Abstract
Type 3 fimbriae produced by Klebsiella pneumoniae are required for initiating infection by attaching to host tissues and responsible to the strong immunogenicity of bacteria. This macromolecular organelle is assembled via the chaperone-usher pathway. The first aim of this study is to identify residues in the major pilin MrkA important for pilus assembly. Site-directed mutagenesis was performed on MrkA and whether the MrkA of these mutants can be assembled into fimbriae were evaluated. The data showed that two amino acids at the middle region of MrkA, Ile-82 and Cys-87, play critical roles in pilus assembly. Six amino acids in the N- and C- terminal regions of MrkA, including Val-32, Phe-34, Asp-40, Val-45, Gly-189 and Tyr-201, disrupt normal pilus functions and may relate to maintaining stability of pilus structures. The second aim of this study is to construct a vaccine displaying fimbriae. Previously, our lab had created 4 insertion mutants containing 18-amino-acid porcine endogenous retrovirus (PERV) envelop epitopes inserted in 4 different positions of MrkA respectively. After confirming the fimbriation and epitope display efficiencies of these mutants, the fimbriae of one mutant strain, D27-E, was purified. Immunization of mice with the purified recombinant fimbriae successfully elicited antibodies that specifically recognized PERV envelope protein, revealing the potential of type 3 fimbriae as vaccine display systems.