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

Abstract

第一型和第三型線毛已被證實是克雷白氏肺炎桿菌的重要的致病因子。分析克雷白氏肺炎桿菌CG43基因體，我們發現這兩型線毛基因組相鄰座落，暗示二者的基因表現有協同調控的可能。本論文中，我們探討MrkH、MrkI、MrkJ、CsgD調控蛋白對此兩型線毛表現的影響。首先，分別構築mrkH、 mrkI 、mrkJ和csgD的基因剔除菌株，再藉由甘露醣競爭酵母菌凝集測試、西方墨點法、生物膜形成能力分析這些基因缺損的影響。結果顯示，mrkI基因剔除菌中第一型線毛表現增加而第三型線毛的表現量減少；mrkH基因的剔除也減少MrkA的表現量及降低生物膜活性，但不影響FimA的表現量；在mrkJ基因剔除株中，MrkA的表現量有些許增加，但生物膜活性卻略微降低；另一方面，csgD基因剔除後降低第三型線毛基因組啟動子活性。而在mrkI基因剔除株中，第三型線毛基因組啟動子的活性降低，但fimS翻轉變為正向的趨勢卻增加，此結果進一步證實MrkI調控蛋白在轉錄層次正向調控第三型線毛、負向調控第一型線毛的表現。此外，實驗顯示MrkJ重組蛋白具有磷酸二酯酶活性，這也暗示二級信使c-di-GMP在第三型線毛表現上扮演重要角色。最後，增加c-di-GMP結合蛋白MrkH表現能提高第三型線毛表現量，此結果更進一步支持了上述的假說。

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.