RcsB蛋白質在克雷白氏肺炎桿菌CG43中抗酸能力所扮演的角色

Abstract

已知Rcs雙分子訊息傳導系統參與調控細菌體中許多的生理反應；如同許多腸內菌，此雙分子系統的反應調控蛋白rcsB的基因缺損，會明顯的降低克雷白氏肺炎桿菌CG43莢膜多醣體的生合成。我們在此研究中探討：是否如同近期報導的大腸桿菌RcsB蛋白，克雷白氏肺炎桿菌CG43的RcsB也具有抗酸調控的功能，並進一步研究其調控機制。我們發現：克雷白氏肺炎桿菌CG43在弱酸 (pH 4.4)適應一小時後，其抗酸逆境 (pH 3)能力大幅提高，而rcsB基因的缺損會降低其抗酸能力，但rcsB的啟動子活性並不受弱酸的誘導。同時，我們利用生物資訊工具在CG43基因體序列 (http://genome.nhri.org.tw/KP/index.php)中，找出六個可能由RcsB所調控的抗酸基因，其中yfdX, hdeD-hdeB1及hdeB2聚集座落於相對大腸桿菌的酸適應島嶼 (acid fitness island)；再經PCR選殖以LacZ來報告這六個啟動子的活性，結果發現cfa及yfdX在rcsB缺損株的表現明顯下降；然而，cfa或yfdX基因缺損突變株的抗酸能力，與野生株差異不大；我們發現只有在靜置培養下，yfdX的基因缺損才會降低克雷白氏肺炎桿菌的抗酸能力。而在靜置培養下分析hdeD-hdeB1, yfdX及hdeB2的啟動子活性，我們發現rcsB或kvhA的基因缺損都會降低上列啟動子的活性。最後，我們利用二維電泳比較分析 CG43和rcsB基因缺損株的蛋白質體，結果發現在中性環境下 (pH 7.5)，沒有看到表現量差異很大的蛋白質點；而在酸適應條件下 (pH 4.4)，發現有2個蛋白質點在CG43S3□rcsB的二維電泳膠上消失，另有3個蛋白質點的表現量，則在CG43S3□rcsB分別下降了2.18, 1.90及1.52倍。我們將進一步鑑定其蛋白質ID，以了解是哪些蛋白質提高了抗酸能力。綜合以上結果，RcsB在克雷白氏肺炎桿菌CG43於震盪培養條件下，會調控未知的蛋白質來提高抗酸能力；而在靜置培養下，則可能藉由調控基因hdeD-hdeB1, yfdX及hdeB2的表現來提高其抗酸能力，同時，位於此可能的酸適應島嶼中的雙分子訊息傳導系統KvhAS也參與調控HdeD, HdeB1, YfdX及HdeB2的抗酸能力。

The Rcs two component signal transduction system controls a variety of physiological functions in bacteria. Deletion of the response regulator gene rcsB in Klebsiella pneumoniae CG43, a highly encapsulated clinical isolate, resulted in reduction of the CPS production as reported for many enterobacteria. Recently, an involvement of RcsB in the acid resistance regulation has been reported in Escherichia coli. If K. pneumoniae RcsB plays a similar role is investigated and the regulatory mechanism also analyzed in this study. The resistance to acidic stress (pH 3.0) apparently increased for K. pneumoniae CG43 after an adaption under the weak acidic environment (pH 4.4). Deletion of rcsB reduced the bacterial survival under the acid stress treatment. However, the acid adaptation had no inducing effect for the expression of rcsB. Homologous gene search in CG43 genome (http://genome.nhri.org.tw/KP/index.php) using bioinformatic tools revealed six putative RcsB-dependent acid resistance genes. Among them, yfdX, hdeD-hdeB1 and hdeB2 genes were found to be clustered within the putative AFI (acid fitness island). The putative promoters were PCR amplified and cloned into the LacZ reporter plasmid pLacZ15. The activity measurement showed that the expression of cfa or yfdX was reduced in the rcsB deletion mutant. Deletion of cfa or yfdX had no effect on acid resistance ability of K. pneumoniae. Only under a statically culture condition, deficiency in the acid resistance ability could be observed for the yfdX deletion mutant. Thus, the promoter activity of yfdX, hdeD-hdeB1, or hdeB2 was analyzed under static culture. The result showed that deletion of rcsB or kvhA reduced the promoter activity of these genes. Finally, comparative proteome analysis of CG43S3 and CG43S3□rcsB using two-dimensional electrophoresis was also employed. No significant change of expression fold was found under the growth at pH 7.5. While under the acidic condition (pH 4.4), 2 protein spots only present on the 2D gel of CG43S3 and 3 proteins with decreased expression level (-2.18, -1.90 and -1.52 fold, respectively) in CG43S3□rcsB were observed. The proteins ID will be resolved in the near future. To sum up, RcsB appears to regulate some unknown proteins for acid resistance response under shaking culture. Under static culture with micro-aeration, expression of yfdX, hdeD-hdeB1 and hdeB2 was positively regulated by RcsB to increase the acid resistance activity. Moreover, the 2CS KvhAS located on the putative AFI is probably also involved in regulation of the acid resistance ability conferred by YfdX, HdeD, HdeB1 or HdeB2 in K. pneumoniae CG43.