- 學位論文
酸逆境下本土分離腸出血性大腸桿菌 O157:H7 TWC01之生理反應及蛋白質表現變化
The physiological response and protein expression of local isolated Escherichia coli O157:H7 TWC01 under acid stress
摘要
O157:H7 型大腸桿菌為重要之食品病原菌之ㄧ，與其他病原菌相比，此菌有較高之酸耐受性。根據前人研究發現，將此菌暴露於中等酸度環境一段時間，其會針對酸逆境進行調控，可能提高此菌對後續強酸 (如胃酸) 或是其他環境壓力之抵抗力，即產生酸耐性反應 (acid tolerance response, ATR)。本研究乃針對本土分離之 O157:H7 型大腸桿菌，觀察其於酸性環境下之生長情形，並對其產生酸耐性反應之蛋白質調控機制進行探討。 本研究使用民國 87 至 93 年間，自臺灣地區分離之 6 株 O157:H7 型大腸桿菌，包括 5 株家畜糞便分離株以及 1 株人體臨床檢體分離株，觀察其於酸性環境下之生長特性，結果顯示此 6 株本土分離之O157:H7 型大腸桿菌彼此間的酸耐受性並無顯著差異。繼而選擇其中人體臨床檢體分離株 TWC01，使用鹽酸及乳酸做為酸化劑，進行酸馴化 (acid adaptation) 處理，使其產生酸耐性反應，之後萃取馴化後菌體之總蛋白質，進行蛋白質體學分析，此外並觀察酸馴化對毒素分泌之影響。實驗結果發現，在毒素分泌方面，臨床分離株 TWC01 經酸馴化處理後有提高其 stx 毒素分泌的趨勢。分析二維電泳後之影像顯示，兩種酸馴化處理後之細胞，其蛋白質表現多為 up-regulation，且乳酸對細胞之影響大於鹽酸。以質譜分析表現量差異明顯的蛋白質發現，其主要為代謝相關之蛋白質，另有一部分與轉譯、蛋白質折疊，以及膜蛋白相關。由這些蛋白質於酸處理下表現量的變化得知，O157:H7 型大腸桿菌於酸處理下會產生許多大腸桿菌間共通之耐酸機制，同時代謝路徑傾向增加能量的生成以及 polypeptide 的合成，供細胞足夠之能量應付酸逆境所造成之生理變化，因而增加細胞對後續酸逆境之抗抵力，產生酸耐性反應；另一方面，O157:H7 型大腸桿菌細胞對環境中鹽酸 (無機酸) 與乳酸 (有機酸) 之感應，以及所引起之調控反應有所差異，顯示細胞需要較多之能量以應付環境中的乳酸。本研究有助於了解O157:H7 型大腸桿菌於酸逆境下之調控反應以及致病能力的影響，於食品安全方面，將可提供做為此菌防治上之參考。
並列摘要
Escherichia coli O157:H7 is one of the important food-borne pathogens, and it has unusually higher resistance to acidic environment than other food-borne pathogens. Some researches revealed that acid adapted cells, by exposure to moderately acidic conditions, are more resistant to a subsequent strong acidic challenge or other stress. This study was conducted to understand the growth and the protein regulatory pathways of acid tolerance response (ATR) of local isolated E. coli O157:H7 induced by acidic environment. Six local isolated E. coli O157:H7 were compared the growth under the acid stress and had no difference in acid tolerance. After that, chose the clinical strain TWC01 for further study. By using hydrochloric acid (HCl) or lactic acid as acidifier, E. coli O157:H7 TWC01 were acid adapted to induce ATR. The total protein of adapted cells were extracted for proteomic analysis. Furthermore, the effect of acid adaptation to stx secretion were examined. The result revealed that after acid adaptation, it tended to increase the stx secretion of E. coli O157:H7 TWC01. Image analysis of the gel indicated that a great part of proteins were up-regulation, and lactic acid had more effect on cell than HCl. Analyzed proteins by mass spectrometer, most of proteins were metabolism related protein, and others were related to translation, folding, and membrane proteins. According to protein expression variety under acid stress has found that the induction of many acid tolerance mechanism of E. coli. The metabolism pathway were toward increasing energy production and polypeptide synthesis, in order to provide enough energy and induce ATR. Furthermore, E. coli O157:H7 needed more energy to deal with lactic acid than HCl, revealed that organic and inorganic acid have different stress sensor and response system. This study provide the information of regulation and virulence of E. coli O157:H7 under acid stress, it may help control of this pathogen and improve food safety.
參考文獻
延伸閱讀
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