摘要：

胃幽門螺旋菌是一種寄宿在人類胃部的主要致病菌.人類在遭受此菌長期感染後會產生胃潰瘍, 十二指腸潰瘍, 胃癌和胃黏膜相關淋巴組織淋巴瘤等疾病.胃幽門螺旋桿菌的外膜上帶有一種普遍存在於革蘭氏陰性菌的脂多醣類 (LPS).LPS提供了細菌外膜的結構完整性,並且會誘發寄主的免疫反應.LPS與寄主細胞之間的交互作用在胃幽門螺旋菌的致病性上可能扮演關鍵的角色.ADP-L-glycero-D-manno-heptose-6-epimerase酵素對LPS的生合成很重要,了解它的生化特性將有助於抗生素的研發.我們從胃幽門螺旋菌26695菌株選殖了和大腸桿菌rfaD同源的HP0859並將其大量表現.它的重組蛋白經過分析顯示其在天然的狀態下可能是一個六聚體.HP0859蛋白的輔助因子不是NAD+,而是NADP+.CD光譜的測試顯示移去NADP+輔助因子將對此蛋白質的二級結構造成顯著的影響.我們同時建構了HP0859基因缺失的菌株,並檢查其生理特徵的改變.此菌株產生了截短的脂多醣體結構,生長速率降低並對novobiocin抗生素有較強的感受性.另外破壞HP0859的表現也降低了胃幽門螺旋菌吸附到AGS細胞的能力,而受感染的細胞也失去了展現典型的蜂鳥狀外形.但當將完整的HP0859基因重新導入此基因剔除菌株時,其衍生的菌株則完全恢復其正常的外表型及生理特性.總結:我們證明了HP0859所表現的蛋白質對LPS生合成的重要性,並指出胃幽門螺旋菌LPS結構的完整性和此菌吸附寄主細胞的能力大有關聯.Helicobacter pylori (H. pylori) is a major pathogen residing in human stomach. Chronic infection of this bacterium can cause gastric ulcer, duodenal ulcer, gastric cancer and mucosa-associated lymphoid tissue (MALT) lymphoma. The outer membrane of H. pylori anchors the lipopolysaccharide (LPS) which is present in many Gram-negative bacteria. LPS not only contributes to the structural integrity of the outer membrane but also induces immune responses from the host cells. The interaction between LPS and the host cell is believed to be related to disease development. ADP-L-glycero-D-manno-heptose-6-epimerase is an enzyme essential for LPS synthesis and understanding of its biochemistry is critical for drug development. We cloned one putative ortholog of Escherichia coli rfaD, HP0859, from H. pylori 26695. Determination of the native molecular weight of the recombinant HP0859 protein suggests that the protein is likely a hexamer. NADP+, instead of NAD+, was proved to be the physiological cofactor for HP0859 protein. Circular dichroism spectrum analysis demonstrated that the secondary structure of this protein is significantly altered when the cofactor is removed. We also constructed an HP0859 knockout mutant and examined its phenotypic properties. The HP0859 knockout mutant exhibited a severe truncation of LPS, a decreased growth rate, and a higher susceptibility to novobiocin. Disruption of HP0859 also reduced the adhesive capacity of H. pylori to AGS cells, and the infected cells failed to display the classic hummingbird phenotype. Complementation of the HP0859 knockout mutation restored these phenotypes completely. In conclusion, we demonstrate that HP0859 codes for a protein essential for the LPS inner core biosynthesis in H. pylori and an intact LPS structure contributes to the adherence ability of this bacterium.

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