臺灣博碩士論文加值系統

沙門氏菌(Salmonella)隸屬於腸內桿菌科(Enterobacteriaceae)，為一兼性厭氧 型革蘭氏陰性菌，由其腸道沙門氏菌亞種的鼠傷寒沙門氏菌血清型(Salmonella enterica serovar Typhimurium, S. Typhimurium)所造成的感染則是重要的人畜共通傳染病之一。S. Typhimurium在小腸上皮細胞的黏附作用被視為其致病機制的初 始階段，與之相關的毒力因子即為第一型線毛(type 1 fimbriae, T1F)。T1F由fim gene cluster所編碼，在fim gene cluster內，fimA編碼T1F的主要結構蛋白，而fimZ是主要的正向調控基因。FimZ可以直接結合於fimA的啟動子部位並增強結構基因的表達。fimY則是另一個正向調控基因，可以直接增強fimZ的表達。先前的研究發現了許多可以調控fimZ的上游基因成分(genetic elements)和環境因子(environmental factors)，而fimY上游的相關研究則十分有限。本研究旨在對T1F調控網絡fimY的部分進行探討，為能夠更完整地闡述S. Typhimurium的致病機理提供相關理論基礎。為了從S. Typhimurium中選殖能夠正向調控fimY的genetic elements，本研究使用質體pACYC184構建了一個平均插入片段大小約為2,000 bp的基因體庫(genomic library)，並最終產生了8,000多個包含重組質體的菌落。將菌落接種於LB agar上培養48小時後，若其可以與酵母菌發生凝集反應，則很有可能是通過上調fimY的表達來增強了第一型線毛的表現，當然這需要反轉錄聚合酶鏈鎖反應的進一步驗證。為了將篩選到特定基因的機率提高至90%以上，根據Carbon-Clarke equation，超過5,591個菌落被挑選並測試，結果均為陰性。後又將可能的調控基因sirA選殖入同樣的質體，並轉形入S. Typhimurium進行測試，結果仍無法產生凝集。為了探測可能影響fimY表達的環境因子，通過將fimY的啟動子區域DNA選殖入載體pMC1403，並轉形至S. Typhimurium，也就是構建了一個fimY-lacZ reporter system於S. Typhimurium。將此菌株分別培養在25 °C、37 °C 和 42 °C的環境中，發現fimY啟動子的活力會隨溫度升高而增強，T1F的表現量也呈現出類似的趨勢。酸性和鹼性的環境壓力對於 fimY 表達的影響也詳加探討，與pH=7的培養環境相比，fimY啟動子在pH=4的環境中幾乎不表 現活力。而在pH=8的情況下fimY啟動子的活力要高於pH=7的情況。在低Mg2+濃度(10 μM)環境壓力下，fimY啟動子的活力與對照組類似，並無明顯差異。環境因素調控fimY表達的具體機制仍須進一步的研究闡明。

Salmonella is a Gram-negative facultatively anaerobic intracellular bacterium and Salmonella enterica serovar Typhimurium remains one of the leading causes of foodborne illness in human. The ability to adhere to intestinal epithelium cells is considered as one of the complicated pathogenesis mechanisms conferred by Salmonella and is a prerequisite step for infection. Type 1 fimbriae (T1F), encoded by the fim gene cluster, is one of the most commonly seen adhesive organelles in Salmonella. In the fim gene cluster, fimA encodes for the major subunit of T1F, while fimZ encodes for a DNA-binding protein which can directly bind to fimA promoter region and activate its expression. fimY acts as another T1F regulatory gene and FimY can directly activate fimZ expression. Previous studies have shown that several genetic elements and environmental factors can regulate fimZ expression, while reports regarding the regulation of fimY are limited. Elucidation of the type 1 fimbriae regulatory network involving fimY could provide an insight to understand the pathogenesis of Salmonella. In order to clone the genetic elements that may upregulate the expression of fimY, a genomic library containing more than 8,000 colonies was constructed by ligating the partially digested genomic DNA fragments into pACYC184 vector and transformed into S. Typhimurium. According to Carbon-Clarke equation, in order to increase the possibility of finding the target genetic elements up to 90%, more than 5,591 colonies were tested for their ability to agglutinate to yeast cells when grown on LB agar. Any transformant that mediates agglutination may indicate the presence of the genetic determinants that activate the expression of type 1 fimbriae, possibly through upregulation of fimY. Nonetheless, this will be confirmed by RT-PCR. However, all the screened colonies turned out to be yeast agglutination negative. A possible regulatory gene sirA was also cloned into the same vector and still cannot
mediate agglutination. As to determine the environmental factors that may influence fimY expression, a fimY promoter-containing DNA fragment was cloned into the pMC1403 vector to construct a fimY-lacZ reporter molecule and transformed into S. Typhimurium. It was revealed that the fimY promoter activity increased as the temperature increased from 25 °C, 37 °C, to 42 °C. In addition, expression of T1F correlated with that of fimY expression. fimY expression in an acid or alkaline environment was also measured and its promoter activity was extremely low in pH=4 as compared to pH=7, while the promoter activity in pH=8 was higher than that in pH=7. fimY promoter activity in the low Mg2+ concentration (10 μM) environment was similar to that as observed when Salmonella was cultured in the rich media. What environmental factors that may regulate fimY expression still needs further investigation.

中文摘要.......... 1
Abstract........... 3

目 錄..........5
表目錄......... 8
圖目錄 ...... 9
附錄......... 10

第一章、文獻回顧..........11
第一節 沙門氏菌......... 11
第二節 S. Typhimurium 的致病機制 ........ 12
2.1 感染過程.......... 12
2.2 毒力因子(virulence determinants)....... 13
第三節 S. Typhimurium 第一型線毛 ........ 15
3.1 S.Typhimurium第一型線毛的受體........15
3.2 相變化(phase variation) ........... 16
3.3 fim gene cluster...... 16
第四節 fimY 在調控 S. Typhimurium 第一型線毛表達的過程中所扮演的角色 ............ 18
第五節 可能調控 fimY 表達的基因成分......... 20
5.1 sirA........20
5.2 lrp.........21
第六節 可能調控 fimY 表達的環境因子....... 22
6.1 溫度...... 22
6.2 pH........22
6.3 Mg2+ .........23

第二章、材料與方法........24
第一節 菌株的保存與培養......... 24
第二節 酵母菌凝集試驗.......... 24
第三節 細菌總 RNA(total RNA)的萃取......... 25
第四節 反轉錄聚合酶鏈鎖反應(reverse transcription PCR, RT-PCR) ....... 25
第五節 LB5010 NB1 菌株的構建......... 26
5.1 細菌菌體 DNA (genomic DNA) 的萃取 ......... 26
5.2 fimY基因的擴增..........26
5.3 pACYC184質體的萃取.......27
5.4 限制酶切割，黏接與轉形............ 28
5.5 LB5010勝任細胞的製備與重組質體的轉形........29
第六節 LB5010 genomic library 的構建........... 29
6.1 LB5010genomicDNA的萃取...........29
6.2 pACYC184質體的萃取...........29
6.3 限制酶切割............ 29
6.4 凝膠電泳，割膠回收與黏接........... 29
6.5 LB5010電穿孔勝任細胞的製備.........30
6.6 電穿孔(electroporation)............ 30
第七節 LB5010 genomic library 的篩選 .......... 31
7.1 菌株培養............. 31
7.2 酵母菌凝集試驗............ 31
7.3 反轉錄聚合酶鏈鎖反應(reverse transcription PCR, RT-PCR).............. 31
第八節 LB5010 NB2 菌株的構建.......... 31
第九節 Reporter system 的構建........... 32
9.1 pMC1403質體的萃取............32
9.2 fimYpromoter序列的擴增.........32
9.3 限制酶切割，黏接與轉形.......... 32
第十節 測量不同環境條件下 fimY promoter 的活力......... 33
10.1 不同溫度下菌株的培養方式........... 33
10.2 不同pH環境下菌株的培養方式......33
10.3 不同 Mg2+濃度環境下菌株的培養方式............. 34
10.4 β-半乳糖苷酶的活力測定(β-galactosidase activity assay)....... 34

第三章、實驗結果........... 36
第一節 LB5010 NB1 可以在 LB agar 上表現出第一型線毛........... 36
第二節 經過 LB5010 genomic library 的構建與篩選，並未找到酵母菌凝集陽性的菌株..........36
第三節 LB5010 NB2 無法在 LB agar 上表現出第一型線毛....... 36
第四節 fimY 的表達隨著溫度升高而增強........... 37
第五節 fimY 的表達隨 pH 變化而改變............ 37
第六節 低 Mg2+濃度的環境並未影響 fimY 的表達.............. 38

第四章、討論............... 39

參考文獻............. 45

Table 1. Bacterial strains used in this study............59
Table 2. Primers used in this study....................60

Figure 1. Phenotypic expression of type 1 fimbriae in LB5010, LB5010 V1, LB5010 NB1 analyzed by yeast agglutination test.......61
Figure 2. RT-PCR analysis for fimY and fimA transcription from LB5010, LB5010 V1 and LB5010 NB1 cultured on LB agar or in static LB broth..........62
Figure 3. Partial digestion of LB5010 genomic DNA.................63
Figure 4. Phenotypic expression of type 1 fimbriae in LB5010 NB2 analyzed by yeast agglutination test.................64
Figure 5. β-galactosidase activity of LB5010 NBP1 and the maximum dilution factor of LB5010 NBP1 that can induce the positive mannose-sensitive agglutination at different temperature..................65
Figure 6. β-galactosidase activity of LB5010 NBP1 and the maximum dilution factor of LB5010 NBP1 that can induce the positive mannose-sensitive agglutination in pH4 and pH7..................66
Figure 7. β-galactosidase activity of LB5010 NBP1 and the maximum dilution factor of LB5010 NBP1 that can induce the positive mannose-sensitive agglutination in pH8 and pH7................67
Figure 8. β-galactosidase activity of LB5010 NBP1 and the maximum dilution factor of LB5010 NBP1 that can induce the positive mannose-sensitive agglutination in limited Mg2+ concentration.........68
Figure 9. A possible regulatory route mediated by fimY in S. Typhimurium......69

Appendix 1. The fim gene cluster of S. Typhimurium....................70

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