The bacterium Bordetella pertussis causes """"""""whooping- cough"""""""", a disease which killed thousands of children in the U.S. prior to a widespread vaccination program. The bacterium elaborates several toxins, including """"""""pertussis toxin"""""""", which are involved in the pathogenesis of the disease. A """"""""whole-cell"""""""" vaccine in current use in the U.S. causes severe, potentially permanent, side-reactions. Thus, efforts are underway to develop safer vaccines relying on specific components, such as pertussis toxin. Once developed, a new pertussis vaccine will be given to millions of babies each year. Given the risk of severe side-effects, it behooves us to carefully define this vaccine. Thus, acquiring knowledge concerning pertussis toxin is important to provide a rational basis for its use in the vaccine.
The Specific Aims of this application are designed to yield information helpful for this purpose. In addition, pertussis toxin is a valuable tool is basic research. Thus, the Aims are also designed to improve our understanding of the toxin in basic terms as a scientific tool.
The Aims stem from the recent discovery that adenine nucleotides, detergents, and dithiothreitol directly stimulate the NADase and ADP-ribosyltransferase activities of pertussis toxin. These activities are the basis of its mechanism of action. The application proposes to: 1. Develop an assay of the ADP-ribosyltransferase activity of the toxin using peptide analogs of known pertussis toxin ADP-ribosylation sites. 2. Determine if activating substances separate the subunits of the toxin. If so, then develop new methods for purification of the subunits. 3. Determine if activating substances allow mild conditions to render the toxin incapable of intoxicating cells. Such a process may produce a stable detoxified toxin that is a better immunogen than those produced by other, harsher, procedures. 4. Describe molecular mechanisms of the activation process both in vitro and in intact cells. The approach includes electrophoresis and immunoblotting. 5. Examine other toxins to help establish which activating mechanisms are unique to pertussis, and which are shared by other toxins.
