Escherichia coli expressing Dr adhesin cause pyelonephritis and related AFA-III cause cystitis. This study seeks to identify the receptor-binding site of DraE and provide insights into the mechanism of renal vs bladder tropism.
Aim 1. To identify and characterize domains of Dr fimbriae that interact with DAF. We propose that N-terminal surface exposed residues of DraE form an assembled epitope that bind DAF. The hydrophilic domain Val30 to Pro42 will be mutated by alanine-scanning mutagenesis and mutants analyzed for binding and invasion of DAF+ CHO cells.
Aim 2. To investigate the pathogenic mechanism that determines kidney vs bladder tropism of Dr+ E. coli. DraE and AWE differ in three amino acids (D54N, T90M, I113T). Dr+E. coli infect the kidneys while AFA-III+ E. coli infect the bladder. Amino acids of DraE will be replaced with those of Afa3E and renal tropism analysed using isogenic mutants of E. coli HI 11128 in mice.
Aim 3. To study attachment and cell invasion by DraE and Afa3E adhesins. DraE is more invasive than Afa3E. We will study invasion using polystyrene beads coated with purified DraE mutants, immunogold staining and electron microscopy. Resolving the binding domain will enable design of peptide inhibitors against the common receptor preventing bacterial dispersion and cell invasion.
