Infectious bovine keratoconjunctivitis (IBK), caused by the bacteria Moraxella bovis, will be used as a model system for human bacterial eye disease. IBK is a widespread, highly contagious ocular disease of cattle causing temporary and, occasionally, permanent blindness. IBK is an especially model system because; 1) its pili are closely related to the pili of human ophthalmic disease bacteria, and 2) well-defined animal model systems exists for testing the pathogenesis of altered bacteria in vivos as well as in vitro. The focus of these studies will be the role of the two different types of pili found within a given strain of M. bovis in the disease process, and particularly the function of the phase variation which occurs between the two pili types alpha, and beta in strain EPP63. The near term goals of these studies will be: 1) to determine the exact position of the inversion-recombination sites by sequence analysis of both alpha and beta expressing DNA's, 2) to produce phase-locked mutants of M. bovis strain EPP63 capable of stably expressing either only alpha or only beta-pili, 3) to test the M. bovis phase-locked mutants in vitro, in mice and in calves, and 4) to investigate the other genes involved in the pilin gene inversion event and potentially the other genes involved in pili assembly and expression. The sequence analysis will be done from lambda gt11 clones of beta-pilin producing DNA and either Charon 4A or lambda gt11 clones of alpha-pilin producing DNA. Phase-locked mutants will be produced by in vitro replacement of the inversion-recombination site distal to the expression site locus by a drug resistance gene marker. DNA from the drug resistant mutants will be used to transform alpha-piliated and beta-piliated M. bovis strains. Transformants unable to undergo phase variation, and containing either only alpha-pili or only beta-pili will be tested for their relative adherence ability in vitro using a corneal tissue assay. The phase-locked M. bovis strains will then be tested in eye inoculation studies in mice and calves. Beta- pilin-lacZ fusions will be constructed to study the control of the inversion event in either E. coli or M. bovis. Deletion, transposition and in vitro mutagenesis will be used to localize the positions of the gene(s) responsible for the inversion event, as well as potential genes involved in pili assembly and expression. The long range goal of this research is to obtain a better understanding of the role played by pili phase variation in ophthalmic bacterial diseases.
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