膽固醇醣苷代謝物標定與分析以揭露它們如何增強胃幽門螺旋桿菌的致病性

Metabolic labeling and profiling of cholesteryl glucosides to unveil how they enhance virulence in Helicobacter pylori

詹皓名(Hau-Ming Jan)

指導教授 ： 林俊宏

國立臺灣大學/生命科學院/生化科學研究所/博士(2016年)

https://doi.org/10.6342/NTU201602064

摘要

世界上大約有一半的人口被胃幽門螺旋桿菌所感染，而這個細菌也是造成各種胃部疾病的主要原因。這種病菌是無法製造膽固醇的，它會吸收宿主細胞身上的膽固醇並將其轉換成各種膽固醇醣苷衍生物，包括膽固醇6'-酰基和6'-磷脂基葡萄糖苷（CAG 和CPG）。由於缺乏靈敏的分析方法，無法得知在CAG 和CPG 上面的酰基(acyl)或是磷脂基(phosphatidyl)的組成和變化，是否會影響在生理上所扮演的角色。在此，我們建立了一個達到飛莫爾(femto-molar)層級的代謝物標記法，並利用這樣的方式將這些衍生物做詳細的定性和定量的分析，得以建立膽固醇醣苷衍生物的質譜/質譜資料庫，並可分析所有的膽固醇代謝衍生物。進一步發現這些細菌能夠獲得宿主表皮細胞的磷脂，進行CAG 的合成。同時，我們也找到負責CAG 生合成的酵素(HP0499)，進一步證實HP0499 在感染宿主細胞時，會透過外膜小囊(OMV)或直接接觸的方式，將酵素及產物轉置到人類細胞上，以產生帶有較長或是較多不飽和酰基的CAG(又稱作含有人類脂質的CAG)，因而促進脂筏(lipid rafts)的形成，以及細胞表面的整合素α5β1 的聚集，導致增加細菌黏附到宿主表皮細胞，因而大幅增加毒素蛋白因子(CagA)轉置進入宿主細胞。這樣的結果支持了一個想法: 幽門螺桿菌演化出特殊的宿主/病原體的交互作用以增加細菌的毒性。我們的研究也詳細闡述了，不同酰基組成的CAG，如何連結到細菌的致病性。

關鍵字

胃幽門螺旋桿菌 ； 膽固醇 ； 代謝物 ； 質譜分析 ； 脂筏 ； 磷脂

並列摘要

Helicobacter pylori, which infects approximately half of the human population, is the main cause of various gastric diseases. This pathogen is auxotrophic for cholesterol, which it converts upon uptake to various cholesteryl alpha-glucoside derivatives, including cholesteryl 6’-acyl and 6’-phosphatidyl alpha-glucosides (CAGs and CPGs). Due to a lack of sensitive analytical methods, it remains unknown whether CAGs and CPGs have distinct physiological roles and how the acyl chain components affect function. Herein we describe a metabolite-labeling method for qualitatively and quantitatively characterizing these derivatives at a femto-molar detection limit. We generated an MS/MS database of CGds that allows for profiling of all cholesterol-derived metabolites. The subsequent analysis led to the unprecedented discovery that these bacteria acquire phospholipids for CAG biosynthesis from the membrane of epithelial cells. Furthermore, we also identified the enzyme responsible for CAG biosynthesis (HP0499). The result suggested that HP0499 and the resulting products are translocated from the bacterium to the host cell via direct contact or delivery by outer membrane vesicles (OMVs) to facilitate formation of longer and/or unsaturated CAG acyl chains (also called human lipid-containing CAGs), which helped to promote lipid raft clustering, induced accumulation of integrin α5β1, and thus enhanced bacterial adhesion. This also explained the enhanced translocation of the virulence factor CagA into the host cell. These findings demonstrate how H. pylori evolves the special host/pathogen interplay to enhance bacterial virulence, and that our research pinpoints an important connection between the CAG composition and bacterial pathogenicity.

並列關鍵字

Helicobacter pylori ； cholesterol ； metabolite ； mass analysis ； lipid rafts ； phospholipid

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