Diphtheria toxin (DT) and the family of heat-labile enterotoxins (LTs) are well studied virulence factors of Corynebacterium diphtheriae and Escherichia coli, respectively. Each toxin has a B domain that binds to plasma membrane receptors and an A domain that mediates toxicity. DT ADP-ribosylates elongation factor 2, thereby inhibiting protein synthesis and killing target cells. LTs ADP-ribosylate the regulatory protein G, and activate adenylate cyclase. The long term objectives are to understand the genetics, regulation, and mode of action of these toxins at the molecular level. Such knowledge is essential for understanding pathogenesis of diphtheria and enterotoxic E. coli diarrhea and for development of improved vaccines, immunotoxins, hybrid toxins, or related biologic products involving these toxins.
The specific aims of this proposal are to: 1) determine the mechanisms by which iron regulates production of DT and the activity of the iron uptake system in C. diphtheriae. The investigators will analyze the tox promoter sequence that interacts with the E. coli Fur protein, clone and characterize the tox repressor from C. diphtheria, isolate and characterize ferric uptake regulation (fur) mutants of C. diphtheriae, and determine whether tox repressor activity is mediated by the putative fur gene product in C. diphtheriae. 2) characterize the interactions of DT with its cellular receptor. Dr. Holmes and colleagues have developed monoclonal antibodies to membrane components of human cells that block intoxication by DT and they will characterize their mode of action. They will also purify DT receptor molecule(s), clone and characterize and corresponding cDNA(s), determine whether DT-resistant cells that lack functional receptors have genes or membrane components homologous with those for DT receptors, and characterize the binding domains of DT and its receptor. 3) analyze structure-function relationships of LTs. They will compare purified type I and type II LTs in intestinal assays in several animal species to determine whether their different receptor specificities correlate with differences in biologic activity. They will use genetic complementation with cloned A and B subunit genes, directed mutagenesis of cloned genes, chemical modification of purified toxins, and synthetic oligopeptides to analyze the molecular determinants of biologic activity of representative type I and type II LTs.
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