| Summary: | 碩士 === 國立清華大學 === 分子醫學研究所 === 95 === 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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