Summary
The influence of the two surface structures of Neisseria meningitidis, capsule and pili, in bacterial interactions with human endothelial cells was investigated. Increased association correlated with the presence of pili on bacteria while capsule type had no apparent effect. Strains expressing both Class I and Class II pili associated with endothelial cells in significantly larger numbers compared with the non-piliated variants of the same strains (>10×). Variants of Neisseria gonorrhoeae strain P9 expressing antigenically distinct pili also associated with endothelial cells in larger numbers (>30×) compared with the non-piliated variant. Electron microscopic studies confirmed these data and showed that gonococci were internalized more frequently compared with meningococci. One consequence of increased association was an increase in the cytopathic effect of bacteria on the target cells.

The role of pili in the interactions of pathogenic Neisseria with cultured human endothelial cells

The development of effective prophylactic agents against gonorrhea and the study of adaptation by Neisseria gonorrhoeae to the urogenital mucosa are hindered by the lack of a well-established animal model of gonococcal genital tract infection. Here, a murine model of long-term gonococcal genital tract infection is described. Female BALB/c mice were treated with 17-beta-estradiol and inoculated intravaginally with wild-type gonococcal strain FA1090 or MS11. N. gonorrhoeae was recovered from vaginal swabs for an average of 12 to 13 days following inoculation with 10(6) CFU of either strain. Inflammation occurred in over 80% of infected mice, and diplococci were associated with epithelial cells and neutrophils in stained vaginal smears. Ascended infection occurred in 17 to 20% of mice inoculated with strain FA1090. An outbred mouse strain (SLC:ddY) previously reported to be naturally susceptible to N. gonorrhoeae was also tested; however, as with BALB/c mice, estradiol was required for prolonged infection. Although piliation was not maintained during experimental murine infection, 46 to 100% of vaginal isolates from four of eight BALB/c mice and three of four SLC:ddY mice expressed one or more opacity (Opa) proteins within 4 days after inoculation with an Opa-negative variant of strain FA1090. The observed selection for and/or induction of gonococcal Opa protein expression during murine infection appears to parallel events that occur during experimental urethritis in volunteers.

https://iai.asm.org/content/iai/67/11/5699.full.pdf

Experimental gonococcal genital tract infection and opacity protein expression in estradiol-treated mice.

When pilus+ Gc were introduced into a male subject's urethra, they gave rise to pilus+ variants whose pilin mRNAs differed from that of input Gc. The differences stemmed from the Gc genome's single complete pilin gene having undergone gene conversion by different partial pilin genes' sequences and by different length stretches of a single partial pilin gene. In some instances, the variant's pilin mRNA appeared to reflect two independent gene-conversion events that used sequences from two different partial pilin genes. The resulting variants' pilins exhibited antigenic differences compared with the pilin polypeptide of input Gc; these differences were discernible by immunoblotting with mAbs. Amino acid and antigenic changes occurred in a segment of the variants' pilin polypeptides that previously was thought to be conserved or constant in sequence.

https://rupress.org/jem/article-pdf/165/5/1344/1097027/1344.pdf

Gonococcal pilin variants in experimental gonorrhea

The human pathogens Neisseria meningitidis and Neisseria gonorrhoeae express a family of variable outer membrane opacity-associated (Opa) proteins that recognize multiple human cell surface receptors. Most Opa proteins target the highly conserved N-terminal domain of the CD66 family of adhesion molecules, although a few also interact with heparan sulphate proteoglycans. In this study, we observed that at least two Opa proteins of a N. meningitidis strain C751 have the dual capacity to interact with both receptors. In addition, all three Opa proteins of C751 bind equally well to HeLa cells transfected with cDNA encoding the carcinoembryonic antigen [CEA (CD66e)] subgroup of the CD66 family, but show distinct tropism for CGM1- (CD66d) and NCA (CD66c)-expressing cells. Because the C751 Opa proteins make up distinct structures via the surface-exposed hypervariable domains (HV-1 and HV-2), these combinations appear to be involved in tropism for the distinct CD66 subgroups. To define the determinants of receptor recognition, we used mutant proteins of biliary glycoprotein [BGP (CD66a)] carrying substitutions at several predicted exposed sites in the N-domain and compared their interactions with several Opa proteins of both N. meningitidis and N. gonorrhoeae. The observations applied to the molecular model of the BGP N-domain that we constructed show that the binding of all Opa proteins tested occurs at the non-glycosylated (CFG) face of the molecule and, in general, appears to require Tyr-34 and Ile-91. Further, efficient interaction of distinct Opa proteins depends on different non-adjacent amino acids. In the three-dimensional model, these residues lie in close proximity to Tyr-34 and Ile-91 at the CFG face, making continuous binding domains (adhesiotopes). The epitope of the monoclonal antibody YTH71.3 that inhibits Opa/CD66 interactions was also identified within the Opa adhesiotopes on the N-domain. These studies define the molecular basis that directs the Opa specificity for the CD66 family and the rationale for tropism of the Opa proteins for the CD66 subgroups.

Critical determinants of host receptor targeting by Neisseria meningitidis and Neisseria gonorrhoeae: identification of Opa adhesiotopes on the N-domain of CD66 molecules.

The complete amino acid sequence of pilin from gonococcal strain MS11 and the sequence of constant and variable regions from strain R10 pilin have been determined in order to elucidate the structural basis for adherence function, antigenic diversity, and polymeric structure. The MS11 pilin sequence consists of 159 amino acids in a single polypeptide chain with two cysteines in disulfide linkage and serine-bonded phosphate residues. TC-2 (31-111), a soluble monomeric pilus peptide prepared by arginine-specific digestion, bound human endocervical, but not buccal or HeLa cells and therefore is postulated to encompass the receptor binding domain. Variable regions of CNBr-3 appear to confer antigenic diversity and comprise segments in which changes in the position of charged residues occur in hydrophilic, beta-turns. Residues 2-21 and 202-221 of gonococcal pilins and lower eucaryotic actins, respectively, exhibit 50% homology. When these residues are arranged at intervals of 100 degrees of arc on "helical wheels," the identical amino acids comprise a hydrophobic face on one side of the helix. This observation, the hydrophobic character of this region and the tendency for TC-1 (residues 1-30) to aggregate in water, suggest that this stretch interacts with other subunits to stabilize polymeric structure.

/pdf/gonococcal-pili-primary-structure-and-receptor-binding-2h4qtzsx57.pdf

Gonococcal pili. Primary structure and receptor binding domain.

The identification and isolation of novel pilus types produced by variants of N. Gonorrhoeae P9 following selection in vivo

Guinea-pig subcutaneous chambers were infected with a mixture of gonococcal variants of defined outer membrane protein profile. Survival within the chambers was a two-stage process. The initial advantage conferred by the lack of opacity-related outer membrane protein was transient and survivors were replaced by opaque colonial variants. Amongst these survivors were variants which produced opacity-related proteins (IId, IIe and IIf) not present in the initial inoculum. Thus, outer membrane protein composition is an important factor in survival in vivo.

/pdf/the-role-of-outer-membrane-proteins-in-the-survival-of-f9exhmay6l.pdf

H. M. McBRIDE

Papers

The identification and isolation of novel pilus types produced by variants of N. Gonorrhoeae P9 following selection in vivo

The role of outer membrane proteins in the survival of Neisseria gonorrhoeae P9 within guinea-pig subcutaneous chambers.