The goal of this proposal is to contribute to the generation of effective acellular vaccines to prevent pertussis and Pseudomonas aeruginosa infections. The focus of this research is to define functional residues and domains of pertussis toxin and Pseudomonas exoenzyme S which will lead to the generation of noncatalytic toxoids that can be tested as vaccine candidates. These studies will also determine the mechanism by which pertussis toxin intoxicates eukaryotic cells and presents strategies for engineering a trivalent a cellular vaccine to prevent diphtheria, pertussis and tetanus.
The specific aims of this proposal are to: (i) define the kinetic constants of mutants of the S1 subunit of PT that express reduced enzymatic activity. These studies will define residues that contribute to NAD binding, G protein binding, and catalysis; (ii) determine how pertussistoxin ADP-ribosylates both plasma membrane- and Golgi- associated G(i) proteins. Experiments will define the rate of ADP- ribosylation of plasma membrane- and Golgi-associated G(i) proteins and correlate this data with the physical localization of S1 during the entry of PT into sensitive cells. These studies will also determine the contribution of the ADP-ribosylation of plasma membrane- and Golgi-associated G proteins in the pathogenesis of pertussis toxin; (iii) identify the minimal amino acid sequence of S1 required for association with B oligomer; (iv) engineer a recombinant trivalent diphtheria-pertussis-tetanus vaccine composed of non-toxic deletion peptides of each respective toxin. These fusion proteins will be analyzed for the elicitation of a neutralizing immune response against their respective toxin; and (v) define the kinetic constants of recombinant Pseudomonas exoenzyme S, which ADP-ribosylates the Ras protooncogene product, and identify residues that contribute to the catalysis of exoenzyme S. Mapping functional residues and domains of pertussis toxin and exoenzyme S will define rational approaches for the production of acellular pertussis and Pseudomonas vaccines. Parallel studies on pertussis toxin and exoenzyme S will define relationships between these two members of the family of bacterial ADP-ribosylating exotoxins and may identify new strategies for engineering effective toxoids against other bacterial pathogens.
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