti-PilA antibody was generated and used to examine the structure of the pil cluster encoded products by transmission electron microscopy (TEM). A short hair-like structure was observed in the wild-type SK36 but not the Pil-deficient mutant strain, indicating that pil cluster is responsible for the synthesis of this structure. Furthermore, the pil-deficient mutant strains exhibited reduced biofilm formation. However, neither the adherence to Hela cells nor the twitching motility was affected by the deletion in pil cluster. Taken together, these results suggest that the pil cluster is responsible for the synthesis of a surface structure, and this structure is associated with biofilm formation. The multiple transcription initiation sties with long 5’ untranslated regions suggested the presence of a complex regulation system for the expression of the pil cluster.

TABLE OF CONTENENTS
指導教授推薦書
口試委員會審定書
授權書 ....................................................................................................................iii
誌謝 .................................................................................................................... iv
中文摘要................................................................................................................... v
ABSTRACT............................................................................................................. vi
TABLE OF CONTENENTS.................................................................................... vii
LIST OF TABLES .................................................................................................. viii
LIST OF FIGURES .................................................................................................. ix
INTRODUCTION..................................................................................................... 1
I Viridans streptococci …………….………………………………………1
II Streptococcus sanguinis …………….……………………………………1
III Bacterial motility ……………….…………………………………………2
IV The function of Tfp and Tfp biogenesis.……………………………………5
V Regulation of Tfp and twitching motility…………….…………………… 8
VI Motive…………….……………………………………….……….………9
MATERIALS AND METHODS.............................................................................. 11
RESULTS................................................................................................................ 19
DISCUSSION ......................................................................................................... 27
REFERENCES........................................................................................................ 33
Tables ................................................................................................................... 39
Figures ................................................................................................................... 51
LIST OF TABLES
Table 1. Bacteria strains and plasmid used in this study ........................................... 39
Table 2. Primers used in this study........................................................................... 40
Table 3. Properties and BlastP search results in S. sanguinis SK36 pil locus............. 45
Table 4. BlastP atlas comparing S. sanguinis to P. aeruginosa PAO1 and C.
perfringens SM101..................................................................................... 47
LIST OF FIGURES
Fig. 1. P. aeruginosa PAO1, C. perfringens SM101 and S. sanguinis SK36 genes
involved in Tfp and the confirmation of the S. sanguinis SK36 operonic
organization................................................................................................ 51
Fig. 2. Promoter mapping of the pil cluster by RACE analysis. ................................ 53
Fig. 3. Construction of the S. sanguinis SK36 pil-deficient mutants. ........................ 55
Fig. 4. Growth curves of the wild-type SK36 and mutant strains. ............................. 57
Fig. 5. Production of the recombinant SSA_2315 protein and antisera. .................... 58
Fig. 6. Piliation of wild-type and YC13 mutant strain analyzed by TEM. ................. 59
Fig. 7. The twitching motility of wild-type S. sanguinis and mutant strains. ............. 60
Fig. 8. The biofilm formation of wild-type S. sanguinis and mutant strains. ............. 61
Fig. 9. The adherence of wild-type S. sanguinis and YC13 to Hela cells. ................. 62

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