The objective of this project is to define structural features of bacterial protein toxins and determine in detail how these proteins interact with the membranes and subcellular organelles of eukaryotic cells to achieve toxicity. Current work focuses on the three proteins that comprise anthrax toxin. Prior work showed that receptor-bound protective antigen (PA) is cleaved at amino acid 167 by a cell surface protease, and that this cleavage is required for the toxin to act. Site directed mutagenesis to alter the sequence at residues 164-167 has now proven that the cell protease recognizes the sequence Arg-X-X-Arg, where X is any amino acid. This finding strongly suggests that the protease is furin, a newly-recognized membrane protease that processes many hormone precursors by cleavage at paired basic amino acids. Other mutagenesis studies estab- lished that the extreme C-terminal residues of PA are part of the structure which recognizes the target cell receptor. Insight into how the toxin proteins escape from endosomes was gained by creating protein fusions of the lethal factor protein (LF) with different domains of Pseudomonas exotoxin A. As little as 50 amino acids near the N-terminus of LF seem to be sufficient to cause binding to PA and internalization of domain III of the exotoxin A. This strongly suggests that the translocation function resides in PA, and that this system may provide a non-specific delivery system by which a variety of polypeptides can be delivered to the cytosol of eukaryotic cells. Efforts were begun to clone the genes involved in control of toxin and capsule production by bicarbonate. A fusion of the PA promoter to the lacZ gene was made and this was shown to respond to the putative positive and negative regulatory genes on the plasmid pXO1. A compatible plasmid was identified that can be used to clone the regulatory genes. A collaboration was established to determine the 3-dimensional structure of the PA protein. Highly purified protein yielded excellent crystals and preliminary diffraction data were obtained.
