Microbiology

CRIS AD-421 Research Reports Submitted for 98-35204-6979 on 12/14/99

Investigators: Strom, M. S.

Termination Date: 11/30/00

Progress Report:
The goal of this research is to clone and characterize the genes that encode factors involved in expression of type IV pili and extracellular protein secretion from the pathogen Aeromonas salmonicida, which causes the disease furunculosis in salmonid fish. Pili are fibers that protrude from bacterial cells that are often involved in the initial attachment or colonization of pathogenic bacteria to host cells. These same bacteria are often capable of secreting a number of toxins and degradative enzymes involved in virulence through a related pathway. A demonstration that these factors are important for the pathogenesis of A. salmonicida could form the basis for an effective vaccine against the pathogen. Cloning and DNA sequencing of the tapABCD gene cluster have been completed. The DNA sequences and encoded protein translations have been deposited in the Genbank Sequence Database (accession numbers AF059248 and AF059249). The genes and their encoded proteins are highly homologous to previously characterized type IV pilins and associated proteins involved in pilus biogenesis from A. hydrophila, Vibrio vulnificus, and Pseudomonas aeruginosa. However, even though amino acid sequences of TapA from A. hydrophila Ah65 and A. salmonicida A450 share 64% identity, antisera specific for the A. hydrophila TapA does not recognize TapA from A. salmonicida. Plasmid constructs carrying copies of insertionally inactivated tapA and tapD genes in a suicide vector have been completed. Specific tapA and tapD mutants of A. salmonicida have been constructed by allelic exchange using these clones. A tapA:FLAG fusion construct was made in order to purify recombinant TapA protein for antisera preparation. An in vitro bacterial adherence assay was adapted for A. salmonicida and the salmonid cell line Chinook Salmon Embryo-214 (CHSE-214). This will be used to measure the role of TapA in colonization of tissue cells.

Impact:
A demonstration that type IV pili are involved in the intial interaction or colonization of the salmonid fish host in infections caused by Aeromonas salmonicida could potentially lead to the development of a safe, efficacious, and easy to administer vaccine against furunculosis.

Publications:
(No publications reported.)