BORDETELLA CYCLASE: STRUCTURE AND BIOLOGICAL ACTIVITY

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Pertussis (whooping cough) is a major childhood illnesses in the developing world and, as a result of concerns about pertussis vaccine reactogenicity, has increased in incidence in many areas of the developed world. In the development of a non-whole cell pertussis vaccine, several important toxins and virulence factors are being considered as potential vaccine antigens. A major candidate among these is the adenylate cyclase (AC) toxin, which is the focus of this research project. Bordetella pertussis AC toxin is a calmodulin-activated enzyme which is novel in its location external to the cytoplasmic membrane of the bacterium and its ability to bind to and enter target eukaryotic cells where it catalyzes the production of cAMP from endogenous ATP. In this laboratory the enzyme/toxin has been partially purified to extremely high enzymatic specific activity while retaining its toxin activity, and monoclonal antibodies have been prepared. In the work proposed herein, the monoclonal antibodies will be characterized further and used to immunopurify the AC toxin. The toxin subunit structure will be evaluated with the use of the monoclonal antibodies and other techniques. The AC toxin gene will be identified and sequenced and the isolated gene employed to obtain in vitro expression of the protein. A major focus of the work will be investigation of the delivery, binding and cell entry mechanisms for the toxin. Immuno-electron microscopy will enable study of these processes and determination of the intracellular fate of the toxin. In line with the development of a new pertussis vaccine, the purified AC toxin (or toxoid thereof) and antibodies directed against the toxin will be tested for their ability to protect against infection with B. pertussis in the mouse intracerebral challenge and the mouse aerosol infection model. Sera from patients convalescent after pertussis will be examined for antitoxin activity. Finally, B. pertussis AC toxin will be compared and contrasted with the only other known AC toxin, from Bacillus anthracis. The homology of DNA sequence, immunologic cross reactivity, relationships between receptors and entry mechanisms will be examined, enabling consideration of the evolutionary development and relatedness of the two. The work proposed in this application and ongoing at present will provide information of use in vaccine development and of importance to the understanding of bacterial toxins and their roles in pathogenesis of bacterial disease.