The presence of pili can be correlated with virulence in many gram- negative bacteria that inhabit mucosal surfaces of man. The expression of these proteinaceous appendages by Neisseria gonorrhoeae, the causative agent of gonorrhea, appears to be a requisite determinant of pathogenicity in gonococcal disease. An analogous association between piliation and virulence can be made for Neisseria meningitidis, the etiologic agent of epidemic menigitis. Together, these two pathogenic species of Neisseria are responsible for an extensive amount of morbidity and mortality world-wide. Although the genetic mechanisms responsible for the antigenic and phase variation of gonococcal pili have received enormous attention, little progress has been made in elucidating the structure / function relationships of gonococcal pilin and pili, the processes of pilus biogenesis and the nature of the correlation between virulence and piliation. The overall goal of the proposed research is to begin to address these questions by the isolation and characterization of mutants of gonococci that are altered in the expression of pili and pilus-associated properties. The selection of these pili mutants will be facilitated by the use of gonococcal strains deficient in homologous recombination functions and therefore stabilized with regard to pilin gene expression; thus avoiding the major impediment that has heretofore precluded their isolation. Based on previous studies and preliminary results, two main classes of pilus mutants are anticipated to arise from the selection protocol: one class resulting from mutations in the pilin subunit gene and the other resulting from mutations within genes that qualitatively or quantitatively influence the biogenesis or expression of pili. These mutants will first be characterized according to the phenotypes that they display and by assessing their behavior using in vitro model systems that are thought to reflect the functional roles served by pili in the pathogenesis of gonorrhea (adherence to human cells and resistance to phagocytosis by human polymorphonuclear leukocytes). Finally, the genes in which spontaneous mutations have occurred leading to the altered expression of pili will be physically and identified.
