A major cause of morbidity in females is urinary tract infections (UTI) caused by Escherichia coli. UTIs are thought to be initiated by the binding of a bacterial adhesin (PapG) to the Gal-alpha (1-4)Gal receptor present in the host. PaG is presented at the distal ends of thin tip fibrillae which are joined to long rigid pilus rods on the bacterial cell surface. These composite adhesive fibers are called P pili. P pilus assembly requires the PapD chaperone, a member of a family of periplasmic chaperones required for the assembly of virulence-associated pili in diverse gram negative pathogens. PapD binds to six P pilus subunit types and escorts them from the cytoplasmic membrane to an outer membrane assembly site called an usher. In the first Aim,the molecular basis of the chaperone-subunit interactions will be investigated. The C-terminus of pilus subunits has been identified as part of a site recognized by papD and a peptide corresponding to the C-terminus of PapG was co-crystallized with PapD. The role of hydrophobic interactions in determining binding specificity will be investigated using an alanine scan and a deletion series of an optimal short peptide and testing the ability of the peptides to bind to PapD. other regions critical for chaperone recognition will be revealed by identifying regions of PapG that when fused to ?(BP result in PapD binding to the MBP-G fusion in vivo and in vitro. Relevant peptides will be co-crystallized with PapD to establish the structural basis of the interaction. In the second Aim, the molecular basis for PapD function will be examined by site directed mutagenesis of amino acids strictly conserved in all pilus chaperones. The mutant chaperones will be analyzed by in vivo and in vitro experiments using X ray crystallography, genetics, biochemistry, and synthetic chemistry to elucidate the structural basis of the chaperone recognition function and chaperone-subunit-usher interactions.
The third Aim i s to investigate the function of chaperone- subunit interactions using an in vivo import model in which expression of pilus subunits in the absence of a chaperone (or in the absence of an interaction with a chaperone) is toxic to the cells.
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