克雷白氏肺炎桿菌CG43中cAMP受體蛋白CRP和二級傳訊分子cyclic di-GMP相互調控之研究

Study of CRP and cyclic di-GMP interacting regulation in Klebsiella pneumoniae CG43

許原臺(Yuan-Tai Hsu)

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(2019年)

摘要

克雷白氏肺炎桿菌CG43是從長庚紀念醫院肝膿瘍病患中分離出的一株革蘭氏陰性、兼性厭氧、有厚重莢膜、不具有鞭毛移動能力的桿菌，能產生肺炎、肝膿瘍、泌尿系統感染、菌血等症狀。CRP-cAMP是細菌的重要調控因子，有研究顯示CRP-cAMP可能藉由調控c-di-GMP的生成量來負向影響第三型纖毛的表現，本論文的重點在釐清此假說並進一步探討其機制。首先，比較crp、cya、cpdA與親本株CG43S3第三型纖毛單位蛋白MrkA的表現量差異，結果發現crp或cya基因剔除使MrkA表現量稍微下降，另外，葡萄糖的添加也會降低MrkA表現量；而CRP對MrkA表現的正向影響在pdeC基因缺失的背景下，也就是在較CG43S3高的c-di-GMP濃度下，較為明顯；接著，以啟動子活性測試分析CRP對生成與降解c-di-GMP的dgcT、blrP1與pdeC的表現影響，結果發現crp基因缺失使三者的啟動子活性升高，而此影響在去除啟動子上CRP的結合位即消失，此顯示CRP負向調控dgcT、blrP1與pdeC的表現，進一步以EMSA試驗分析，然而，因為試驗條件有待改進而無法證明CRP是否直接結合三者啟動子上的序列；另外，啟動子活性測試也發現crp基因缺失使mrkA表現量下降但不影響mrkH的啟動子活性；最後，改以分析胞外多醣的生成來佐證CRP對c-di-GMP濃度的調控，結果發現crp或cyaA剔除後，會使胞外多醣表現上升，此暗示CRP-cAMP負向影響c-di-GMP濃度，為了確認其調控機制，進一步利用啟動子活性測試CRP對胞外多醣PNAG生合成的相關基因pgaA與kva15表現的影響，結果顯示crp基因剔除會提升pgaA與kva15啟動子活性，顯示其為負向調控。綜而言之，CRP-cAMP可能經由調控D364_RS17070轉錄因子表現，進而影響第三型纖毛的活性，而CRP-cAMP對c-di-GMP濃度的負向調控也可由胞外多醣PNAG生成量來佐證。

關鍵字

克雷白氏肺炎桿菌 ； 環腺苷酸受體蛋白 ； 環二鳥苷酸 ； 第三型纖毛 ； 胞外多醣

並列摘要

Klebsiella pneumoniae CG43, a Gram-negative, facultative anaerobic, encapsulated and non-motile bacillus was isolated from a diabetic patient with liver abscess at Chang Gung Memorial Hospital. It can cause nosocomial diseases such as bacteremia, pneumonia, urinary and respiratory tract infection. CRP-cAMP, a master regulator in bacteria, had been reported a negative role on the expression of type 3 fimbriae via regulating the c-di-GMP level. The study aims at demonstrating this hypothesis and further exploring the regulatory mechanism. At first, by comparing the expression of major pilin MrkA of type 3 fimbriae, we found that MrkA production slightly decreased by the deletion of crp or cya. Addition of glucose also decreased the MrkA expression. The positive effect of CRP on the MrkA expression was more apparent under the pdeC deletion background with higher level of c-di-GMP than that of CG43S3. Then, promoter activity analysis was employed to study the CRP regulation on the c-di-GMP levels. We found that crp deletion increased the dgcT, blrP1 or pdeC promoter activity, and the effect was eliminated when the CRP binding site on the promoter was truncated. This suggests a negative effect of CRP on dgcT, blrP1 and pdeC expression. Futhermore, EMSA we applied to analyze the presence of a direct binding of CRP to the promoter region of dgcT, blrP1 and pdeC. Nevertheless, the optimal binding condition remained to be identified for a conclusion. In addition, we also found that crp deletion decreased mrkA expression but had no change on the mrkH promoter activity. Finally, we use EPS production assess the CRP regulation on the c-di-GMP level. Deletion of crp or cyaA significantly increased EPS production suggesting CRP-cAMP negatively regulated c-di-GMP level. To explore the regulatory mrchanism, CRP deletion effect on the expression of the PNAG biosynthesis genes, pgaA and kva15, was analyzed and the results showed that crp deletion increased pgaA and kva15 promoter activity, indicating a negative regulation of CRP on the expression of pgaA and kva15. In summary, D364_RS17070 transcriptional factor may mediate the CRP-cAMP dependent type 3 fimbriae expression. In addition, CRP-cAMP negative regulation on the c-di-GMP levels could be assessed by the EPS production levels.

並列關鍵字

Klebsiella pneumoniae ； cAMP receptor protein ； cyclic di-GMP ； type 3 fimbriae ； extracellular polysaccharides

參考文獻

1. Keynan, Y, & Rubinstein, E. The changing face of Klebsiella pneumoniae infections in the community. Int J Antimicrob Agents. 2007; 30 (5): 385-389.

2. Hoyos-Orrego A, Rivera-Rivera O, Hoyos-Posada C, Mesa-Restrepo C, Alfaro-Velasquez JM. Características clínicas, epidemiológicas y de susceptibilidad a los antibióticos en casos de bacteriemia por Klebsiella pneumoniae en neonatos. Rev CES Med. 2007; 21 (2): 31-39.

3. Fung CP, Lin YT, Lin JC, et al. Klebsiella pneumoniae in gastrointestinal tract and pyogenic liver abscess. Emerg Infect Dis. 2012; 18 (8): 1322-1325.

4. Siu LK, Yeh KM, Lin JC, Fung CP, Chang FY. Klebsiella pneumoniae liver abscess: a new invasive syndrome. Lancet Infect Dis. 2012; 12 (11): 881-887.

5. Tsai FC, Huang YT, Chang LY, Wang JT. Pyogenic Liver Abscess as Endemic Disease, Taiwan. Emerg Infect Dis. 2008; 14 (10): 1592-1600.