The hypothesis proposed in grant R01 DK42029 stated that renal interstitial attachment of Dr adhesin bearing Escherichia coli creates structural basis for development of chronic interstitial nephritis. Exploration of consequences of Dr-adhesin Dr-tissue-ligand interaction resulted in the development of an experimental model of ascending chronic pyelonephritis (ChP) that fulfills histological requirements for human ChP. In this project, the investigators will attempt to understand the molecular mechanisms involved in and to prevent the development of ChP. They will attempt the following projects. Map receptor-binding epitopes on short consensus repeat-3 (SCR-3) domain of decay accelerating factor (DAF) for Dr fimbriae. (a) Map Dr binding epitopes on DAF-SCR-3 by synthetic peptides by competitive inhibition. (b) Map Dr binding epitopes on DAF-SCR-3 by antipeptide antibodies. (c) Replace single amino acids within the SCR-3 region using site-directed mutagenesis. (d) Test in vitro attachment of BN406 on Chinese hamster ovary (CHO) cells that express constructed DAF (SCR-3) mutants. (2) Map functional regions within the dra operon necessary for interstitial colonization and developing ChP on a mouse C3H/HeJ in vivo model. (a) Characterize the nucleotide sequence of the draA, draB. draC, draD, and draE mutants on a ChP model to dissect involvement of adhesive, and renal interstitial tropism properties in the development of ChP. (c) Examine virulence of draA substitution mutants defective in type-IV collagen binding on a ChP mouse model. (d) Study route and kinetics of dra mutants spread leading to interstitial colonization on a SCID mouse model. (3) Test the effect of vaccination with recombinant Dr adhesin on development of ChP in C3H/HeJ mice.
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