幽門螺旋桿菌在高鹽壓力下的蛋白質體分析:嗜中性細胞激活蛋白的物化性質研究

Abstract

中文摘要 幽門螺旋桿菌是一種微耗氧性(5% O2)的革蘭氏陰性桿菌，外觀主要呈螺旋狀，菌體長約2至4微米，寬約0.5微米，其中一端具有數根鞭毛，可幫助菌體以螺旋狀方式移動至胃部，並穩定地寄居在人體胃部的黏膜層。根據統計，目前幽門桿菌約感染全世界一半以上的人口，但在絕大多數的受感染者體內都只造成症狀輕微的慢性胃炎，只有部份感染毒性較強菌株的病患才會發生胃潰瘍或甚至是胃腺癌等胃部疾病。然而雖然目前研究已知感染幽門桿菌是導致胃癌與消化性潰瘍的關鍵因素，但也有部份研究認為，當這項因素單獨存在並不足以致病。根據先前流行病學及動物模式的實驗發現，在已感染幽門螺旋桿菌的狀況下，高鹽的飲食習慣可能會促進胃炎的惡化及提高胃癌的發生率。 在這篇研究當中，我們想以蛋白質體學的方式去討論在高鹽環境下，幽門桿菌的蛋白質表現量是否會受到調節。在實驗中，我們分別將由臨床胃癌及十二指腸潰瘍病患身上分離出的幽門桿菌菌株培養在含有0.5%或2%氯化鈉的腦心浸出物培養液(BHI broth)中培養24小時，接著利用二維電泳、液相層析串聯質譜儀的結果與生物資訊資料庫作搜尋比對，我們找出數個在高鹽濃度的培養液中表現量會上升的蛋白質，其中包含嗜中性細胞激活蛋白(Neutrophil Activating Protein, NapA)、過氧化氫分解酶(Catalase)、尿素分解酶A、B次單元體(UreaA, UreaB)，與50S核糖體蛋白質L7/ L12 (50S ribosomal proteins L7/L12)等蛋白。 NapA蛋白是屬於Dps family (DNA protection during starvation)蛋白的一員，是由約17 kDa左右的相同單體聚合成的十二聚體蛋白(dodecamer)，是幽門桿菌一個重要的致病因子(virulence factor)之一。目前研究認為NapA對於幽門桿菌有兩個主要的功能：第一個功能是吸引嗜中性細胞(neutrophils)與單核球(monocytes)，並促使這些免疫細胞產生活性氧物質(reactive oxygen species, ROS)，引發局部發炎反應；而另一個可能的功能則是與鐵離子結合，避免細胞中的游離鐵離子與過氧化氫進行Fenton reaction而生成會對細胞造成傷害的氫氧自由基(OH•)。 本論文的研究重點在於探討幽門桿菌臨床胃癌菌株HC28的NapA之重組蛋白(HC28-NapA)及十二指腸潰瘍菌株HD30的NapA之重組蛋白(H30-NapA)在高鹽壓力下其物化性質的變化，我們利用了分析級超高速離心機(Analytical Ultracentrifuge, AUC)及快速液態層析儀(Fast Performance Liquid Chromatography, FPLC)去分析NapA蛋白在高鹽壓力下的變化，結果發現在2000 mM NaCl濃度下，NapA蛋白的分子量會由原態大小約200 kDa，下降至150 kDa左右，表示NapA蛋白在高鹽濃度下，其蛋白質次單元體(subunit)可能會發生解離(dissociation)。同時我們也利用圓雙色偏光吸收光譜儀(Circular Dichroism, CD)及螢光光譜(Fluorescence Spectroscopy)去測定NapA蛋白在高鹽濃度下的二級和三級結構變化。並利用恆溫滴定熱卡計ITC (Isothermal Titration Calorimetry)去測量NapA蛋白與鐵離子結合的化學計量數(stoichiometry, n)，並討論高鹽狀態下NapA蛋白與鐵離子的結合能力是否受到影響。

Helicobacter pylori (H. pylori) is a helix-shaped, microaerophilic Gram-negative bacterium, about 2 to 4 μm long with a diameter of about 0.5 μm. The bacterium can move through the stomach by means of its flagella, forming long-term stable colonies in the mucosa of stomachs. According to previous epidemiological studies, H. pylori infected stomachs of more than 50% of human population. Infection by H. pylori usually causes chronic gastritis without obvious symptoms; but in some strains producing specific virulence factors, they may lead to the development of ulcers or even gastric adenocarcinoma. It is known that H. pylori infection is the leading cause of gastric cancer and peptic-ulcer diseases. However, the factors involved in diseases caused by this bacterium are known to be complex and multi-factorial. Some human epidemiologic studies and animal models have confirmed that a high-salt dietary intake significantly increase the risk of gastritis and enhance the development of gastric carcinoma caused probably by H. pylori infection. To address the influence of high osmotic stress, we focus on the protein expression profile of H. pylori under high salt concentration by a proteomic approach. Clinical isolates of H. pylori from patients of gastric cancer (HC28) and duodenal ulcer (HD30) were grown in a normal BHI medium (0.5% NaCl) and hyper-osmotic stress (BHI medium supplemented with 2% NaCl) conditions for 24 h, followed by 2D electrophoresis, LC-MS/MS, MALDI-TOF-MS and bioinformatics database search/peptide-mass comparison. In this study, we identify some H. pylori proteins that were altered in response to high salt concentration in liquid culture media, including conspicuously a virulence factor of H. pylori, neutrophil activating protein (NapA), plus catalase, UreaA, UreaB, and 50S ribosomal proteins L7/L12. NapA is a dodecameric protein consisting of 17 kDa monomers, and belongs to a member of Dps protein family. It acts as one of the major virulence factors in H. pylori infection. It has also been shown to play dual roles inside affected cells. First, it can elicit the cellular responses for the recruitment of human neutrophils and monocytes, the activation upon which immune cells would generate reactive oxygen radicals released from neutrophils and cause the inflammation locally. Secondly, NapA can sequester free ferrous ions in the cell to prevent the production of toxic hydroxyl radicals from Fenton reaction. In this study, we have also cloned and purified recombinant NapA by RT-PCR and overexpression of this protein factor. We characterized and compared the molecular sizes of NapA obtained from H. pylori of gastric cancer strain HC28 (HC28-NapA) and duodenum ulcer strain HD30 (HD30-NapA) under high-salt stress by using analytical ultracentrifuge (AUC) and fast performance liquid chromatography (FPLC). We found that the molecular mass of NapA can be reduced from 200 kDa to about 150 kDa in the presence of 2000 mM NaCl, suggesting that the subunits of NapA may dissociate into smaller aggregates under high-salt concentration. Furthermore, we also characterized the secondary and tertiary structures of NapA under different salt concentrations by circular dichroism (CD) and fluorescence spectroscopy. The binding stoichiometry between iron and NapA under different salt concentrations was also analyzed by using isothermal titration calorimetry (ITC) in order to reveal the quantitative binding relationship between iron and NapA.