幽門桿菌感染造成人類六碳胺糖和岩藻糖水解酶的酵素活性提升：探討它們所扮演的角色

胃幽門螺旋桿菌是一種感染胃上皮細胞的病菌，會造成許多上消化道疾病，包括胃潰瘍、十二指腸潰瘍、胃炎、胃癌和黏膜相關淋巴組織淋巴癌等等，但實際上致病的因子仍不甚清楚。在本篇論文中，我們利用分子探針去偵測當胃幽門螺旋桿菌感染時，表皮細胞所增加的岩藻糖水解酶和活性。以便了解宿主和致病菌之間的關係。此外我們同時偵測到岩藻糖水解酶和己醣胺水解酶，在細菌感染時，兩者活性大幅提升；這兩個酵素屬於醣水解酶，分別有非還原端水解岩藻糖和乙醯葡萄糖胺乙醯半乳糖胺。在本實驗室先前的研究中，發現當胃幽門螺旋桿菌感染時，會促使宿主細胞釋放出第二型岩藻糖水解酶，並進而影響到細菌的貼附、生長及發病。利用產生醌甲基化物的活性探針，我們進一步地觀測到當胃幽門螺旋桿菌感染時，人類第一型岩藻糖水解酶的活性有增強的現象。在探討各種不同的細菌表面分子中，脂多醣為影響人類第一型岩藻糖水解酶活性增強的主因之一。另外，第二型岩藻糖水解酶和己醣胺水解酶也會在胃幽門螺旋桿菌感染時分泌。其中，兩者產生共同作用，會降低胃幽門螺旋桿菌的生存力。在更進一步的實驗中，利用掃描式及穿越式電子顯微鏡，發現酵素的殺菌效果是藉由破壞細菌表面的脂多醣和肽聚醣。在表面受到破壞後，巨噬細胞呈現出更容易吞噬受到醣水解酶處理後的胃幽門螺旋桿菌。

關鍵字

醣水解酶；幽門螺旋桿菌；脂多醣；醌甲基化物；殺菌

並列摘要

Helicobacter pylori, a Gram-negative bacterium found on the luminal surface of gastric epithelium, is the main cause to the development of three important upper gastrointestinal diseases: duodenal or gastric ulcers, gastric cancer, and gastric mucosaassociated lymphoid-tissue (MALT) lymphoma. Despite the known pathology, factors leading to disease progress still remain ambiguous. In this thesis we aim at examining how the pathogen interacts with gastric host, which includes the application of a synthetic probe to detect the enhanced α-L-fucosidase activity upon bacterial infection and the role of upregulated α-L-fucosidase and human β-D-hexosaminidase in H. pylori infection. The two glycosidases are in-charge of removal of fucose and GlcNAc/GalNAc residues from non-reducing termini of glycans, respectively. We previously indicated that human fucosidase 2 (Fuca2) was secreted upon H. pylori infection, and that the enzyme is critical to bacterial adhesion, growth and pathogenesis. By using a quinone methide-generating, activity-based probe, we additionally observed enhanced activities of human fucosidase 1 (Fuca1, a lysosomal enzyme) upon bacterial infection. Further studying the effect of several bacterial stimulants on the enhanced Fuca1 activity, we identified the lipopolysaccharides (LPS) to be one major factor. In addition to Fuca2, β-D-hexosaminidase is another major glycosidase secreted upon H. pylori infection. The enzyme was found to reduce viability of H. pylori, which was aggravated by the presence of Fuca2. Further studies, including the imaging analyses of scanning electron microscopy and transmission electron microscopy, supported the idea that the bactericidal effect was due to the damage on the cell surface, and that LPS and the adjacent peptidoglycans appeared to be the targets of enzymatic degradation. Additionally, the released enzyme activities were shown to render H. pylori more vulnerable to the phagocytosis by macrophages.