Down-regulation of CD46 by piliated Neisseria gonorrhoeae.

Human membrane cofactor protein (CD46) protects host cells against complement attack and may function as a receptor for pathogenic Neisseriae. We assessed CD46 expression in the human cervical cell line ME-180 after exposure to Neisseria gonorrhoeae. Piliated but not nonpiliated gonococci adhered to cells and produced up to an 80% reduction in CD46 surface expression by 6 h that persisted for at least 24 h. This response required a minimum multiplicity of infection of 10 and was not prevented by antibodies to CD46. CD46 down-regulation was not attributable to intracellular retention or a global or specific shutdown of mRNA or protein synthesis. Substantial quantities of CD46 were found in the supernatants, indicating a specific shedding of this protein. Adherent gonococci lacking the pilus retraction protein PilT did not down-regulate CD46 but de-repression of pilT expression restored CD46 down-regulation. After experimental infection of human volunteers with a gonococcal variant incapable of inducing CD46 down-regulation, variants of this strain were reisolated that exhibited CD46 down-regulation. Pilus-mediated interactions of gonococci with human epithelial cells results in a pathogen-induced manipulation of the host cell environment in which a membrane protein is removed from epithelial cells by liberation into the surrounding milieu.

A co-operative interaction between Neisseria gonorrhoeae and complement receptor 3 mediates infection of primary cervical epithelial cells.

Little is known about the pathogenesis of gonococcal infection within the lower female genital tract. We recently described the distribution of complement receptor 3 (CR3) on epithelia of the female genital tract. Our studies further indicate that CR3-mediated endocytosis serves as a primary mechanism by which N. gonorrhoeae elicits membrane ruffling and cellular invasion of primary, human, cervical epithelial cells. We have extended these studies to describe the nature of the gonococcus-CR3 interaction. Western Blot analysis demonstrated production of alternative pathway complement components by ecto- and endocervical cells which allows C3b deposition on gonococci and its rapid conversion to iC3b. Anti-iC3b and -factor I antibodies significantly inhibited adherence and invasion of primary cervical cells, suggesting that iC3b covalently bound to the gonococcus serves as a primary ligand for CR3 adherence. However, gonococcal porin and pili also bound to the I-domain of CR3 in a non-opsonic manner. Binding of porin and pili to CR3 were required for adherence to and invasion of cervical epithelia. Collectively, these data suggest that gonococcal adherence to CR3 occurs in a co-operative manner, which requires gonococcal iC3b-opsonization, porin and pilus. In conjunction, these molecules facilitate targeting to and successful infection of the cervical epithelium.

Antigenic variation of gonococcal pilin expression in vivo: analysis of the strain FA1090 pilin repertoire and identification of the pilS gene copies recombining with pilE during experimental human infection.

Antigenic variation of gonococcal pilin involves a family of variable genes that undergo homologous recombination, resulting in transfer of variant sequences from the pilS silent gene copies into the complete pilE expression locus. Little is known about the specific recombination events that are involved in assembling new variant pilin genes in vivo. One approach to understanding pilin variation in vivo is to carry out experimental human infections with a gonococcal strain having a fully characterized repertoire of pilin genes, so that the specific recombination events occurring in vivo can be determined. To this end, the authors cloned, sequenced and mapped the pilin genes of strain FA1090 of Neisseria gonorrhoeae. This strain contains one pilE locus and 19 silent gene copies that are arranged in five pilS loci; the pilE locus and four of the pilS loci are clustered in a 35 kb region of the chromosome. The general features of the pilin loci in FA1090 are similar to those in strain MS11, in which the mechanism of pilin variation has been extensively studied. However, none of the silent copy sequences are identical in the two strains, which emphasizes the extreme variability in this gene family among gonococci. Three male volunteers were inoculated with the same variant of strain FA1090 and developed urethritis within 2--4 d. The pilE gene sequences from a total of 23 colonies cultured from the subjects were analysed, determining which pilS silent copy donated each portion of the expressed pilE genes. There were 12 different pilin variants, one of which was the original inoculum variant, among the in vivo-expressed pilE gene sequences. The pilE of the inoculum variant was derived entirely from a single silent copy (pilS6c1). However, the pilE genes in the majority of the colonies cultured from the infected subjects were chimeras of sequence derived from two or three silent copies. Recombination to generate new pilE sequences involved exchange of single variable minicassettes, multiple minicassettes, entire silent gene copies, or (rarely) recombination within a minicassette.

Recombinational reassortment among opa genes from ET-37 complex Neisseria meningitidis isolates of diverse geographical origins.

Opacity (Opa) proteins are a family of antigenically variable outer-membrane proteins of Neisseria meningitidis. ET-37 complex meningococci, defined by multilocus enzyme electrophoresis, have been isolated on different continents. Twenty-six different Opa proteins have been observed within strains of the ET-37 complex isolated between the 1960s and the 1980s, although individual strains have only four opa genes per chromosome. In this work the opa genes of four closely related ET-37 complex N. meningitidis strains recently isolated from Mali, West Africa were characterized and compared with the opa genes of strain FAM18, an ET-37 complex isolate from the USA. DNA sequence analysis and Southern blot experiments indicated that recombinational reassortment, including gene duplication and import by horizontal genetic exchange, has occurred in the opa genes within the ET-37 complex, resulting in two partially different Opa repertoires being present in FAM18 and the Mali isolates. Using synthetic peptides derived from the hypervariable (HV) regions of opa genes, the epitopes for nine mAbs were mapped. These bacteria, isolated on different continents, contain both shared and unique opa HV regions encoding epitopes recognized by mAbs and show evidence of recombinational reassortment of the HV regions.