Gene Cloning and Protein Characterization of Fucosyltransferase in Klebsiella pneumoniae (II)

Previously, our study discovered that the capsule of Klebsiella pneumoniae (KP) that caused liver abscess (LA) contained high amount of fucose. This bacterium caused high fatality rate in infected mice. In contrary, the KP that caused urinary tract infection (UTI) did not contain fucose and caused no fatality in mice. Both strains caused bacteremia. This led us to suspect that fucose (also a component in human tissue) enabled the LA-KP to evade host immune reaction. Our experiment of bacteria-phagocytes interaction in mice peritoneum revealed that the LA-KP rarely interacted with macrophages while the UTI-KP readily attached to macrophage surface indicating the LA-KP could survive and multiply to high concentration in blood without being cleared by phagocytosis to invade various organs. In human colon cancer, studies revealed that cancer cells expressed Lewis antigens are easily metastasize to liver. Lewis antigens contain fucose on their polysaccharide surface. We therefore hypothesize that LA-KPs may contain Lewis antigen for them to migrate to liver and cause liver abscess. Fucosyltransferase (FucT) is needed to form Lewis antigens. Our preliminary results discovered the LA-KP contained Lewis antigen. However, this type of FucT has not been reported in KPs. Our goal in this proposal is to design primers of FucT using human or Helicobacter pylori FucT gene sequences or from FucT protein conserved region, to amplify from KP genome by polymerase chain reaction (PCR), to clone the amplified fragment into yT&A vector for DNA sequencings, to compare the sequence with bacterial or plasmid gene sequences on gene bank, to design new primers, to amplify the complete coding region by PCR, to determine the cloned DNA sequence again, eventually to clone the coding region into expression vector and purify the protein. The protein will be screened for enzyme activity, chemical properties and crystallized for structure analysis (This part will collaborated with Dr. Lin CH in Academia Sinica). The FucT sequence will be compared with FucT of other source to investigate the origin of the gene. Creation of knock out bacterial mutant strain and examination of mice survival rate with the mutant strain will also be tested. This project may enable us to understand the virulence factor in KP and may serve as a therapeutic target or vaccine bacterial strain development.

Project ID:PC9907-2508
External Project ID:NSC99-2320-B182-003