The proposed research will study structure function relationships in the Dr family of adhesins. These adhesins are associated with diarrheal disease and urinary tract infections caused by E. coli. The adhesins are exceptional among E. coli adhesins in that they recognize protein receptors, rather than glycoconjugates. We will focus on one member of this family, the Dr hemagglutinin, and its adhesive subunit DraE, as a model. The receptors for this adhesin are the complement regulatory protein decay accelerating factor (DAF) and type [V collagen. We propose to characterize mutants we have isolated with altered DAF binding phenotypes for the ability to bind to the two known cell-associated form of DAF. We will carry out refined structural predictions which will form the basis of new mutagenesis experiments and deletion analysis of the adhesive subunit. We will study type IV collagen binding by the DraE adhesin, and test the hypothesis that the collagen binding domain is a conformational epitope comprised of amino acids from two neighboring subunits in the polymer. Common neutralizing epitopes of DraE which induce antibodies that inhibit the binding of other Dr adhesins will be sought. We will test the hypothesis that binding by the Dr hemagglutinin is enhanced by shear forces. The receptor domains of DAF and type IV collagen will be investigated by analysis of the ability of synthetic peptides to inhibit binding, and by crosslinking of the adhesin-receptor complex, followed by proteolysis and identification of crosslinked peptides by mass spectrometry. Strains will be constructed for the expression of chaperone-adhesive subunit complexes for crystallization studies. The evolution of divergence in the Dr family, a family characterized by exceptional variability considering the conservation of binding specificity, will be investigated to determine functional pathoadaptation. These studies will contribute to the understanding of diarrheal disease and urinary tract infections caused by E. coli, and will provide knowledge applicable to the development of new strategies for therapy and prevention.
