克雷白氏肺炎桿菌CG43S3預測為孤兒反應調控蛋白MrkI和CsgD的功能性探討

Study of the putative orphan response regulator MrkI and CsgD in Klebsiella pneumoniae CG43S3

林小倩(Siao-Cian Lin)

指導教授 ： 彭慧玲

國立交通大學/生物科技學院/生物科技學系暨研究所/碩士(2018年)

摘要

細菌的訊息傳遞系統中，孤兒感應調控蛋白是指一群帶有磷酸根接收序列功能區但其激酶未明的感應蛋白，本論文針對克雷白氏肺炎桿菌CG43兩個功能未明的孤兒感應調控蛋白MrkI和CsgD做探討。
本論文的第一部份分別以qPCR和啟動子活性測試來研究MrkI是否和與其同一控制組的MrkH一樣參與纖毛基因組表現或二次訊息分子c-di-GMP水平的調控。克雷白氏肺炎桿菌CG43有11套纖毛基因組，分別為第一型(fim)、第三型(mrk)、kpa, kpb, kpd, kpe, kpf, kpg, kph, kpi和ecp 纖毛基因組。以qPCR分析的結果顯示：在CG43S3中，主要偵測到第三型纖毛單位蛋白mrkA基因的表現，第一型纖毛fimA單位蛋白基因的轉錄水平顯著低於mrkA，而其它九套纖毛的表現幾乎無法被偵測；而在剔除mrkI或mrkH後，mrkA轉錄水平顯著降低而相對提升fimA的轉錄，此與已被報導的結果相吻合；另外，kpaA、kpbA及kpdA的轉錄水平因mrkI或mrkH基因的缺失而顯著下降；啟動子序列分析後發現在mrkH、mrkABCDF、fimE、kpbA、kpeA的啟動子區皆有MrkH辨識序列TATCAA，然而，mrkI或mrkH基因缺失除了明顯抑制mrkA表現外，其他啟動子活性均不受影響。另外，qPCR分析顯示mrkI或mrkH基因缺失對幾個在啟動子區有MrkH辨識序列的c-di-GMP磷酸二酯酶或環化酶的表現影響相反，此暗示MrkI與MrkH在c-di-GMP水平調控上扮演不同角色。
第二部份以活體外磷酸根傳遞分析以推斷CpxA是否為傳遞磷酸根給CsgD的激酶:以Pro-Q Diamond磷酸化蛋白質螢光染劑檢測結果顯示CsgD可被染上螢光，此支持其為孤兒反應蛋白的可能性；在 CG43S3[pQE81L-CsgD]以IPTG誘導CsgD表現後，其FimA量及酵母菌凝集力增加，而此影響會因cpxA基因缺失而消失，相對的，剔除rcsC或rcsD基因不影響CsgD表現對第一型纖毛表現的影響，此暗示CpxA可影響CsgD對於第一型纖毛的調控；雖然IPTG誘導CsgD表現後無法增加其胞外多醣的生成，卻發現CG43S3[pQE81L-CsgD-N]在剛果紅培養基上所呈現之紅色沉澱會因cpxA基因缺失而消失，而 CpxA是否為影響CsgD-N磷酸化的激酶還待確認。

關鍵字

克雷白氏肺炎桿菌 ； 孤兒反應調節蛋白 ； 磷酸化分析

並列摘要

In the bacterial signal transduction system, the orphan response regulator refers to a group of sensory proteins with a phospho-acceptor sequence functional region but whose kinase is unclear. In this thesis, the functionally unknown orphan response regulator, MrkI and CsgD of Klebsiella pneumoniae CG43 were explored.
For the first part of this thesis, qPCR and promoter activity tests were used to study whether MrkI as well as MrkH that residing in the same operon was involved in the regulation of fimbrial gene clusters expression or the level changes of the second messenger c-di-GMP. Klebsiella pneumoniae CG43 has eleven distinct of fimbrial gene clusters, namely type 1 (fim), the type 3 (mrk), kpa, kpb, kpd, kpe, kpf, kpg, kph, kpi and ecp fimbriae. The results of qPCR analysis showed that in CG43S3, the expression of the type 3 fimbriae major pilin gene mrkA was mainly detected, and the transcription level of the type 1 fimbriae major pilin gene fimA was significantly lower than mrkA, while the expression of the other nine fimbriae were barely detectable. Consistent with the previous findings, deletion of mrkI or mrkH significantly decreased the transcription level of mrkA, but increased the transcription of fimA. In addition, the transcriptional levels of kpaA, kpbA and kpdA were significantly decreased by the deletion of the mrkI or mrkH. The promoter sequences analysis revelaed that the MrkH recognition sequence TATCAA, could be found in the putative promoter region of mrkH, mrkABCDF, fimE, kpbA, and kpeA. Deletion of mrkI or mrkH was found to significantly inhibit mrkA expression, while other promoter activities were unaffected. Moreover, the deletion effects of mrkI or mrkH on the selected c-di-GMP cyclase and phosphodiesterase-encoding genes of which the putative promoter carrying MrkH recognition element, were analyzed by qPCR, and the results showed that MrkI and MrkH differentially affected the expression and hence MrkI and MrkH may play different regulatory role in the c-di-GMP regulatory pathways.
For the second part of the thesis, an in vitro phosphorelay assay was employed to infer whether CpxA was the kinase that transmited phosphate to CsgD. Firstly, the recombinant CsgD was shown to be able to be stained by the Pro-Q Diamond fluorescent dye which supporting the possibility that CsgD was an orphan response regulator. The induced expression of CsgD by IPTG in CG43S3[pQE81L-CsgD] increased expression of FimA and its yeast agglutination activity. However, this effect was not observed by deleting cpxA suggesting that CpxA affected the regulation of CsgD on the type 1 fimbriae. By contrast, deletion of rcsC or rcsD did not affect the CsgD activated expression of type 1 fimbriae. Although the induction of CsgD could not increase the production of extracellular polysaccharides (EPS), the expression of CsgD-N significantly increased the EPS production and the effect was no longer observed in ∆cpxA. If CpxA is the kinase that affected CsgD-N phosphorylation remains to be investigated.

並列關鍵字

Klebsiella pneumoniae ； Orphan response regulator ； Phosphorelay assay

參考文獻

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3. 鄭崴云 (2011) 克雷白氏肺炎桿菌CG43中MrkH、 MrkI、MrkJ和CsgD在調控第一型與第三型線毛表現所扮演的角色，國立交通大學碩士論文。

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