HdeB、HdeB1、HdeD蛋白質參與克雷白氏肺炎桿菌CG43抗酸反應的探討

Study of the involvement of HdeB, HdeB1, HdeD in the acid stress response of Klebsiella pneumoniae CG43

曾品瑄

指導教授 ： 彭慧玲 ； Hwei-Ling Peng

國立交通大學/生物科技學院/生物科技學系暨研究所/碩士(2011年)

https://doi.org/10.6842/NCTU.2011.00802

摘要

克雷白氏肺炎桿菌是一株伺機性的格蘭氏陰性菌，常造成院內感染，包含：肺炎、化膿性感染、尿道感染、敗血症等等。在侵入人體到達腸道前，必須經過強酸環境的胃，細菌如何從強酸的環境中存活下來，是一個重要的課題。已知，大腸桿菌有五種抗酸系統和具有十二個基因的酸適應島嶼（AFI）。我們利用生物資訊工具搜尋現有的CG43基因體序列，結果發現一類似酸適應島嶼的區域，其上的hdeB、hdeB1、hdeD和大腸桿菌AFI的hdeA、hdeB、hdeD序列相近。大腸桿菌的HdeA及HdeB蛋白存在其膜間質中，在酸性環境中活化成為伴隨蛋白（Chaperone）來保護被酸破壞的膜間質蛋白。我們在此研究中探討hdeB、hdeB1、hdeD基因產物是否也扮演類似抗酸的角色，並且進一步分析HdeB及HdeB1是否也具有Chaperone的功能。本研究室曾發現在靜置培養下，雙分子訊息傳遞系統調控基因rcsB或kvhA的缺損突變菌株會降低他們的啟動子活性，而在震盪培養下，我們發現在rcsB、kvhA、fur、phoP的缺損突變菌株中hdeDB和hdeB1啟動子活性下降，而rpoS的缺損反而對hdeDB啟動子活性有小幅度的提升；利用LacZ報告系統發現在弱酸環境中，hdeDB和hdeB1的啟動子活性都比中性環境略高；而hdeB、hdeB1或hdeD基因缺損突變株的抗酸能力較野生株低，其中hdeB1基因缺損突變株的抗酸能力最弱；最後，重組的HdeB蛋白被證實能夠保護ADH不被強酸破壞，此暗示著HdeB有伴隨蛋白的功能。

關鍵字

抗酸 ； 克雷白氏肺炎桿菌 ； HdeB

並列摘要

K. pneumoniae which is a gram-negative, opportunistic pathogen often causes diseases in hospitals including pneumonia, suppurative infection, urinary tract infections and septicemia. Before reaching the intestinal tract, they need to pass through the stomach an extremely acidic environment. How to survive this deadly acidic condition is an important issue for the bacteria to establish an infection. There are 5 acid resistance (AR) systems and an acid fitness island (AFI) containing 12 protein-encoding genes reported in Escherichia coli. Using bioinformatics tool, we have found an AFI-like DNA region which contains hdeB, hdeB1 and hdeD in the partially sequenced K. pneumoniae CG43 genome. E. coli HdeA and HdeB, activated by acidic stress, have been shown to function as chaperone in the periplasmic space to protect the proteins from damage. Here we study if HdeB, HdeB1 and HdeD play protective roles in K. pneumoniae CG43 resistance to acid stress, and HdeB and HdeB1 also act as chaperone to prevent the protein from damages. The two-component system (2CS) regulators RcsB and KvhA have been previously reported to positively regulate the promoter activity of hdeDB and hdeB1 under static-cultured condition. While under shaking-cultured condition, besides the regulation of RcsB and KvhA, the hdeDB and hdeB1 promoter activity were found to be also positively affected by the 2CS regulator PhoP and iron uptake repressor Fur. By contrast, the promoter activity of hdeDB slightly increased in the rpoS mutant. Using LacZ reporter system, we have observed that the promoter activity of hdeDB and hdeB1 in mild acid condition (pH 4.4) were slightly higher than in pH 7.0 condition. We have found that deletion of hdeB, hdeB1 or hdeD from the genome reduced the bacterial survival under acid treatments. Among them, the hdeB1 deletion mutant appeared to most sensitive to the acid-induced damages. Finally, the recombinant HdeB appeared to be able to protect ADH from the damage by the extreme acid treatment suggesting HdeB is a chaperone.

並列關鍵字

Klebsiella pneumoniae CG43 ； acid stress response

參考文獻

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