The pilus is a major virulence factor common to many bacterial pathogens including Neisseria gonorrhoeae, the causative agent of gonorrhea. Pilus fibers (pili) aid attachment to host epithelial cells and are composed primarily of the 18,000 KD pilin protein.
We aim to complete studies on the structure, assembly, function, antigenicity, and immunogenicity of the gonococcal pilus at the atomic level. We will accomplish this overall goal by 1) further analysis of our first MS11 gonococcal pilin structure followed by determination of mutant and strain variant pilin structures, 2) defining the nature of the pilin assembly into fibers by antibody labeling and cryo-electron microscopy of gonococcal-assembled pili complemented by crystal packing studies of other pilin crystal forms, 3) determining gonococcal pilus antigenicity and immuno-genicity by peptide mapping of pilus antisera specificities combined with testing the binding of site-directed antibodies. This work will focus on the structure, assembly, and antigenicity of gonococcal pilin but where feasible will also include studies on the role of important accessory proteins such as PilC. We will complete analysis of our atomic structure of N. gonorrhoeae MS11 pilin and then use molecular replacement techniques to determine three dimensional structures for additional gonococcal mutant and variant pilins. We will use electron microscopy and image reconstruction of whole pili, site-directed antibody mapping, and structurally designed site-directed gonococcal pilin mutants to establish the molecular basis for pilin self-association to form the pilus fiber and for pilin structure-function relationships. Expert collaborators will test implications from our results for gonococcal biology and pilus function. Epitopes we have identified from the human, rabbit and mouse immune response to pili will be mapped onto the pilin and pilus structures. Pilin and pilus surface shape and electrostatic potential, along with the crystallographically observed heptane-triol binding sites, will be analyzed to identify and characterize functionally important pilus binding sites. From these different pilin structures, we will define sequence and structural conservation and variation in the context of the folded pilin subunit and assembled pilus fiber including the structural basis allowing pilin's antigenic variation and its effect on assembly parameters. Taken together, these results aid integration of numerous biochemical, immunobiological, genetic, and functional studies on pathogenic pili and thereby provide an in-depth understanding of the pilus virulence factor. These studies will thus increase basic understanding of pilus structure-function relationships with long term potential applications for drug and vaccine design against gonorrhea and several other bacterial diseases representing major threats to public health.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI022160-11
Application #
2671818
Study Section
Bacteriology and Mycology Subcommittee 2 (BM)
Project Start
1985-07-01
Project End
2000-01-31
Budget Start
1998-06-01
Budget End
2000-01-31
Support Year
11
Fiscal Year
1998
Total Cost
Indirect Cost
Name
Scripps Research Institute
Department
Type
DUNS #
City
La Jolla
State
CA
Country
United States
Zip Code
92037
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