克雷白氏肺炎桿菌CG43中 RcsFCDB訊息傳遞所扮演的角色

Abstract

RcsFCDB磷酸根傳遞系統最早被發現可調控細菌莢膜的生合成，近年來陸續有研究報導此訊息傳遞系統也參與了鞭毛的形成、泳動力及O抗原鏈的生成等與細菌毒性相關的調控。RcsF脂質蛋白位於外膜；RcsC及RcsD位於內膜，分別具有組氨酸激酶和組氨酸轉移酶活性；RcsB是細胞質中的反應調節蛋白。前期實驗室成員的研究發現克雷白氏肺炎桿菌CG43其rcsB基因缺損會降低莢膜多醣體含量、酸性逆境存活率、膜間質扮隨蛋白YfdX及第三型線毛單位蛋白MrkA的生成量，本論文探討RcsF、RcsC及RcsD蛋白對此磷酸根傳遞途徑下游的RcsB調控功能的影響。首先，我以同源互換原理建構了克雷白氏肺炎桿菌CG43rcsC、CG43rcsD、CG43rcsD-hk、CG43rcsD-hpt、CG43rcsF、CG43rcsCrcsF突變株，並進一步分析比較這些突變菌株與CG43rcsB表現型的差異後發現：CG43rcsD與CG43rcsB有相同的表現型，而CG43rcsC與CG43rcsF的莢膜多醣與MrkA的生成量沒有明顯變化；過度表達RcsC會增加莢膜多醣生成量；rcsF基因缺失會使細菌失去偵測多黏菌素壓力的能力，CG43rcsD與CG43rcsB酸壓力下存活率下降。接著，我以LacZ報導系統及定量PCR分析啟動子活性，結果顯示啟動子PrcsB活性遠高於PrcsC、PrcsDB及PrcsF三個啟動子，而PrcsB、PrcsDB、PrcsF會被弱酸誘導；PrcsB、PrcsC、PrcsF會被多黏菌素所誘導。最後，體外磷酸轉移系統建立的結果顯示：磷酸根染劑ProQ將來可以做為探討磷酸根自RcsC經由RcsD至RcsB傳遞路徑的標記。

The RcsFCDB phosphorelaying system originally identified as a regulator of capsule synthesis (Rcs) is also involved in regulation of the expression of other virulence properties including flagella production, swimming motility, and O-antigen chain length control. RcsF is a periplasmic lipoprotein; RcsC and RcsD are two inner membrane proteins respectively exerting histidine kinase (Hk) and histidine phosphotransfer (Hpt) activity; RcsB is a cytoplasmic response regulator. We have previously shown that the deletion of rcsB from Klebsiella pneumoniae CG43S3 reduced the levels of not only the capsular polysaccharide (CPS) biosynthesis but also the type 3 fimbriae major pilin MrkA production and the acid stress response. Here we investigate if the regulatory activity of RcsB could be correlated to the RcsFCD phosphorelay. Firstly, we generate K. pneumoniae CG43S3ΔrcsF, CG43S3ΔrcsC, CG43S3ΔrcsD, CG43rcsD-hk, and CG43rcsD-hpt, and compare their deletion effects with those of CG43S3ΔrcsB. The results showed that CG43S3ΔrcsD exerted a similar phenotype as that of CG43S3ΔrcsB. However, the deletion of rcsF or rcsC had no apparent effect on the production of CPS or MrkA; overexpression of RcsC increased the CPS production; deletion of rcsF rendered the bacteria insensitive to polymyxin B stimulation. We have also measured the gene expression using promoter reporter assay and qRT-PCR analysis. The results revealed that PrcsB had much higher activity than the activity of PrcsB, PrcsDB, and PrcsF could be induced by weak acid while PrcsC, PrcsB, and PrcsF were polymyxin B inducible. Finally, the set up in vitro phosphorelay system revealed that the non-specific dye ProQ could be used to determine the phosphorelaying activity between the signal transferred from RcsC, RcsD to RcsB.