The biogenesis of diverse fibrous organelles by Gram-negative bacteria plays a critical role in the pathogenesis of many diseases. Uropathogenic E. coli assembles P and type 1 pili on their surfaces via the conserved chaperone-usher pathway. These pili mediate attachment to host tissues, a key early event in the development of disease. Pilus subunits have immunoglobulin-like (Ig) folds that lack their canonical C-terminal b-strand. The Ig-like periplasmic chaperone transiently donates a b-strand to complete the fold of the subunit, via a mechanism termed donor strand complementation. Subunits do not fold in the absence of a chaperone and are proteolytically degraded. During pilus assembly, the Ig fold of a subunit is thought to be more permanently completed by the exposed N-terminal extension of its neighbor, which displaces the chaperone in a mechanism termed donor strand exchange. Pilus subunits with complete Ig folds will be generated by genetically linking the relevant N-terminal extension to the C-terminus of the subunit to create stable, monomeric donor strand complemented (Dsc) subunits. Dsc subunits will be tested for their ability to fold in the absence of a chaperone to determine the role of the missing strand in subunit folding. Dsc and N-terminal deleted (Ntd) subunits, which have their N-terminal extensions removed and therefore cannot interact with other subunits, will be used with x-ray crystallography and in vivo and in vitro assembly systems to elucidate the structural basis of donor strand exchange. Stable Dsc adhesins will be crystallized with their saccharide receptors to determine the structural basis of microbial colonization and will be tested as vaccines to treat and prevent urinary tract infections. Gram-negative pathogens also assemble surface fibers termed curli via the nucleation-precipitation pathway. Curli share the diagnostic properties of amyloid fibers, which characterize a variety of human diseases, including Alzheimer's disease. The nucleation, formation, and structure of amyloid-like curli fibers will be studied. These studies will reveal general principles that govern the fundamental processes of protein folding and organelle biogenesis, shed light on bacterial attachment and its role in pathogenesis, and contribute to the development of new methods to treat and prevent a variety of diseases.
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