This renewal aims to characterize structure-function relationships for type IV pili fibers, which key virulence factors for pathogenic Gram-negative bacteria. Structural analyses for type IV pilin subunits will be integrated with electron microscopy (EM), fiber diffraction, and small angle x-ray scattering (SAXS) structures of native fibers via objective Fourier correlation methods. These proposed studies, which span atomic to subcellular resolutions, will focus upon type IV pili from Neisseria gonorrhoeae, the causative agent of gonorrhea. Successful methods and results on gonococcal pili will allow complementary structural and mutational studies on pili from Pseudomonas aeruginosa, the causative agent on deadly opportunistic nosocomial infections, and Vibrio cholerae, the causative agent of cholera, to define conserved and variable aspects of type IV pili. Key questions concerning pilus structure-function relationships will be addressed including whether the N. gonorrhoeae pilin fold is representative of all type IV pilins, how extreme antigenic variation avoids disrupting the pilin fold and fiber assembly, the nature and significance of post-translational modifications, structural changes associated with fiber formation, species-specific conservation of surface regions acting in target cell recognition and accessory protein binding, the structural chemistry controlling bundling, structural characteristics of immunodominant regions, and optimal approaches to the design of cross- species vaccines. Structural results and hypotheses will be experimentally tested by quantitative correlations among diffraction and electron microscopy results and by mutational analyses. The proposed integrated multi-disciplinary studies provide innovation in determining challenging fiber-forming protein structures and in bridging the huge resolution gap between protein crystal structures and EM image reconstructions of subcellular organelles. Overall, these structural and mutational results will promote integration of ongoing biochemical, immunobiological, genetic, and functional studies to decipher the structural chemistry governing pilus actions in pathogenicity: host cell surface attachment, twitching motility, bacteriophage absorption, modulation of transformation efficiency and toleration of extreme sequence variability while retaining structural integrity and flexibility. This understanding of pilus structure-function relationships has long-term potential applications for drug and vaccine design against major bacterial diseases now showing increasing antibiotic resistance and threats to public health.
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