綠膿桿菌尿嘧啶雙磷酸葡萄醣去氫酵素缺損株致病性之探討

Abstract

綠膿桿菌屬於伺機性感染的革蘭氏陰性細菌，特別容易感染免疫系統不全的人，例如燒傷患者或肺纖維囊腫病人。 尿嘧啶雙磷酸葡萄醣去氫酵素催化尿嘧啶雙磷酸葡萄醣合成尿嘧啶雙磷酸葡萄醣醛酸。 尿嘧啶雙磷酸葡萄醣及尿嘧啶雙磷酸葡萄醣醛酸具多種重要的代謝功能，例如組成細菌的脂多榶、莢膜和參與半乳糖的代謝等。 我們實驗室先前的研究證明尿嘧啶雙磷酸葡萄醣去氫酵素基因和尿嘧啶雙磷酸葡萄醣聚磷酸化酵素基因位於同一基因組，同時尿嘧啶雙磷酸葡萄醣聚磷酸化酵素基因缺損會導致綠膿桿菌致病力降低。 為了研究尿嘧啶雙磷酸葡萄醣去氫酵素對綠膿桿菌的重要性，我們構築綠膿桿菌尿嘧啶雙磷酸葡萄醣去氫酵素基因缺損株，來探討此酵素在綠膿桿菌生理及致病因子中所扮演的角色。 首先，在尿嘧啶雙磷酸葡萄醣去氫酵素基因上插入抗卡那黴素基因，經細菌接合作用將此質體送入綠膿桿菌，再藉由同質性重組作用，使完整的尿嘧啶雙磷酸葡萄醣去氫酵素基因被缺損的基因所置換，最後以南方墨點法確認，我們得到了兩株基因缺損株。 我們發現不管是在野生株或缺損株中都無法測得尿嘧啶雙磷酸葡萄醣去氫酵素的活性，顯示此酵素在綠膿桿菌體中可能因為表現量過低而無法偵測。 進一步分析純化之重組尿嘧啶雙磷酸葡萄醣去氫酵素，證明具有酵素活性。 不同於尿嘧啶雙磷酸葡萄醣聚磷酸化酵素之缺損株，尿嘧啶雙磷酸葡萄醣去氫酵素缺損株的遊走運動、對血清的敏感度、對過氧化氫的抗性、及其外膜蛋白和脂多醣的結構，和野生株都無顯著的差異。 再進一步利用西方轉印免疫法觀察A-band及B-band脂多醣，發現也和野生株相似，顯示尿嘧啶雙磷酸葡萄醣去氫酵素可能對於脂多醣的合成沒有影響。因此對於尿嘧啶雙磷酸葡萄醣去氫酵素在綠膿桿菌中參與的角色，仍待進一步的探討。

Pseudomonas aeruginosa is an opportunistic pathogen that causes fatal infections in immunocompromised patients including those suffered from burn wounds and cystic fibrosis. The UDP-glucose dehydrogenase (Ugd) converts UDP-glucose to UDP-glucuronate. We have previously shown that ugd and UDP-glucose pyrophosphorylase encoding gene (galU) were organized as an operon. The galU mutant that lost the ability to produce UDP-glucose was less virulent. In this study, we have constructed the ugd mutant of P. aeruginosa (PAO1) to investigate the role of ugd in the bacterial physiology and pathogenesis. The ugd was disrupted by insertion of a Kanamycin-resistant-gene cassette and the mutants obtained through homologous recombination. Two ugd mutants were obtained and verified by Southern blotting. We found that the Ugd activities of wild type P. aeruginosa PAO1 and the ugd mutants were nondetectable indicating that the Ugd expression level is low. We further purified the recombinant Ugd and confirmed it possesses a Ugd activity. Unlike the galU mutant, we did not find discernible differences in swarming, serum sensitivity, susceptibility to reactive oxygen and antibiotics, and profiles of outer membrane proteins and the lipopolysaccharides between wild type PAO1 and the ugd mutants. The A-band and B-band lipopolysaccharides of the ugd mutants were also similar with those of PAO1 by Western immunoblotting using specific monoclonal antibodies. Therefore, the role of ugd in P. aeruginosa remains to be investigated.