B. pertussis secrete a calmodulin (CaM) sensitive adenylyl cyclase (AC) toxin that elevates cAMP in eukaryotic cells. The structural gene (cyaA) for the AC toxin encodes a 200 kDa bifunctional protein containing both AC and hemolysin (HLY) enzymatic domains. Although the AC toxin is crucial for pathogenesis, the mechanism by which the toxin enters animal cells is not understood. B. pertussis cultured in media with high levels of BSA secretes a cell invasive 200 kDa cyaA toxin that has both AC and HLY functions. In the absence of BSA, the AC activity of the secreted toxin is associated with a 45 kDa catalytic subunit (C) that corresponds to the N- terminus of cyaA which does not enter cells without the addition of other secreted bacterial polypeptides (IF) that originate from the HLY domain of the toxin. A common cell entry mechanism may exist for these two forms of the toxin. We hypothesize that this mechanism involves a Ca2+-promoted interaction between the AC catalytic domain of the toxin and IF. In addition, we propose that the energy for membrane translocation may be derived from high affinity binding to intracellular CaM and/or the interaction of ATP with a secondary ATP binding site.
The specific aims of this proposal are to define the role of the IF domain for cell entry, determine if there is an IF interaction site within the a AC catalytic domain that is required for toxin entry, examine the relationship between Ca2+-dependent conformational changes of the cyaA toxin and its cell entry properties, examine the role of high-affinity CaM binding for membrane translocation of cyaA and virulence against newborn mice, determine if a second putative ATP-binding site in cyaA is required for cell entry, and attempt to crystallize the catalytic subunit of AC for X-ray crystallography.
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