克雷白氏肺炎桿菌 CG43 對多黏菌素的抗性調控分析

Regulation of polymyxin resistance in Klebsiella pneumoniae CG43

陳顗峰(Yi-Fong Chen)

指導教授 ： 彭慧玲

國立交通大學/生物科技學院/分子醫學與生物工程研究所/碩士(2009年)

https://doi.org/10.6842/NCTU.2009.00462

摘要

已知，由七個基因所組成的pmr操縱子和ugd所轉譯的蛋白共同參與4-aminoarabinose的合成，進而修飾脂多醣體的磷酸根以降低其陰電性，此修飾行為能保護細菌免受陽離子性抗生素多黏菌素的攻擊。比較克雷白氏肺炎桿菌、沙門氏菌、大腸桿菌以及鼠疫桿菌的pmr操縱子的上游序列，只在克雷白氏菌及鼠疫桿菌的相對位置發現PhoP和PmrA的結合位；不同於鼠疫桿菌，克雷白氏菌還擁有pmrD同源基因。首先，我們證實在添加2 units/ml多黏菌素的培養環境下，pmrH、 pmrF、或ugd 基因的缺損，會使克雷白氏菌存活率下降；而PhoP、PmrA或PmrD的基因缺損時，菌株對多黏菌素的抗性也會降低。進一步以LacZ為報導子分析PpmrH的活性，結果顯示在phoP或pmrA突變株中其活性均會降低。然而，PhoP、PmrA或RcsB的基因缺損，並不影響ugd的啟動子Pugd-1的活性。同時，我們利用反轉錄聚合酶鏈鎖反應證實ugd和上游相鄰的manC、manB基因屬同一操縱子，暗示另一ugd啟動子的存在，我們命名為Pugd-2。雖然Pugd-2的活性在rcsB突變株中有明顯下降，但是rcsB基因的缺損並不影響菌株對多黏菌素的抗性。最後，細胞內存活率測試顯示pmrF、phoP或pmrA的基因缺損不利克雷白氏菌在已分化的THP-1或RAW264.7細胞內的存活。

關鍵字

克雷白氏肺炎桿菌CG43 ； 雙分子系統 ； 脂多醣體 ； 多黏菌素

並列摘要

It has been demonstrated that the seven-gene pmr operon and ugd respectively encode the proteins for the synthesis of 4-aminoarabinose to modify the phosphate residues of lipopolysaccharide (LPS). The decrease of the net negative charge by the LPS modification allows protection of the bacteria from the attack of the cationic antimicrobial peptide polymyxin B. In comparison of the pmr upstream sequences of Klebsiella pneumoniae, Salmonella enterica, Esherichia coli and Yersinia pestis, the putative PhoP and PmrA binding boxes were identified in the counterpart of K. pneumoniae and Y. pestis. Different from Y. pestis, the pmrD homologue was only present in the genome of K. pneumoniae. Deletion of either pmrH, pmrF or ugd reduced the resistance to 2 units/ml polymuxin B. The deletion mutants of phoP, pmrA, or pmrD also appeared to decrease their resistance to polymyxin B. Promoter activity measurement using LacZ as reporter revealed a reduction of PpmrH activity in phoP or pmrA mutant. However, deletion of phoP, pmrA or rcsB had no apparent effect on the expression of Pugd-1 indicating a different regulation of polymyxin B resistance in K. pneumoniae from that in S.enteric. RT-PCR analysis demonstrated that the ugd with the upstream manC and manB are organized as an operon indicating the presence of another promoter of ugd, namely Pugd-2. The activity of Pugd-2 reduced in the rcsB mutant. However, the deletion of rcsB had no apparent effect on the polymyxin B resistance. Finally, deletion of pmrF, phoP or pmrA was found to reduce the bacterial survival in the cells of either THP-1 or RAW264.7.

並列關鍵字

Klebsiella pneumoniae CG43 ； Two-component system ； lipoploysaccharide ； Polymyxin B ； PmrA/PmrB ； PhoP/phoQ ； PmrHFIJKLM ； 4-amino-4-deoxy-L-arabinose

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