第三型線毛為克雷白氏肺炎桿菌(Klebsiella pneumoniae)重要的毒力因子之一。組成克雷白氏肺炎桿菌的第三型線毛至少需四個相連的基因，分別為mrkA, mrkB, mrkC和mrkD。為了辨識主要單體MrkA蛋白質中影響MrkA-MrkA相互作用的重要區域，我們建立了MrkA隨機插入突變株。我們發現Class І pilins保留性胺基酸區域，對線毛主體(pilin)分子間的相互作用扮演重要角色。此外，我們發現片段插入MrkA低保留性的第140到第150個胺基酸之間，對線毛的功能也有影響。為了測試功能性，我們檢視MrkA插入突變株透過離心沉澱速率、菌液黏度、人類紅血球凝集、黏附膠原蛋白和生物膜形成測試，證實這些插入突變株會影響第三型線毛的生物活性。進一步利用穿透式電子顯微鏡，直接證實有四株MrkA插入突變株可在大腸桿菌細胞表面表現線毛。此外，我們挑選此四株仍可表現線毛的MrkA插入突變株，並於MrkA蛋白質塞入豬內源反轉錄外套蛋白病毒的抗原決定位。我們進一步證實，塞入抗原決定位的雜合型MrkA聚合體能在大腸桿菌中表現。克雷白氏肺炎桿菌為人類重要的病原菌，我們期望這些結果可以幫助了解第三型線毛的組裝機制，進而阻止此細菌的聚集與感染。未來，第三型線毛也有潛力可在大腸桿菌中當作呈現外來抗原決定位的一種工具。

Type 3 fimbriae is one of the major virulence properties associated with the pathogen Klebsiella pneumoniae. At least four linked genes, mrkA, mrkB, mrkC, and mrkD are required to produce type 3 fimbriae in K. pneumoniae. To identify regions on major pilin MrkA important for MrkA-MrkA interaction, random insertion mutagenesis was performed on this protein. Our results showed that amino acid regions, which are conserved among all subunits of Class І pilins, play a critical role in intermolecular interaction of MrkA. In addition, insertion at a variable region from their Asn-140 to Ile-150 affects pilus function. These mrkA insertion mutants were functionally assessed for the ability to agglutinate human erythrocytes, binding to type Ⅳ collagen, sedimentation rate, viscosity and biofilm formation, and the results showed that these insertion mutations affect the activity of type 3 fimbriae. Moreover, analysis using transmission electronic microscopy has demonstrated that four of the 5 aa-insertion mrkA mutants could express fimbriae on E. coli cell surface. At the insertion sites, we further inserted an Env protein antigenic determinant of porcine endogenous retrovirus. Our results demonstrated that the recombinant MrkA polymer with an insertion of the viral antigenic determinant is expressed in E. coli. K. pneumoniae is important human pathogen. We anticipate that the results obtained here will help to understand the type 3 pilus assembly mechanism in K. pneumoniae and employed in blocking the bacterial colonization and infections. In the future, the MrkA fimbriae may also be developed into a potent tool for the presentation of foreign antigenic determinants in E. coli.

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