The obligate human pathogen N. gonorrhoeae (the gonococcus) is the causative agent of the common sexually transmitted disease gonorrhea. One essential virulence factor is the gonococcal pilus -- a fiber-like structure that mediates adherence to the genital epithelium, and undergoes antigenic variation. During antigenic variation, amino acid sequence changes occur in the major pilus subunit, pilin. These events produce antigenically variant pili on the cell surface that allow for immune evasion, and may also provide different functional characteristics to the bacterial cell surface. We have previously characterized pilin antigenic variation in strain FA1O90 during infection of human volunteers. This collaborative project with Drs. Cannon and Cohen of UNC Chapel Hill, made use of gonococcal isolates from human volunteers to compare pilin variation in vivo to variation during in vitro growth. We observed multiple pilin variants at every time point sampled, with different variants detected at each time point. These data support two hypotheses: either there is a stimulation of the frequency of antigenic variation during infection of volunteers and/or there is selection for new subsets of pilin variants during infection. This proposal will test predictions of these hypotheses.
The specific aims are: (1) Analyze pilin variation in strain FA1090 during infection of human volunteers, (2) Isolate and characterize monoclonal antibodies raised against different FA109O pilins, (3) compare the adherence properties of different FA1090 pilin variants to eukaryotic cells, (4) determine the rate of FA109O pilin variation during laboratory culture, and (5) use human volunteer studies to test the role of different pilin variants in infectivity. Reisolates from different volunteers will be examined to determine the range of variation that occurs within volunteers. Monoclonal antibodies will be isolated that define common and type-specific epitopes of FA109O pili, and used to help define portions of the protein required for adherence. In vitro studies will determine whether the isolates from volunteers exhibit variable adherence and whether that can be correlated to changes in primary amino acid sequences. The rate of variation will be measured under different growth conditions to test whether the frequency of antigenic variation is constant or variable. New volunteer studies using pilin variants expressing variable adherence will determine whether the expressed pilin gene influences infection. These studies will examine the environmental and selective conditions that may account for the observed variation of gonococcal pili during inoculation of human volunteers. They will establish the contributions of selection to variation. This information is required to understand why pilin antigenic variation occurs, and will aid in determining the feasibility of pilus-based vaccines.
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