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標題: Stenotrophomonas maltophilia MopB 外膜蛋白之分析
Characteristics of MopB protein in Stenotrophomonas maltophilia
作者: 吳漢強
Wu, Han-Chiang
關鍵字: Stenotrophomonas maltophilia;非醱酵性的嗜氧革蘭氏陰性桿菌;outer membrane protein (Omp);MopB;外膜蛋白
出版社: 生命科學院碩士在職專班
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非醱酵性的嗜氧革蘭氏陰性桿菌Stenotrophomonas maltophilia (S. maltophilia),普遍存在於自然環境中,如土壤、污水等。近年研究發現,癌症、免疫力缺失和長期使用抗生素等之病患的院內感染率持續提高,可能與此菌之感染有關。細菌之外膜蛋白 (outer membrane proteins, Omps)負責維持外膜的完整性,且擔任黏著素 (adhesins) 和運輸者 (transporter) 的角色,並與抗藥性 (antimicrobial resistance) 及致病性因子 (virulent factors) 的釋出有關。已知MopB為 S. maltophilia 最主要之外膜蛋白。為探討細菌感染動物細胞時此蛋白所扮演之角色,本實驗室曾經突變S. maltophilia ATCC13637 (Sm 13637)之 mopB基因,獲得突變株,命名為SmMopB。本研究中首先比較野生株Sm 13637與突變株SmMopB 在生理及病理學上之一些特性。結果發現,於穿透式和掃瞄式電子顯微鏡觀測下,突變株菌體的細胞外膜與肽聚醣層(peptidoglycan layer)之間有明顯分離現象,且菌體表面粗糙,細胞之間出現較多不明的細絲狀 (filament-like)物質。但在抗生素敏感性測試 (antibiotic susceptibility test),細胞毒性分析(lactate dehydrogenase released assay ),及組織病理學研究方面,突變株與野生株之間並無明顯差異。為進一步探討MopB蛋白在菌體與動物細胞作用上之影響,本研究利用小鼠皮膚 L929 細胞株 (mouse skin L929 fibroblast tissue),進行細菌貼附測試 (adhesion assay) 與吞噬測試 (phagocytosis assay),評估菌株的內化作用 (internalization)。研究指出經較長時間共培養後,SmMopB 附著於L929的能力增加,且被吞噬進入細胞的數目較多。本研究證實MopB 在維持 S. maltophilia細胞外膜結構穩定性上扮演重要角色。雖然突變株與野生株之細胞毒性無顯著差異,MopB蛋白缺失卻可能直接或間接促進細菌與細胞的黏附作用,並增加細菌被吞噬進入宿主細胞,相關機制則尚待探討。

Widespread in the environment Stenotrophomonas maltophilia (S. maltophilia) is a motile, nonfermentative, Gram-negative bacillus which was rarely reported to cause direct infection to human before. However, reports of nosocomial infection by this bacterium have increased rapidly in the last decade, especially on cancer patients, immunosuppression, and prolonged antibiotic therapies. Outer membrane proteins (Omps) play important roles in maintaining the structural integrity of outer membrane of bacteria, and function as adhesins, transporters, important components of antimicrobial resistance, and virulence related proteins. MopB protein is an abundant Omp in S. maltophilia ATCC13637, and the mopB mutant strain, SmMopB, has been constructed. In this study, SmMopB was used to analyze the physiological and pathological role of MopB protein in S. maltophilia, especially the effects on the infected cells and animals. Based on the morphology examination under Electron Microscopes (Transmission and Scanning), we observed that the outer membrane was not closely associated with the peptidoglycan layer and the outer surface became rough in SmMopB, and some obvious filaments were found among cells. On the other hand, the cytotoxicity of SmMopB strain seemed to have no significant differences from that of the wild type strain according to the results of histopathological study, assay of LDH released and antibiotic susceptibility test. However, the increasing adherence ability of SmMopB which might improve the internalization of bacteria into animal cells had been noticed along with longer incubation with L929 cells. Thus, MopB protein is demonstrated to be an important structural protein that stabilized the outer membrane of S. maltophilia. Although the cytotoxicity of SmMopB was not significantly changed, the increasing internalization and adhesion ability of these bacteria into L929 cells could be directly or indirectly related to the deficiency of MopB protein.
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