克雷白氏肺炎桿菌第三型纖毛組成蛋白MrkD與MrkF之功能探討

Abstract

本實驗室在過去的研究中，將17 株克雷白氏肺炎桿菌腦膜炎臨床分離株基因序列加以分析，並定出四種第三型纖毛黏附蛋白的變異型，分別命名為MrkDv1, MrkDv2, MrkDv3, MrkDv4。將帶有不同黏附蛋白變異型的第三型纖毛分別表現在大腸桿菌JM109中，發現帶有MrkDv3黏附蛋白的纖毛具有最高的膠原蛋白黏附能力以及紅血球凝集能力。比較這四種黏附蛋白的胺基酸序列發現MrkDv3具有一段與其他三種黏附蛋白顯著不同的胺基酸序列位於第120到第140個胺基酸之間，顯示出此段胺基酸序列似乎與MrkDv3的高黏附能力相關。此外，基因重組大腸桿菌HB101[pmrkABCF]與HB101[pmrkABCD]皆能產生纖毛，顯示MrkF可能參與纖毛組裝過程。本實驗製作三種不同長度的MrkDv3截短蛋白分別帶有前170個胺基酸(MrkDv3NL)、前150個胺基酸(MrkDv3N)以及前120個胺基酸(MrkDv3NS)。不可溶性重組蛋白大量表現於重組大腸桿菌中並可藉由蛋白質電泳觀察出來。經由尿素處理加以純化而得的重組蛋白dMrkDv3N 可以抑制帶有pmrkABCDv3F重組質體的大腸桿菌對第四型膠原蛋白的黏附力。此外，透過西方墨點法以及穿透式電子顯微鏡的觀察顯示MrkF是第三型纖毛的組成分子並且與調控纖毛長度有關。利用共沉澱分析進一步證實MrkF與MrkA於纖毛之中相互連結並且透過膠原蛋白黏附測試、生物膜形成測試、以及細菌聚集測試證實MrkF參與調控第三型纖毛的生物活性。

Our previous analysis of type 3 fimbriae in 17 Klebsiella pneumoniae meningitis isolates has identified four type 3 fimbrial adhesin variants namely MrkDv1, MrkDv2, MrkDv3, and MrkDv4. The recombinant Escherichia coli JM109 displaying type 3 fimbriae with mrkDv3 allele exerted the highest level of activity in collagen binding and mannose resistant hemagglutination. Sequence comparison with the other three variants revealed a most variable region from Gly120 to Gln140, suggesting a responsible sequence for the strongest adhesion activity. In addition, the recombinant E. coli HB101[pmrkABCF] expressed fimbriae as well as E. coli HB101[pmrkABCD] indicating the involvement of MrkF in fimbriation. In the study, the recombinant clones expressing with three truncated forms of MrkDv3 including MrkDv31-170aa (MrkDv3NL), MrkDv31-150aa (MrkDv3N), and MrkDv31-120aa (MrkDv3NS) were generated. Overexpression of all the recombinant proteins could be observed in E. coli BL21(DE3), however, appeared to be insoluble. Purification via urea denaturalization of the inclusion body was carried out and the purified protein MrkDv3N was shown to be able to inhibit the collagen binding activity of E. coli JM109[pmrkABCDv3F]. Moreover, analysis using western blotting hybridization, transmission electronic microscopy (TEM) with anti-MrkA and anti-MrkF sera demonstrated that MrkF is a component of type 3 fimbriae and likely a regulator for the length of the fimbriae. Co-immunoprecipitation analysis with anti-MrkF and anti-MrkA antibodies has proved an interaction of MrkA and MrkF. Finally, the assessment by the assays of collagen binding, biofilm formation, and autoaggregation, also showed that MrkF likely plays a regulatory role in the activity of type 3 fimbriae.