A family of exotoxins (RTX) produced by a variety of important human and animal pathogens, such as Escherichia coli, Bordetella pertussis, Actinobacillus actinomycetemcomitans and Pasteurella haemolytica has been recently recognized. The RTX toxins with the possible exception of the B. pertussis RTX toxin, form pores in target cell plasma membranes which lead to cell lysis. The RTX toxins can be distinguished from one another by differences in cell- and host-specificity. The main objective of the research is to understand the bases for RTX toxin pore formation and cell- and host-specificity. The most thoroughly studied member and prototype of the family is the E. coli hemolysin (HlyA). hlyA mutations and reciprocal hybrids of different RTX structural genes will be isolated. The HlyA mutant and hybrid proteins will analyzed in a variety of functional tests that will examine their ability to form channels in cells and artificial lipid bilayers and determine their target-cell specificity characteristics. A particular class of HlyA mutants will be studied in detail. These mutants retain hemolytic activity but have lost the ability to lyse nucleated cells. The mutants will allow an assessment of the relative contribution of the hemolytic versus leukotoxic activities to virulence by testing them in a rat model of E. coli intra-abdominal sepsis and in vitro tests of HlyA activities (inhibition of macrophage antigen processing and presentation, channel formation, target-cell binding etc.). The topography of HlyA inserted into plasma membranes will be studied by protease- protection experiments and flow cytometry using a panel of anti-HlyA monoclonal antibodies with epitopes mapped to specific HlyA regions. HlyC is necessary for production of cytolytically active HlyA. To study this poorly understood event, a monoclonal antibody (D12) specific for """"""""active"""""""" HlyA will be used to screen HlyA mutants. A second long term objective of the work is the identification and characterization of additional RTX determinants among pathogenic bacteria. Exoproteins produced by Neisseria meningitidis and Hemophilus influenzae type b (Hib) have been identified which are antigenically related to RTX toxins. The N. meningitidis RTX- like protein will be characterized in a collaborator's laboratory. To isolate the RTX-like Hib genes, an existing recombinant library of Hib genomic DNA will be screened with anti-RTX antibodies and RTX gene probes. to assess the significance of the Hib RTX-like protein in Hib pathogenesis, isogenic Hib strains altered in their ability to produce the putative RTX protein will be constructed and tested in an infant rat intranasal- challenge model of sepsis and meningitis. The Hib RTX-like exoprotein will be also tested in in vitro assays of cytoxicity and pore formation. The structure and function of HlyA and its RTX relatives and the role of the RTX toxins in the pathogenesis of significant human and animal diseases are not understood. The information gathered from the research proposed in this application will be valuable in the development of selective strategies for the prevention and treatment of a large number of serious bacterial diseases.
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