The preliminary research objective of this laboratory is the determination of the immunogenic and functional domains of the exotoxin A molecule from Pseudomonas aeruginosa. To accomplish this, we have taken a combined immunochemical, biochemical and genetic approach. Specifically, we have produced a library of toxin-specific monoclonal antibodies which are being used as site-specific probes for the detailed analysis of this protein. Several of these antibodies inhibit specific functions of the toxin molecule. Using specific monoclonal reagents in conjunction with genetically altered toxin molecules we are conducting a comparative analysis of toxin structure and function. We hope to determine: (1) the location of the immunogenic epitopes required to develop host immunity to toxin; (2) the location of the receptor-specific epitope(s) of toxin; (3) the NAD- dependent ADP-ribosyltransferase active site of toxin; (4) location of the membrane translocation domain of toxin. The project outlined above has very specific objectives, but the potential applications of the expected results are numerous and significant. First, a detailed knowledge of functional regions of this protein will allow an in-depth analysis of the correlation between structure and function for microbial toxins, since the complete amino acid sequence of this protein is known. Knowledge of the specific immunogenic epitopes of the toxin will provide the basis for the development of synthetic peptides corresponding to regions known to elicit the formation of anti- toxin in vivo. As such, the system described (a lethal toxin well- characterized with respect to susceptible cell lines and animal model systems) will provide the means to effectively test the plausibility of the use of synthetic peptides in vaccine development.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Modified Research Career Development Award (K04)
Project #
5K04AI000876-03
Application #
3070914
Study Section
Bacteriology and Mycology Subcommittee 2 (BM)
Project Start
1988-06-01
Project End
1993-05-31
Budget Start
1990-06-01
Budget End
1991-05-31
Support Year
3
Fiscal Year
1990
Total Cost
Indirect Cost
Name
Ohio State University
Department
Type
Schools of Arts and Sciences
DUNS #
098987217
City
Columbus
State
OH
Country
United States
Zip Code
43210
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Kessler, S P; Galloway, D R (1992) Pseudomonas aeruginosa exotoxin A interaction with eucaryotic elongation factor 2. Role of the His426 residue. J Biol Chem 267:19107-11
Peters, J E; Park, S J; Darzins, A et al. (1992) Further studies on Pseudomonas aeruginosa LasA: analysis of specificity. Mol Microbiol 6:1155-62
Galloway, D R (1991) Pseudomonas aeruginosa elastase and elastolysis revisited: recent developments. Mol Microbiol 5:2315-21
McGowan, J L; Kessler, S P; Anderson, D C et al. (1991) Immunochemical analysis of Pseudomonas aeruginosa exotoxin A. Analysis of the His426 determinant. J Biol Chem 266:4911-6
Seo, Y; Galloway, D R (1990) Purification of the pyocin S2 complex from Pseudomonas aeruginosa PAO1: analysis of DNase activity. Biochem Biophys Res Commun 172:455-61
Peters, J E; Galloway, D R (1990) Purification and characterization of an active fragment of the LasA protein from Pseudomonas aeruginosa: enhancement of elastase activity. J Bacteriol 172:2236-40
Galloway, D R; Hedstrom, R C; McGowan, J L et al. (1989) Biochemical analysis of CRM 66. A nonfunctional Pseudomonas aeruginosa exotoxin A. J Biol Chem 264:14869-73
Wozniak, D J; Hsu, L Y; Galloway, D R (1988) His-426 of the Pseudomonas aeruginosa exotoxin A is required for ADP-ribosylation of elongation factor II. Proc Natl Acad Sci U S A 85:8880-4