Bordetella pertussis, the causative agent of whooping cough, producess several biologically active products whose mechanisms of action and roles in bacterial virulence and pathogenicity remain unclear. One of these, Bordetella extractyoplasmic adenylate cyclase (ECAC), is receiving increasing attention as a novel bacterial product which is able to modify the function of selected eukaryotic cells. This bacterial enzyme is predominantly extracytoplasmic in location and is activated by the calcium-dependent regulatory protein, calmodulin, which is produced only by eukaryotic cells. In recent studies from this laboratory and others, it has been shown that extracts of B. pertussis and B. parapertussis are able to increase cAMP levels in human lymphocytes, monocytes and macrophages and inhibit several immunologic functions. Because of the crude preparations involved, however, it has not been possible to determine specifically if the ECAC is solely responsible for these effects and what the mechanisms are. Therefore, the goals of this renewal project are to purify the ECAC from B. pertussis and B. parapertussis, characterize its structure, and investigate its mechanism of action. Because previous purification of ECAC yielded a product with enzymatic, but no biological activity, it is essential that both paramenters be followed concurrently throughout purification. Biological activity will be monitored by ability to increase cAMP levels in target cells and to inhibit monocyte oxidative responses. ECAC binding and entry into susceptible target cells will be investigated. A novel approach to study ECAC is being provided by the mouse lymphoma cell line S49, which is killed by increased cAMP levels, ECAC receptor (binding) and entry mutants are being selected by exposure to the enzyme. These studies proposed herein are important to bacterial toxinology in that a similar ECAC has recently been identified from B. pertussis. Furthermore, the possibility that immunologic defects induced by Bordetella ECAC may contribute to the morbidity and mortality from whooping cough make the study of this mechanism and design for specific pharmacologic intervention essential. Finally, better understanding of the tissue specificity and mechanism of action may enable this novel bacterial product to be used as a probe in other areas of biomedical research.
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