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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI030162-06
Application #
2003625
Study Section
Bacteriology and Mycology Subcommittee 2 (BM)
Project Start
1990-07-01
Project End
1999-11-30
Budget Start
1996-12-01
Budget End
1997-11-30
Support Year
6
Fiscal Year
1997
Total Cost
Indirect Cost
Name
Medical College of Wisconsin
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
073134603
City
Milwaukee
State
WI
Country
United States
Zip Code
53226
Zuverink, Madison; Barbieri, Joseph T (2018) Protein Toxins That Utilize Gangliosides as Host Receptors. Prog Mol Biol Transl Sci 156:325-354
Zuverink, Madison; Barbieri, Joseph T (2017) Protein Structure Facilitates High-Resolution Immunological Mapping. Clin Vaccine Immunol 24:
Kroken, Abby R; Blum, Faith C; Zuverink, Madison et al. (2017) Entry of Botulinum Neurotoxin Subtypes A1 and A2 into Neurons. Infect Immun 85:
Chen, Chen; Barbieri, Joseph T (2017) When Escherichia coli doesn't fit the mold: A pertussis-like toxin with altered specificity. J Biol Chem 292:15159-15160
Chen, Sheng; Barbieri, Joseph T (2016) Solubility of the catalytic domains of Botulinum neurotoxin serotype E subtypes. Protein Expr Purif 118:18-24
Chen, Chen; Przedpelski, Amanda; Tepp, William H et al. (2015) Heat-Labile Enterotoxin IIa, a Platform To Deliver Heterologous Proteins into Neurons. MBio 6:e00734
Zuverink, Madison; Chen, Chen; Przedpelski, Amanda et al. (2015) A Heterologous Reporter Defines the Role of the Tetanus Toxin Interchain Disulfide in Light-Chain Translocation. Infect Immun 83:2714-24
Zuverink, Madison; Barbieri, Joseph T (2015) From GFP to ?-lactamase: advancing intact cell imaging for toxins and effectors. Pathog Dis 73:ftv097
Simon, Nathan C; Aktories, Klaus; Barbieri, Joseph T (2014) Novel bacterial ADP-ribosylating toxins: structure and function. Nat Rev Microbiol 12:599-611
Simon, Nathan C; Barbieri, Joseph T (2014) Exoenzyme S ADP-ribosylates Rab5 effector sites to uncouple intracellular trafficking. Infect Immun 82:21-8

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