鼠傷寒沙門氏菌fimY基因在第一型線毛表現調控可能扮演的角色

The role that fimY may play in the regulatory system of the type 1 fimbriae in Salmonella enterica serotype Typhimurium

黃怡寧(Yi-Ning Huang)

指導教授 ： 葉光勝

臺北醫學大學/醫學院/醫學科學研究所/碩士(2011年)

摘要

鼠傷寒沙門氏菌與大多數腸內菌科的細菌一樣，常具有線毛的結構。線毛位於細菌表面，為類似毛髮的蛋白質結構，主司吸附作用。第一型線毛為鼠傷寒沙門氏菌中最常見的線毛種類，會吸附到具甘露糖結構的接受體。第一型線毛的表現牽涉到 fim 基因組中的 fimY 、 fimZ 、 fimW 及 fimU 等基因間複雜的調控作用。先前研究發現， FimZ 及 FimY 為第一型線毛的正向調控因子。 FimZ 可直接結合於線毛結構單位基因 fimA 的啟動子上，活化 fimA 基因表現，使細菌產生第一型線毛，但活化fimA基因有賴於 FimY 的協同作用。 FimZ 亦可和負向調控因子 FimW 產生蛋白質與蛋白質之間的結合作用。fimU 是一 arginine tRNA ，可透過轉譯的過程調控 FimY，進而影響第一型線毛表現。由於經胺基酸序列比對，發現 FimY 的 C 端具有與 LuxR family 相似的 helix-turn-helix 的結構，因此擬探討FimY是否具DNA結合能力，結合至哪一個基因的啟動子，以及FimY是否會與FimZ產生蛋白質之間的結合等有趣課題。 藉由RT-PCR分析，fimZ及fimY基因剔除的鼠傷寒沙門氏菌突變株，在fimA基因的表現都明顯下降，而 fimZ 基因剔除的突變株 fimY 基因表現下降，同樣的fimY基因剔除的突變株fimZ基因表現也下降，佐證fimY及fimZ彼此可互為影響，且與fimA表現有關。實驗結果也說明fimZ與fimY基因並不屬於同一條順反子 (cistron)。純化的 FimY-His 融合蛋白可和 605-bp 的 fimZ 啟動子區域的 DNA 片段結合而造成凝膠位移，但FimY不會與一266-bp的fimW的啟動子區域DNA以及與一232-bp的fimA啟動子區域DNA結合。 除此之外，藉由 pull-down assay 分析，證實FimY及FimZ有蛋白質間的結合情形。我們的實驗結果說明FimY是一個DNA結合蛋白質，可結合在fimZ基因的啟動子上，且FimY與FimZ有蛋白質間的結合作用，對闡明鼠傷寒沙門氏菌第一型線毛調控的機制有所助益。

關鍵字

鼠傷寒沙門氏菌 第一型線毛 DNA結合蛋白質

並列摘要

Like many members of the Enterobacteriaceae family, Salmonella enterica serotype Typhimurium (S. Typhimurium) produces fimbrial appendages. Fimbriae are hair-like protein structures whose function is primarily to mediate adherence. Type 1 fimbriae with the binding specificity to the mannose residues is the most commonly found fimbrial type conferred by S. Typhimurium. The phenotypic expression of type 1 fimbriae is the result of the interaction and cooperation of fimZ, fimY, fimW and fimU within the fim gene cluster. Previous studies revealed that FimZ and FimY are both positive regulators for type 1 fimbriae. FimZ can bind to the promoter region of the fimA gene (the fimbrial major subunit gene) and active the gene to produce type 1 fimbriae. However, activation of fimA also requires cooperation of fimY. FimZ was demonstrated to interact with a negative regulator FimW. fimU is an arginine tRNA and controls fimY at the translational level. Similarity search indicated that the C-terminal part of FimY polypeptide possesses a helix-turn-helix motif, like LuxR family proteins. Therefore, it is interesting to further explore research topics like if FimY possesses the capability to bind DNA, what region it may bind and if FimY binds FimZ protein. In the present study, reverse transcriptase polymerase chain reaction (RT-PCR) analysis indicated that a S. Typhimurium fimY and fimZ mutant strains exhibited less fimZ and fimY, respectively. The fimA expression levels of both strains were also decreased. These findings confirmed that fimZ and fimY can activate each other and both are required for the fimA expression. Our result also indicated that fimY and fimZ did not belong to one cistron. A purified FimY-His fusion protein caused mobility shift when incubated with a 605-bp DNA fragment possessing the fimZ promoter, whereas FimY did not cause a 266-bp DNA fragment containing the fimW promoter and a 232-bp DNA fragment containing the fimA promoter DNA to shift. Pull down assay demonstrated that FimY and FimZ may have protein/protein interaction. The present study indicated that FimY is a DNA binding protein and binds to the fimZ promoter region. In addition, there is protein/protein interaction between FimY and FimZ. These findings may shed light on the elucidation of the regulatory system of the type 1 fimbriae in S. Typhimurium.

並列關鍵字

Salmonella enterica serotype Typhimurium, type 1 fimbriae, DNA binding protein

參考文獻

第六章 參考文獻

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