Pseudomonas and diphtheria toxins (PE and DT) are virulence factors produced by Pseudomonas aeruginosa and Corynebacterium diphtheriae respectively. These distinct protein toxins both inhibit mammalian cell protein synthesis by inactivating cytoplasmic elongation factor 2. Evidence from our laboratory shows that PE and DT enter sensitive cells by receptor mediated endocytosis, following clustering over specialized clathri-coated areas on the cell surface, move in membrane bound vesicles to the Golgi region and then to the lysosomes. This pathway is essential for expression of toxicity.
The specific aims of the research are: l. To definitively characterize organelles involved in intracellular processing of DT and PE. 2. To determine the site of conversion from proenzyme to enzyme active form. 3. To initiate purification and characterization of DT and PE binding proteins. 4. To visualize toxin-receptor uncoupling, and entry of toxin into cytoplasm. Biochemical and electron microscopic studies will be carried out in parallel to compare the movement of PE and DT in both sensitive and resistant cells. Subcellular fractionation studies also will be used to localize iodinated toxins in cells. Agents which stop the intoxication process at different steps will be used to determine where the toxins are activated and where they, or an enzyme active fragment, enter the cell cytoplasm. Immunocytochemistry will be used to determine where toxin and ligand dissociate, and toxin enters the cytoplasm.
