Diphtheria toxin(DT) is the major virulence factor of the disease diphtheria. The recent reappearance of this disease in a number of countries of Europe, Asia, Africa, and South America, underscores it's continuing significance as a worldwide health problem. The long-term goals of this proposal are to characterize fully the cell surface receptor which DT utilizes to gain illicit entry into toxin-sensitive host cells and to understand completely the interaction between the toxin and its receptor. The investigators earlier discovered that DT binds to the transmembrane/precursor form of heparin-binding EGF-like growth factor (HB-EGF). They have recently identified the EGF-like domain of the cell surface of HB-EGF precursor as the region where the toxin binds and have shown that the soluble/mature form of HB-EGF inhibits binding of the toxin to the native toxin receptor on cells. The first specific aim is to determine the role of the cytoplasmic domain of the toxin receptor. The second specific aim is to characterize the functional interactions of the extracellular domain of the receptor with the toxin and with other receptor-associated proteins. The third specific aim is to characterize the toxin-binding site of the receptor employing a combination of site-directed mutagenesis and X-ray crystallography. The fourth specific aim is to produce transgenic mice bearing the HB-EGF precursor in order to determine whether this precursor can act an a DT receptor in vivo and to test the ability of recombinant mature HB-EGF to ameliorate intoxication by DT in this novel DT-sensitive experimental animal model. The results of this proposed project, in addition to extending our knowledge on toxin:receptor interactions, may provide a new kind of therapy for clinical diphtheria in which mature HB-EGF would replace equine horse anti-DT serum as the antidote of choice.
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