- 學位論文
探討胃幽門螺旋桿菌26695 O抗原生合成蛋白HP1581, HP1206及HP1039缺失對其脂多醣合成及感染之影響
Studies on Helicobacter pylori 26695 proteins HP1581, HP1206, HP1039 involved in the O-antigen biosynthetic pathway and their effects on lipopolysaccharide biosynthesis and infection
摘要
胃幽門螺旋桿菌 (Helicobacter pylori) 為螺旋狀、微耗氧、革蘭氏陰性細菌。全世界超過50%的人口受此菌感染，是一種人類重要的病原菌。受感染者可能會衍生多種胃部相關之疾病，例如：胃及十二指腸潰瘍、胃癌、黏膜相關淋巴組織淋巴瘤等疾病。脂多醣 (Lipopolysaccharide, LPS) 是革蘭氏陰性菌外膜的主要成分，可以維持細菌外膜的完整性，並且保護細菌不受毒性和疏水性物質攻擊，例如：清潔劑以及脂溶性抗生素等物質。脂多醣由三部分組成，分別是：脂質A (lipid A) 、核心多醣 (core oligosaccharide) 以及O-抗原 (O-antigen polysaccharide) 。在胃幽門螺旋桿菌脂多醣中的O-antigen具有Lewis antigen，其可以模仿人類胃部細胞的多聚醣結構，幫助細菌逃脫人類的免疫系統並且造成慢性感染。 在本篇研究中，我們研究三種被認為與胃幽門螺旋桿菌脂多醣中O抗原之生合成有關的基因，分別是wecA、wzk以及waaL。在近期被提出的模型中，胃幽門螺旋桿菌脂多醣的生合成可能是透過一種需要Wzk參與的路徑；其中WecA (HP1581) 是一個可以將UDP-GlcNAc轉移到UndP脂質載體上的起始酵素，而整個O抗原的合成發生在細胞質。隨後Wzk (HP1206) 翻轉酶會將O抗原轉位到周質，其後WaaL (HP1039) 連接酶會將O抗原與核心多醣-脂質A結合起來產生完整的脂多醣。生物資訊分析的結果指出HP1581是屬於內膜蛋白質，能使 UDP-GlcNAc殘基轉移到UndP脂質載體上；HP1206和位在內膜的Wzk翻轉酶具有高度同源性，而且與其他革蘭氏陰性菌中脂多醣生合成的Wzk-dependent路徑有所關聯性；此外胃幽門螺旋桿菌26695中的HP1039可能是一個具有特殊功能的內膜蛋白質。為了探討HP1581、HP1206及HP1039對於脂多醣之生合成和感染的功能特性，我們建構了相對應的突變菌株並且研究其表型特性。利用銀染分析以及免疫墨點法去偵測Lewis X抗原的結果顯示這些突變菌株皆有O抗原的缺失。利用免疫墨點法去偵測Lewis Y抗原的結果則暗示在胃幽門螺旋桿菌26695中某些酵素活性會去彌補缺失的酵素功能。研究也發現這三株功能缺失突變菌株在生長曲線的死亡期時會降低生存能力。此外，與正常菌株相比，HP1206及HP1039突變菌株對SDS和抗生素novobiocin較敏感，而HP1581突變菌株則沒有顯著差異。總而言之，本篇研究報告中所獲得的知識將有助於發現新標的以利於往後開發對抗胃幽門螺旋桿菌感染的藥物。
並列摘要
Helicobacter pylori is a spiral-shaped microaerophilic gram-negative bacterium and is recognized as a notorious human pathogen that infects a half of the world’s human population. Several human stomach diseases such as gastric ulcer, gastroduodenal ulcer, gastric adenocarcinoma and mucosa-associated lymphatic tissue (MALT) lymphoma are related with H. pylori infection. Lipopolysaccharide (LPS) is a major component on the surface of gram-negative bacteria including H. pylori and plays an important role in maintaining the integrity of bacterial outer membrane as well as providing a protective barrier against the entry of toxic hydrophobic compounds such as detergents and lipophilic antibiotics. LPS is composed of lipid A, core oligosaccharide and O-antigen polysaccharide. O-antigen of H. pylori LPS contains Lewis antigens which mimic glycan structures produced by human gastric cells leading to escape from host immune response and chronic infection. In this study, we investigated three genes including wecA, wzk and waaL which are putatively related to H. pylori LPS O-antigen biosynthesis. In a recently proposed model, H. pylori LPS biosynthesis follows a novel Wzy-dependent pathway and WecA (HP1581) is an initiating enzyme to transfer UDP-GlcNAc onto the UndP lipid carrier. The synthesis of entire O-antigen occurs in the cytoplasm which later is translocated to the periplasm by flippase WzK (HP1206) and ligated with core-lipid A by ligase WaaL (HP1039) to form the complete LPS. These bioinformatic results indicated that HP1581 is an inner membrane protein and responsible for transferring a UDP-GlcNAc residue onto the UndP lipid carrier; HP1206 has high homology with wzk flippase located in the inner membrane which is related to the Wzy-dependent pathway of LPS biosynthesis in other gram-negative bacteria; and HP1039 is likely an inner membrane protein with an unique function in H. pylori 26695. To further characterize the functional role of HP1581, HP1206 and HP1039 in the LPS biosynthesis and infection, the corresponding H. pylori knockout mutants, ΔHP1581, ΔHP1206 and ΔHP1039 are constructed and their phenotypic properties were characterized. These H. pylori 26695 knockout mutants all showed the deficiency of O-antigens when analyzed by silver staining and immunoblotting for the anti-Lewis X determinant. The immunoblotting result with anti-Lewis Y determinant suggested that the lost enzymes may be compensated by certain enzymatic activities in H. pylori 26695. The three knockout mutants have reduced the survival ability in the death phase of growth. In addition, ΔHP1206 and ΔHP1039 have increased the sensitivity to SDS and novobiocin while ΔHP1581 shows no significant difference compared to that of the wild-type H. pylori 26695. In conclusion, we hope that the obtained knowledge from this study would be useful to discover attractive targets for the development of inhibitors or antimicrobial agents to manage H. pylori infection.