標題: 克雷白氏肺炎桿菌 CG43 雙分子系統 CpxAR 調控路徑之探討
Study of the two component system CpxAR-dependent regulatory pathways in Klebsiella pneumoniae CG43
作者: 劉亭伊
彭慧玲
Liu, Ting-Yi
分子醫學與生物工程研究所
關鍵字: 雙分子調控系統;第三型纖毛;纖維素;克雷白氏肺炎桿菌;two component system;type 3 fimbriae;Cellulose;Klebsiella pneumoniae
公開日期: 2016
摘要: CpxAR 雙分子調控系統由組胺酸激酶 CpxA 與反應調控蛋白 CpxR 所組成,可幫助細菌偵測外在環境的變化而採適當應對反應,已知可活化 cpx 基因組的訊號包括鹼性 pH、滲透壓、溫度、金屬、細胞膜組成改變與錯誤摺疊的蛋白。我們已經發表的報導顯示:克雷白氏肺炎桿菌 CG43S3 在剔除 cpxAR 基因後,其第一型與第三型纖毛的表現會顯著增加;並以核醣核酸定序技術與即時定量聚合酶連鎖反應來分析比較 CG43S3、CG43S3ΔcpxAR 與 CG43S3ΔcpxR 轉錄體的差異,來了解 CpxAR 系統如何影響第三型纖毛的表現。本論文首先以 cpxAR 與 cpxP 啟動子活性分析來尋找可活化 cpx 基因組的訊號,結果發現其活性會被鋅離子與過氧化氫所抑制,而不受尿酸鹽的影響;此外,我們發現 cpxAR 基因缺損會明顯增加其對抗生素的感受性,而以回補實驗測試確認 CpxAR 在抗生素抗性作用中可能扮演重要角色;接著,將前述轉錄體資料分析整理後,我們在這些 CpxAR 調控途徑的可能成員中選擇三個可能影響第三型纖毛表現的基因做進一步探討,其一為負責編碼雙鳥苷酸環化酶的 yfiN,第二個是莢膜生成的轉錄因子 rcsA,第三個是活化第三型纖毛的轉錄因子 mrkH。經啟動子活性分析顯示 cpxAR 基因缺損會提升 yfiRNB 表現,以凝膠電泳位移分析發現純化後的重組 CpxR 可以專一結合 yfiRNB 的啟動子 DNA 片段;再者,由西方免疫墨點法分析發現 rcsA 基因剔除會增加其第三型纖毛的主要結構蛋白 MrkA 的表現,而以纖維素生成量分析實驗指出 rcsA 基因缺損可使纖維素生成量增加,而在 rcsA 基因缺損中增量表現分解 c-di-GMP 的酵素基因 yjcC 可以降低纖維素的生成,進一步以生資軟體搜尋,結果發現在影響 c-di-GMP 濃度的基因 yjcC 與 ycdT 啟動子序列上均可找到 RcsAB 的結合序列,此暗示 RcsA 可能透過調控 c-di-GMP 濃度進而影響纖維素的生成與第三型纖毛的表現;最後,在 cpxAR 或 yjcC 基因缺損株中分別剔除 mrkH 基因後,均可降低其纖維素的生成,而在六個影響 c-di-GMP 濃度的 D364_24605、D364_06025、D364_08130、yoaD、D364_14405 及 D364_13985 啟動子上均可找到 MrkH 的結合序列,此暗示 MrkH 可能藉由調控這些基因表現而影響 c-di-GMP 濃度;另外,西方免疫墨點法分析顯示 MrkH 只有在 cpxAR、yjcC 雙基因缺損株中有微量表現,此暗示 MrkH 的表現只有在 c-di-GMP 濃度夠高時才可被偵測到。總而言之,CpxAR 可能會透過調控 YfiN、RcsA 和 MrkH 蛋白進而影響 c-di-GMP 濃度而調控第三型纖毛表現與纖維素生成。
The envelope stress two component system CpxAR, which is composed of the sensor kinase CpxA and the cognate response regulator CpxR, allows bacteria monitor the ever changing environments and respond with proper adjustments. The known cpx activation signals include alkaline pH, osmotic pressure, temperature, metals, alterations of membrane composition and misfolded proteins. We have previously reported that deleting cpxAR gene from Klebsiella pneumoniae CG43S3 significantly enhanced the expression of type 1 and type 3 fimbriae. A comparative transcriptomic analysis of CG43S3, CG43S3ΔcpxAR, CG43S3ΔcpxR by RNA sequencing and qRT-PCR were subsequently employed to understand how Cpx system affects the type 3 fimbriae expression. In this thesis, we firstly analyzed the cpxAR and cpxP promoter activity and found that activities could be repressed by zinc ion and hydrogen peroxide, but had no apparent response to the addition of urate. The deletion of cpxAR increased the sensitivity to antibiotics and the defects could be complemented by introducing a expression plasmid into the mutant which further confirmed that CpxAR may play an important role in the resistance to antibiotic. Next, after analysis and intergration of the aforementioned transcriptome data, we selected three potential members of the CpxAR regulatory pathways for further study. One is yfiN which coding for diguanylate cyclase (DGC), the second is rcsA that encodes the major transcription regulator for the capsular polysaccharides synthesis, the third is mrkH which is an activator for the expression of type 3 fimbriae. Promoter activity analysis revealed that the deletion of cpxAR increased the expression of yfiRNB and EMSA analysis indicated that the recombinant CpxR was able to specificity bind to the yfiRNB promoter. Furthermore, we have found that the rcsA deletion increased the production of MrkA (the major pilin of type 3 fimbriae) as assessed using western blot analysis, and the deletion also increased the cellulose production. Moreover, the rcsA deletion effects could be restored by transforming a YjcC expression plasmid into the mutant, and bioinformatic analysis revealed that the putative promoter region of yjcC and ycdT contain RcsAB binding sequences. These results suggest the RcsA-mediated regulation is through controlling the c-di-GMP levels thereafter affecting the production of type 3 fimbriae and cellulose. At last, deletion of mrkH from ΔcpxAR or ΔyjcC decreased the cellulose production, and MrkH binding elements could be identified on the puatative promoter of six c-di-GMP level modulators D364_24605, D364_06025, D364_08130, yoaD, D364_14405 and D364_13985, implying a MrkH dependent expression of these genes. In addition, western blot analysis showed that MrkH expression could only be detected in ΔcpxARΔyjcC. Taken together, we speculate that CpxAR regulates the activity of YfiN, RcsA, and MrkH thereby influencing the c-di-GMP levels and eventually affects the type 3 fimbriae expression and cellulose production.
URI: http://etd.lib.nctu.edu.tw/cdrfb3/record/nctu/#GT070357129
http://hdl.handle.net/11536/138970
Appears in Collections:Thesis