Pseudomonas exotoxin A (ETA), one of several potential virulence products produced by Pseudomonas aeruginosa is a three domain bacterial toxin that kills mammalian cells by gaining entry to the cytosol and inhibiting protein synthesis. The toxin binds to a surface receptor on susceptible cells, is internalized via coated pits and endosomes, and enters the cytosol. During this journey, the toxin is cleaved to generate an enzymatically active fragment which ADP-ribosylates elongation factor 2. The pathway ETA takes to reach its target is only partially elucidated. Recently the ETA receptor has been isolated and shown to be similar to the alpha2macroglobulin receptor/LDL related protein (alpha2MR/LRP) The long-term goals of this research are to fully understand the cellular action of ETA, including how it is processed, and to determine its interaction with its receptor on toxin-sensitive cells.
The specific aims of this proposal are: 10 Confirm the relationship between the ETA receptor and the alpha2MR/LRP. The investigator will identify sequences in human alpha2MR/LRP. The investigator will identify sequences in human alpha2MR/LRP. The investigator will identify sequences in human alpha2MR/LRP receptor that correspond to ETA binding sites; 2) Determine the basis for cellular susceptibility to toxins. This includes a) determining ETA receptor level in toxin sensitive (LM and Chang) and resistant cells (OVCAR and Hela); b) establishing the level of alpha2MR/LRP receptor associated protein (RAP) in various mammalian cells; c) identifying and localizing the compartments where ETA is proteolytically processed, is activated, and enters the cytosol. Where feasible, these studies will include diphtheria toxin (DT), to determine if a second bacterial toxin is processed in a manner similar to ETA. 3) Define the intracellular pathway taken by the ETA receptor in sensitive cells. This work is significant because it will increase understanding of the pathophysiology of pseudomonas infections, help in the construction of efficient chimeric toxins, and help to answer the question of how toxins have adapted to take advantage of well conserved cellular components such as the alpha2MR/LRP.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI017529-13
Application #
2060528
Study Section
Bacteriology and Mycology Subcommittee 2 (BM)
Project Start
1980-12-01
Project End
1996-03-31
Budget Start
1995-04-01
Budget End
1996-03-31
Support Year
13
Fiscal Year
1995
Total Cost
Indirect Cost
Name
University of Cincinnati
Department
Genetics
Type
Schools of Medicine
DUNS #
City
Cincinnati
State
OH
Country
United States
Zip Code
45221
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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