标题: | 克雷白氏肺炎杆菌CG43中MrkH、 MrkI、MrkJ和CsgD在调控第一型与第三型线毛表现所扮演的角色 Regulatory roles of MrkH, MrkI, MrkJ and CsgD on the expression of type 1 and type 3 fimbriae of Klebsiella pneumoniae CG43 |
作者: | 郑崴云 Cheng, Wei-Yun 彭慧玲 Peng, Hwei-Ling 生物科技学系 |
关键字: | 克雷白氏肺炎杆菌;第一型线毛;第三型线毛;Klebsiella pneumonia;type 1 fimbriae;type 3 fimbriae |
公开日期: | 2010 |
摘要: | 第一型和第三型线毛已被证实是克雷白氏肺炎杆菌的重要的致病因子。分析克雷白氏肺炎杆菌CG43基因体,我们发现这两型线毛基因组相邻座落,暗示二者的基因表现有协同调控的可能。本論文中,我们探讨MrkH、MrkI、MrkJ、CsgD调控蛋白对此两型线毛表现的影响。首先,分别构筑mrkH、 mrkI 、mrkJ和csgD的基因剔除菌株,再藉由甘露醣竞争酵母菌凝集测试、西方墨点法、生物膜形成能力分析这些基因缺损的影响。结果显示,mrkI基因剔除菌中第一型线毛表现增加而第三型线毛的表现量减少;mrkH基因的剔除也减少MrkA的表现量及降低生物膜活性,但不影响FimA的表现量;在mrkJ基因剔除株中,MrkA的表现量有些许增加,但生物膜活性却略微降低;另一方面,csgD基因剔除后降低第三型线毛基因组启动子活性。而在mrkI基因剔除株中,第三型线毛基因组启动子的活性降低,但fimS翻转变为正向的趋势却增加,此结果进一步证实MrkI调控蛋白在转录层次正向调控第三型线毛、负向调控第一型线毛的表现。此外,实验显示MrkJ重组蛋白具有磷酸二酯酶活性,这也暗示二级信使c-di-GMP在第三型线毛表现上扮演重要角色。最后,增加c-di-GMP结合蛋白MrkH表现能提高第三型线毛表现量,此结果更进一步支持了上述的假说。 Klebsiella pneumoniae type 1 and type 3 fimbriae have been reported to be important virulence factors. Analysis of the Klebsiella pneumoniae CG43 genome revealed the two fimbriae encoding gene clusters are physically linked, which suggesting a coordinated -regulation for their expression. In the study, regulatory roles of the MrkH, MrkI, and MrkJ and CsgD on the expression of the two fimbriae were investigated. Firstly, the K. pneumoniae CG43S3-derived mutants respectively with a gene-deletion of mrkH, mrkI, mrkJ, and csgD were generated. The deleting effects on the fimbrial activities were then determined using analysis of mannose-sensitive yeast agglutination, western blot hybridization, and biofilm formation. An increased expression of type 1 fimbriae and a decreased expression of type 3 fimbriae were found for the □mrkI strain. The deletion of mrkH also decreased the expression of MrkA and biofilm formation activity but had no apparent effect on FimA expression. On the other hand, the mrkJ deletion appeared to slightly increase the MrkA expression but decrease the biofilm formation activity while the csgD deletion reduced the promoter activity of MrkA. The mrkI deletion reduced the mrkA promoter activity but increased OFF-to-ON inversion of the fimS suggesting MrkI is the transcription regulator for their reciprocal expression. Moreover, a phosphodiesterase activity of the recombinant MrkJ was demonstrated implying the second messenger c-di-GMP (bis-(3′-5′)-cyclic dimeric GMP) plays a regulatory role on the expression of type 3 fimbriae. The possibility was further supported by the notion that overexpression of the c-di-GMP binding protein MrkH apparently increased the type 3 fimbriae activity. |
URI: | http://140.113.39.130/cdrfb3/record/nctu/#GT079828507 http://hdl.handle.net/11536/47716 |
显示于类别: | Thesis |
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