克雷白氏肺炎桿菌CG43 Fur、RcsB及cyclic di-GMP 對第三型纖毛表現之調控探討

Abstract

生物膜是形成微生物感染過程中重要的一環，細菌的二級訊號分c-di-GMP已被證實是調控生物膜形成的重要分子，而第三型纖毛是決定克雷白氏肺炎桿菌生物膜形成的主要因子，其表現受c-di-GMP與PilZ domain蛋白MrkH所正向調控；另外 Fur及RcsB也會正向調控克雷白氏肺炎桿菌CG43中第三型纖毛的表現。本論文藉著分析特定基因的缺損對於第三型纖毛主要單位蛋白MrkA表現的影響，進一步探討Fur、RcsB和c-di-GMP間交互作用的可能性。我們發現在限鐵或低鐵環境中(LB中添加鐵螯合物deferoxamine、M9或DMEM培養條件下)，fur和小RNA ryhB基因同時缺損下，可以回復MrkA的生成量，進一步測試其啟動子活性和生物膜生成量，結果也顯示Fur活化MrkA表現的方式可能是透過抑制小RNA ryhB；有趣的是，當細菌在靜置培養條件下，fur基因缺損對於MrkA的表現沒有影響，然而，在此培養條件下，fur和rcsB基因同時缺損卻可測得Fur對MrkA表現的影響。在LB或M9震盪培養、或LB靜置培養下，Fur的表現量變化不大，此暗示在微氧環境中，RcsB負向調控Fur。此外，進一步藉測試第一型纖毛的活性及單位蛋白FimA的生成量，結果發現RcsB負向調控第一型纖毛活性，而Fur可能正向影響第一型纖毛的表現；有趣的是，在rcsB基因缺損下，生物膜生成明顯提高，然而，rcsB或fur基因缺損對纖維素的生成沒有明顯影響，此顯示除了第三型纖毛和纖維素外，還有其它決定生物膜生成的重要因子。進一步利用西方墨點法分析RcsB對第三型纖毛表現的調控機制，結果發現RcsB可能與RmpA2作用而影響第三型纖毛的表現。最後，在fur及rcsB雙基因缺損的狀態下，c-di-GMP依然可以活化MrkA表現，只有在mrkH基因缺損下才失去c-di-GMP的活化功能，此結果顯示c-di-GMP與Fur/RcsB對第三型纖毛表現的調控機制是分開獨立的。

Biofilm formation is an important stage for many microbial infections. Cyclic di-GMP (c-di-GMP), a bacterial second messenger, has been shown as a key signaling molecule to modulate the biofilm formation. In Klebsiella pneumoniae, type 3 fimbriae is the major determinant for biofilm formation and the expression is dependent on the c-di-GMP levels. It is speculated that MrkH, a PilZ domain protein, is responsible for the c-di-GMP dependent expression. We have previously demonstrated that Fur (Ferric uptake regulator) and RcsB (regulator of capsule synthesis) positively regulates the expression of type 3 fimbriae in K. pneumoniae CG43. Here we analyze further the specific gene deletion effects on the major pilin MrkA production to explore if interacting regulation is present between Fur, RcsB, and c-di-GMP. In low iron conditions which include LB with deferoxamine, M9 or DMEM medium, deletion of fur and ryhB can restore MrkA production. The promoter activity analysis and biofilm formation measurement also revealed that Fur activation of the MrkA production is probably mediated by repression of the small RNA ryhB. Interestingly, deletion of fur had not affected the MrkA production when the bacteria grown under static condition. However, under the same cultured condition, the fur deletion effect is again observed when the rcsB gene was removed. This implies that RcsB negatively regulates the expression of Fur under microaerobic environment. Meanwhile, type 1 fimbriae activity assessment and FimA production analysis indicated that RcsB negatively affects the expression of type 1 fimbriae while Fur probably plays a positive role on type 1 fimbriae expression. Interestingly, deletion of rcsB increased biofilm formation, however, fur or rcsB deletion had no effect on the cellulose synthesis. This indicates that, in addition to type 3 fimbriae and cellulose, other important determinant(s) is present for the biofilm formation. Furthermore, the analysis of the deletion effects of rcsB, rmpA, rmpA2, and fur on MrkA production suggests that RcsB interacts with RmpA2 for the regulation of type 3 fimbriae expression. Finally, increase c-di-GMP levels still can activate MrkA production in the absence of rcsB or fur, while the c-di-GMP dependent MrkA production is no longer observed when mrkH is deleted. This implies that the regulatory mechanisms of c-di-GMP and Fur/RcsB for the control of type 3 fimbriae expression are independent.