以定量蛋白體學分析胃癌相關之幽門螺旋桿菌生物標記及研究其GroES引發發炎反應之機制

Abstract

幽門螺旋桿菌是一種寄生於人類消化道的革蘭氏陰性菌，並且與慢性胃炎、消化性潰瘍、胃癌等胃部疾病之發生高度相關。雖然已經有許多幽門螺旋桿菌的毒性因子被發表，但其對於胃癌篩檢及致病的關聯性仍不足。本篇論文的第一部分研究目的為以臨床上不同走向的十二指腸潰瘍與胃癌臨床幽門螺旋桿菌菌株為基礎，利用定量蛋白質體學之方法鑑定與胃癌相關之幽門螺旋桿菌生物標記。在鑑定到的107個於胃癌菌株表現量高於十二指腸潰瘍菌株的蛋白中(>1.5倍)，43個可依分子間交互作用被KEGG pathway分類，其中AlpA、OipA、BabA、SabA被分屬於與人類疾病相關之分類，於是我們進一步檢視並比較這四個黏著因子在胃癌及十二指腸潰瘍病人血清辨認之差異。在臨床血清檢體篩檢下發現OipA、BabA、SabA的血清陽性率在胃癌檢體中相對高於十二指腸潰瘍檢體及胃炎檢體，且具有統計上之差異。另外，經統計分析發現同時辨認此三個抗原時，區分胃癌與其他胃部疾病之效果更為提升。為了將所找到的抗原發展成快速又便利的胃癌檢驗平台，我們利用蛋白質晶片技術建立出包含此三個抗原之胃癌相關蛋白質晶片。結果發現此胃癌相關蛋白質晶片能有效區分胃癌病人與十二指腸潰瘍病人或正常人血清，顯示幽門螺旋桿菌感染情形下OipA、BabA、SabA具有做為胃癌相關血清生物標記之潛力，且應用多個生物標記之蛋白質晶片可做為快速篩檢胃癌病人抗體種類組合之平台。 本篇論文的第二部分著重於先前探討過的胃癌相關抗原幽門螺旋桿菌GroES引發胃上皮細胞發炎反應之機制。幽門螺旋桿菌GroES為一個分泌性毒性因子，比其他物種之GroES多了一段羧基端稱為domain B。已知幽門螺旋桿菌GroES會造成宿主細胞釋放前發炎因子並可能參與胃部癌化過程。我們發現幽門螺旋桿菌GroES是透過活化MAPK及NF-κB pathways導致胃上皮細胞釋放前發炎因子IL-8，且不具domain B的幽門螺旋桿菌GroES則失去此能力。進一步發現TLR4抑制劑大幅的抑制了幽門螺旋桿菌GroES引發之IL-8的釋放及MAPK的活化，而幽門螺旋桿菌GroES引發小鼠胃上皮細胞釋放IL-8的現象在TLR4剔除小鼠之胃上皮細胞中則無法觀察到，顯示幽門螺旋桿菌GroES是透過TLR4引發發炎反應。我們同時也發現惟有domain B存在時幽門螺旋桿菌GroES才能與TLR4結合，於是我們針對domain B進行了截斷或針對domain B上會形成雙硫鍵之硫胺酸進行點突變。結果發現domain B被截斷或domain B上之兩對雙硫鍵同時被破壞時，幽門螺旋桿菌GroES會失去與TLR4結合及引發IL-8釋放之能力。總結我們的研究發現具有由雙硫鍵形成特殊羧基端套索結構之幽門螺旋桿菌GroES會透過TLR4引發前發炎因子IL-8之釋放。

Helicobacter pylori (H. pylori) is a Gram-negative, microaerophilic bacterium that selectively colonizes the human stomach. H. pylori infection is a major cause of chronic gastritis and peptic ulcer disease and is highly related to gastric adenocarcinoma and MALT lymphoma. Although many H. pylori-associated factors have been identified, the presence of these factors is not sufficient for gastric cancer (GC) screening. To identify GC-related H. pylori antigens with high seropositivity in GC patients, differences in protein expression levels of H. pylori from GC and its clinical divergent duodenal ulcer (DU) were analyzed by iTRAQ. In the quantified proteins, 107 showed increased expression in GC compared with DU (>1.5-fold) and 43 was categorized by KEGG pathway. Among them, AlpA, OipA, BabA, and SabA were related to human diseases and their seroreactivity were examined. OipA, BabA, and SabA were identified as GC-related antigens with higher seropositivity and the discrimination between GC and other gastric diseases was even improved with the three antigens combined together. A GC-related protein array was developed using multiple biomarkers for rapid diagnosis of GC and we found that OipA, BabA, and SabA were capable of screening GC from DU and normal individuals. We concluded that serologic markers of H. pylori infection including OipA, BabA, and SabA can serve as potential biomarkers for GC, and a GC-related protein array with the combination of these antigens could provide a rapid and convenient diagnosis of H. pylori-associated GC. In the second part of the studies, a previous identified GC-related antigen was investigated. Helicobacter pylori GroES (HpGroES), a potent immunogen, is a secreted virulence factor that stimulates production of proinflammatory cytokines and may contribute to gastric carcinogenesis. HpGroES is larger than other bacterial orthologs because of an additional C-terminal region, known as domain B. We found that the HpGroES-induced IL-8 release by human gastric epithelial cells was dependent on activation of the MAPK and NF-κB pathways. HpGroES lacking domain B was unable to induce IL-8 release. In addition, a TLR4 inhibitor significantly inhibited IL-8 secretion and reduced HpGroES-induced activation of MAPKs. Furthermore, HpGroES-induced IL-8 release by primary gastric epithelial cells from TLR4-/- mice was significantly lower than from wild-type mice. We also found that HpGroES bound to TLR4 in cell lysates and co-localized with TLR4 on the cell membrane only when domain B was present. We then constructed two deletion mutants lacking C-terminal regions and mutants with point mutations of two of the four cysteine residues, C111 and C112, in domain B and found that the deletion mutants and a double mutant lacking the C94-C111 and C95-C112 disulfide bonds were unable to interact with TLR4 or induce IL-8 release. We conclude that HpGroES, in which a unique conformational structure, domain B, is generated by these two disulfide bonds, induces IL-8 secretion via a TLR4-dependent mechanism.