Exotoxin A of Pseudomonas aeruginosa, a protein of molecular weight 66,583, is an important virulence factor in Pseudomonas infections. The toxin is also a model system for preparation and characterization of hybrid toxins that may have clinical importance in cancer chemotherapy. There are four steps in the mechanism of toxin action. First, the toxin binds to cell surface receptors on mammalian cells. Second, the toxin is endocytosed and exposed to a low pH within the endocytic compartment. Third, a portion of the toxin that carries the catalytic center penetrates the membrane of a vesicle and enters the cytoplasm. Fourth, the toxin catalytically arrests protein synthesis. Three lines of research into the molecular and cell biology of exotoxin A are proposed here: 1. Investigation of exotoxin A gene expression. A binding site for a putative activator of toxin gene expression is present upstream of the structural gene. We propose to identify the activator and characterize its mechanism of action. 2.Preparation and characterization of structurally altered variants of exotoxin A. We are particularly interested in variants that fail to interact with receptors because they should be useful components of hybrid toxins. We also intend to search for variants that bind to receptors but are nevertheless not cytotoxic because they fail to penetrate a membrane vesicle after endocytosis. 3.Construction and characterization of hybrid toxins. Exotoxin A variants that fail to interact with receptors will be coupled to alternative receptor-binding ligands. The properties of the hybrid toxins will be studied and their potential to eliminate tumor cells will be assessed.
