- 學位論文
胃幽門螺旋菌之毒性因子：amidase及鞭毛調控因子之研究
Investigation of Helicobacter pylori virulence factors: amidase and flagella-associated regulators
摘要
胃幽門螺旋菌為微需氧的革蘭氏陰性菌，可抵擋胃部強烈胃酸的環境，並藉由鞭毛的移動可幫助細菌群聚與確立至胃上皮黏膜細胞上，進而使細菌侵入到細胞內。本論文主要以結構-功能關聯性研究兩種毒性因子：amidase 及兩個鞭毛調控因子。第一部份為 aliphatic amidase：AmiE 可以產生氨，使得胃幽門螺旋菌具有抗酸的能力。AmiE 為 cytoplasmic acylamide amidohydrolase （AmiE，EC 3.5.1.4），對短鏈的 amides 受質催化生成 carboxylic acid 及氨，且屬於 nitrilase superfamily 其中的一員。為了了解胃幽門螺旋菌 AmiE 結構-功能關聯性及在 nitrilase superfamily 演化上的意義，我們選殖 H. pylori amiE 基因。AmiE 整體結構為 homodimer，每個單體呈現 α-β-β-α 三明治摺疊結構，與 nitrilase superfamily 蛋白質結構摺疊方式非常類似。AmiE 延伸之 C 端是形成 dimmer 最主要相互作用界面，緊密地以 α-β-β-α-α-β-β-α三明治摺疊結構呈現。與 AmiF（Cys166、Glu60 及 Lys133）空間上相互堆疊，AmiE 活性催化殘基被鑑定為由 Cys165、Glu59 及 Lys133 所組成，其在 nitrilase superfamily 中皆具高度保留性。未來我們將繼續探討 AmiE 與 AmiF在 nitrilase superfamily 中所扮演的結構-功能相關性，決定 AmiE 活性中心突變的 apo 及 ligand 形式的晶體結構，並且針對活性中心突變點進行生化及酵素特性功能探討。第二部份為研究調控胃幽門螺旋菌鞭毛基因的表現之 FleS 及 FleR 鞭毛調控因子，我們選殖 fleS 及 fleR 基因並且藉由 allelic exchange mutagenesis 方法構築 fleS- 及 fleR- 缺陷菌株。穿透式電子顯微鏡的實驗觀察，顯示 fleS- 及 fleR- 缺陷菌株其鞭毛型態結構喪失。E-test 測試分析，顯示缺陷菌株對 amoxicillin、clarithromycin 及 metronidazole 抗生素具有高度敏感性。未來我們將繼續探討 FleS 及 FleR 在訊息傳遞調節鞭毛運動扮演的結構-功能相關性，並決定其晶體結構及蛋白質分子之間的相互作用特性進行探討。
並列摘要
Helicobacter pylori is a microaerophilic Gram-negative bacterium which is adapted to the low pH environment of the stomach. It is a flagellated motile organism that penetrates the gastric mucus layer and colonizes the surface of the gastric epithelial cells. This investigation focuses on the structure-function study of two virulence factors: amidase and two flagella-associated regulators. The first part is aliphatic amidase AmiE that is responsible for ammonia production and may be related to selective pressure in acidic environment. AmiE is a cytoplasmic acylamide amidohydrolase (EC 3.5.1.4) that catalyzes the conversion of short-chain amides to corresponding ammonium and carboxylic acids and belongs to a member of the nitrilase superfamily. To understand the structure-function relationship and evolution of H. pylori aliphatic amidase among members of the nitrilase superfamily, we have cloned the gene encoding amiE from H. pylori. The structure demonstrates as a homodimer in which each subunit has a four-layerα-β-β-αarchitecture characteristic that highly resembles the nitrilase superfamily enzymes. AmiE C -terminal tail extension is essential for interacting dimeric interfaces, forming a tight α-β-β-α-α-β-β-αdimer. The putative active-site residues are identified as Cys165, Glu59, and Lys133, which superimpose relatively well with those (Cys166, Glu60, and Lys133) of formamidase AmiF. This catalytic triad is well conserved in the nitrilase superfamily. Future work will be focused on the structure-function relationship of AmiE as well as the relationship with AmiF in the nitrilase superfamily. To this end, structures of active-site mutants in their apo and liganded forms will be determined. We will also perform biochemical and enzymatic characterization for each mutant. The second part is two flagella-associated regulators, FleS and FleR that regulates the expression of the flagella system of H. pylori. We have cloned the gene encoding fleS and fleR and constructed isogenic mutants by allelic exchange mutagenesis. Transmission electron microscopy showed that fleS- and fleR- mutants respectively had no flagella. E-test analysis of fleS- and fleR- isogenic mutants reveals that both mutants are sensitive to amoxicillin, clarithromycin, and metronidazole. Future work will be focused on the structure-function relationship of FleS and FleR and its molecular communication.