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Mouse liver contains a Pseudomonas aeruginosa exotoxin A-binding protein
J J Forristal1, M R Thompson, R E Morris
1Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, Ohio 45267.
Abstract:
The opportunistic pathogen Pseudomonas aeruginosa produces several potential virulence factors, including the ADP-ribosylating toxin, exotoxin A (PE). Studies using a burned mouse model have shown that PE consistently inhibits protein synthesis and depletes elongation factor 2 in mouse liver and variably in other organs. One reason for toxin sensitivity could be the presence of a PE receptor on the surface of cells. Therefore we examined detergent extracts of mouse tissues for the presence of toxin-binding proteins. Proteins which specifically bind PE were present in extracts from liver, kidney, lung, spleen, and heart. Because liver appears to be a prominent target for the toxin in a burned animal, we choose to isolate the PE-binding protein from mouse liver and compare this protein to the recently characterized toxin-binding protein from toxin-sensitive mouse LM fibroblasts. The toxin-binding proteins from both sources have a molecular mass of approximately 350 kDa, share similar protease digestion profiles, and are glycosylated. However the glycosylation patterns for the two species are quite different. Both glycoproteins bind toxin with high avidity. The toxin-binding moiety is located, at least in part, on the plasma membrane and thus could represent the receptor involved in internalization of toxin molecules responsible for cell death.
Insights
Pseudomonas aeruginosa exotoxin A (PE) binds to a 350 kDa glycoprotein found on mouse cell membranes. This protein may act as a receptor, facilitating toxin entry and causing cell death.
Area of Science:
- Microbiology
- Toxicology
- Cell Biology
Background:
- Pseudomonas aeruginosa is an opportunistic pathogen that produces exotoxin A (PE), a key virulence factor.
- PE inhibits protein synthesis by depleting elongation factor 2, primarily in the liver of burned mice.
- Cellular sensitivity to PE may be mediated by specific cell surface receptors.
Purpose of the Study:
- To investigate the presence and characteristics of PE-binding proteins in mouse tissues.
- To isolate and compare the PE-binding protein from mouse liver with that from mouse fibroblasts.
- To determine if these binding proteins function as receptors for PE.
Main Methods:
- Extraction of detergent-soluble proteins from various mouse tissues.
- Affinity purification of PE-binding proteins.
- Molecular characterization including molecular mass determination and protease digestion profiling.
- Comparison of binding proteins from liver and LM fibroblasts.
Main Results:
- PE-binding proteins were identified in liver, kidney, lung, spleen, and heart extracts.
- A ~350 kDa glycoprotein that binds PE with high avidity was isolated from mouse liver.
- This protein shares similarities with, but has distinct glycosylation patterns from, the PE-binding protein in LM fibroblasts.
- The binding moiety is associated with the plasma membrane.
Conclusions:
- A ~350 kDa glycoprotein in mouse liver specifically binds exotoxin A.
- This glycoprotein is glycosylated and located on the plasma membrane, suggesting it functions as a PE receptor.
- Differences in glycosylation between tissue sources indicate potential variations in receptor structure or processing.
- The identified protein is a strong candidate for mediating PE internalization and subsequent cell death.