Infection with Bordetella pertussis remains a cause of pediatric morbidity and mortality and adult morbidity worldwide. Adenylate cyclase (AC) toxin from B. pertussis is a major virulence factor hypothesized to be involved in evasion of host defenses and development of local pathology. In this project, AC toxin structure and function will be studied for three purposes: 1) understanding its mechanism of action; 2) considering it as a vaccine antigen in acellular pertussis vaccine; and 3) contributing to the overall knowledge of bacterial toxins which enter target cells (traverse intact cell membranes). AC toxin is a calmodulin-regulate protein which catalyzes the production of cyclic AMP. Its novel features include its ability to enter intact eukaryotic cells to produce supraphysiologic levels of cAMP and its ability to hemolyze erythrocytes. AC toxin is synthesized in an inactive form which requires modification by a separate protein (product of the cyaC gene) for expression of full toxin and hemolytic activities. The c-gene product (CyaC) will be expressed, isolated and characterized. Monoclonal antibodies will be prepared against CyaC to facilitate study of its site and mechanism of action. The activation of AC toxin by the product of CyaC will be studied in vitro, in order to determine site of protein modification and structural consequences. Activated AC toxin binds calcium and undergoes a conformational change which allows it to enter eukaryotic target cells. This conformational changes does not occur in toxin from mutants missing CyaC, despite the fact that the toxin from such strains binds calcium. The role of CyaC-dependent activation in the structural response to calcium will be explored. Using purified, activated AC toxin the binding and entry processes will be dissected using functional techniques as well as electron microscopy. The study of toxin structure and translocation across target cell membranes will be used to formulate a model of toxin entry which will be specific to AC toxin as well as generally applicable to toxins which cross host-cell membranes. The apparent production of a transmembrane pore by the toxin molecule will be investigated by electro-physiologic methods in intact cells and artificial membranes. Serum from children immunized with whole cell pertussis vaccines or convalescent from pertussis will be evaluated for antibody response to AC toxin. In addition, purified AC toxin will be tested for its capacity to protect mice from B. pertussis infection and death in the intracerebral and aerosol challenge models. Inactive mutants of AC toxin will be evaluated as genetically toxoided antigens if AC toxin protective activity is demonstrated. In summary, these structure/function studies will facilitate better understanding of the mechanism of AC toxin as well as bacterial toxin action in general and will provide an antigen potentially useful in the prevention of clinical pertussis.
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