Diphtheria toxin (DT) and pseudomonas exotoxin A (PE) are potent cytotoxic proteins produced by Corynebacterium diphtheriae and Pseudomonas aeruginosa. Both toxins are virulence factors for the organisms secreting them, as well as serving as useful probes in Cell Biology. Both proteins exert their toxic effect by stopping protein synthesis in mammalian cells; both are enzymes and act by ADP ribosylation of cytoplasmic elongation factor 2. The structures of these two enzymatically identical proteins however are different. In order to exert a biological effect on sensitive cells, both toxins bind to specific cell surface receptors, are internalized via coated areas into endosomes, delivered to the Golgi region and then to lysosomes. During this intracellular trafficking the toxins are converted to an enzyme active form, presumably in endosomes or Golgi associated vesicles. The site of escape into the cytoplasm is unknown. In the next grant period, we propose to: (1) Definitively define the sites and mechanisms for processing both PE and DT in sensitive cells, and determine if they are similar or different in resistant cells. This understanding will allow us to determine if the manner in which a mammalian cell handles a toxin dictates the cell's susceptibility to that toxin. (2) Isolate and characterize the receptor for PE on mouse LM fibroblasts. Once the receptor has been purified, partially sequenced, and antibodies made to it, the cDNA for the receptor can be cloned. (3) Define the intracellular pathway for the PE receptor in LM cells. We will determine if receptor follows the same pathway in the presence and absence of PE, and the site of toxin-receptor dissociation. Experiments on toxin trafficking and processing involve subcellular fractionation and identification of toxin by ELISA, or Induction of ADP ribosyl-transferase activity with gradient fractions. Toxin movement on the ultrastructural level will be followed using biotinyl-toxin-streptavidin-gold, or by pre-embedding immunochemical techniques. Receptor movement will be characterized both with electron microscopy and with biochemical probes.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI017529-09
Application #
3127265
Study Section
Bacteriology and Mycology Subcommittee 1 (BM)
Project Start
1980-12-01
Project End
1991-06-30
Budget Start
1989-07-01
Budget End
1990-06-30
Support Year
9
Fiscal Year
1989
Total Cost
Indirect Cost
Name
University of Cincinnati
Department
Type
Schools of Medicine
DUNS #
City
Cincinnati
State
OH
Country
United States
Zip Code
45221
Mucci, D; Forristal, J; Strickland, D et al. (1995) Level of receptor-associated protein moderates cellular susceptibility to pseudomonas exotoxin A. Infect Immun 63:2912-8
Kounnas, M Z; Morris, R E; Thompson, M R et al. (1992) The alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein binds and internalizes Pseudomonas exotoxin A. J Biol Chem 267:12420-3
Morris, R E; Ciraolo, G M; Saelinger, C B (1992) Validation of the biotinyl ligand-avidin-gold technique. J Histochem Cytochem 40:711-21
Thompson, M R; Forristal, J; Kauffmann, P et al. (1991) Isolation and characterization of Pseudomonas aeruginosa exotoxin A binding glycoprotein from mouse LM cells. J Biol Chem 266:2390-6
Morris, R E; Ciraolo, G M; Saelinger, C B (1991) Gold enhancement of gold-labeled probes: gold-intensified staining technique (GIST). J Histochem Cytochem 39:1585-91
Forristal, J J; Thompson, M R; Morris, R E et al. (1991) Mouse liver contains a Pseudomonas aeruginosa exotoxin A-binding protein. Infect Immun 59:2880-4
Saelinger, C B; Morris, R E (1987) Intracellular trafficking of Pseudomonas exotoxin A. Antibiot Chemother 39:149-59
Morris, R E; Saelinger, C B (1986) Reduced temperature alters Pseudomonas exotoxin A entry into the mouse LM cell. Infect Immun 52:445-53
Saelinger, C B; Morris, R E; Foertsch, G (1985) Trafficking of Pseudomonas exotoxin A in mammalian cells. Eur J Clin Microbiol 4:170-4
Morris, R E; Gerstein, A S; Bonventre, P F et al. (1985) Receptor-mediated entry of diphtheria toxin into monkey kidney (Vero) cells: electron microscopic evaluation. Infect Immun 50:721-7