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  • 學位論文

可能調控鼠傷寒沙門氏菌fimY基因表現的基因選殖與環境因子之探討

Determination of the genetic elements and the environmental factors that control fimY expression in Salmonella enterica serovar Typhimurium

指導教授 : 葉光勝

摘要


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。

並列摘要


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.

參考文獻


1. Fàbrega A, Vila J: Salmonella enterica serovar Typhimurium skills to succeed in the host: virulence and regulation. Clin Microbiol Rev 2013, 26(2):308-341.
2. Pui CF, Wong WC, Chai LC, Nillian E, Ghazali FM, Cheah YK, Nakaguchi Y, Nishibuchi M, Radu S: Simultaneous detection of Salmonella spp., Salmonella Typhi and Salmonella Typhimurium in sliced fruits using multiplex PCR. Food Control 2011, 22(2):337-342.
3. Eng SK, Pusparajah P, Ab Mutalib NS, Ser HL, Chan KG, Lee LH: Salmonella: a review on pathogenesis, epidemiology and antibiotic resistance. Front Life Sci 2015, 8(3):284-293.
4. Brenner FW, Villar RG, Angulo FJ, Tauxe R, Swaminathan B: Salmonella nomenclature. J Clin Microbiol 2000, 38(7):2465.
5. Kozak GK, MacDonald D, Landry L, Farber JM: Foodborne outbreaks in Canada linked to produce: 2001 through 2009. J Food Prot 2013, 76(1):173- 183.

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