The rise in antibiotic resistant pathogens, emergence of new diseases, and involvement of bacterial pathogens in diseases formerly thought to be due to non-infectious agents has rekindled the need to understand the """"""""molecular logic"""""""" of virulent bacteria. Extracellular fibers made by Gram-negative bacteria can effect bacterial interactions with their environment or host tissue. This grant focuses on the mechanisms of assembly of two such fibers, adhesive pili and curli. Pili are composite structures consisting of an adhesive heteropolymeric distal tip fibrillum joined to a rigid helical rod. We discovered that periplasmic chaperones serve as folding templates for pilus subunits, actively shaping the final structure of pilus subunits. Further, we discovered that pilus assembly occurs by a mechanism that we termed donor strand exchange wherein the N terminal extension of every subunit completes the immunoglobulin fold of its neighboring subunit. The resulting pilus structures initiate host-pathogen interactions critical in the pathogenic processes of a wide range of bacteria by binding to host receptors. We found that type 1 pilus mediated entry of uropathogenic Escherichia coli into superficial cells lining the bladder activates a complex genetic cascade leading to the formation of intracellular bacterial communities that undergo a defined maturation and differentiation program necessary to subvert innate host defenses. We will use a multidisciplinary approach including molecular genetics, biochemistry, microscopy, physical techniques and in vivo and in vitro systems to better understand pilus assembly and function. We will investigate the molecular basis of the donor strand exchange interactions that determine the specificity of the subunit types in the pilus and the mechanism by which pilus assembly is initiated and terminated. We will investigate the function of the specific architecture of the plus fiber in pilus strength and function, including mediating biofilm formation and host pathogen interactions. We will also investigate the biogenesis of another type of fiber called curli. Curli fibers are assembled via a nucleation/precipitation pathway and we have shown that they share all the biochemical characteristics of amyloid proteins involved in human diseases such as Alzheimer's, Huntington's, and Parkinson's. We will elucidate the structural basis of how two periplasmic chaperone-like proteins, CsgE and CsgF, facilitate the protein-protein interactions necessary for amyloid formation. This work is spawning new insights into the most basic principles of molecular biology related to protein folding and macromolecular assembly and is providing a paradigm to understand infectious diseases that will lead to better strategies for treatment and prevention.
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