Role for the RecBCD recombination pathway for pilE gene variation in repair-proficient Neisseria gonorrhoeae

The role of the RecBCD recombination pathway in PilE antigenic variation in Neisseria gonorrhoeae is contentious and appears to be strain dependent. In this study, N. gonorrhoeae strain MS11 recB mutants were assessed for recombination/repair. MS11 recB mutants were found to be highly susceptible to DNA treatments that caused double-chain breaks and were severely impaired for growth; recB growth suppressor mutants arose at high frequencies. When the recombination/repair capacity of strain MS11 was compared to that of strains FA1090 and P9, innate differences were observed between the strains, with FA1090 and P9 rec⁺ bacteria presenting pronounced recombination/repair defects. Consequently, MS11 recB mutants present a more robust phenotype than the other strains that were tested. In addition, MS11 recB mutants are also shown to be defective for pilE/pilS recombination. Moreover, pilE/pilS recombination is shown to proceed with gonococci that carry inverted pilE loci. Consequently, a novel RecBCD-mediated double-chain-break repair model for PilE antigenic variation is proposed.     

Biochemical comparison of pili from variants of Neisseria gonorrhoeae P9

Four different types of pili produced by variants of Neisseria gonorrhoeae P9 were isolated and characterized. The pili differed in subunit molecular weight with SDS-PAGE and in subunit isoelectric point on agarose gels. Isoelectric points of the major molecular species in Triton-agarose gels of octylglucoside solubilized pili were: delta, pI 6.5; alpha, pI 6.0; beta, pI 5.3 and gamma, pI 5.5. Amino acid analyses of pili showed close homology between different types but a reduction in the content of aspartate and serine was notable in the low molecular weight delta pili; also beta and gamma maps of tryptic/chymotryptic digests of pili with several major peptides apparently common to all four pilus types.     

Nucleotide sequence of the structural gene for class I pilin from Neisseria meningitidis: homologies with the pilE locus of Neisseria gonorrhoeae

The nucleotide sequence has been determined for the expressed pilin (pilE) locus of Neisseria meningitidis strain C311 which produces class I pili that are antigenically and structurally similar to those of gonococci. The deduced amino acid sequence of the N. meningitidis pilE translation product contains a 7 amino acid N-terminal pre-pilin leader sequence which is identical to that found in gonococcal pilin and which is characteristic of N-methylphenylalanine pili in general. The succeeding N-terminal 53 amino acids are identical to those found in the equivalent position in antigenically variant gonococcal pilins and confirm direct peptide sequencing of the amino-terminus of at least one type of meningococcal pilin. Other regions that are conserved in variant pilin polypeptides from Neisseria gonorrhoeae are conserved at the amino acid level in the class I meningococcal pilin but the coding DNA contains numerous base substitutions when compared with the equivalent gonococcal pil sequence. Sequences extending downstream for about 140 bp on the 3' side of the coding region for both pilin genes are only about 85% homologous.     

Antigenic variation of outer membrane protein II in colonial variants of Neisseria gonorrhoeae P9

Antibodies were detected by an enzyme-linked immunosorbent assay (ELISA) in sera from rabbits immunized wtih outer membranes from colonial opacity variants in Neisseria gonorrhoeae P9. ELISA-inhibition experiments with purified antigens revealed approximately equal proportions of antibodies directed against each of the three major surface antigens, lipopolysaccharide, the major outer membrane protein (protein I) and protein II, the variable protein associated with colonial opacity. Inhibition experiments with intact gonococci showed considerable antigenic diversity which could be correlated with differences between the protein II species present. Despite their considerable structural homology, different protein II species from colonial variants of the same strain showed little cross-reactivity with specific anti-protein II sera, thus demonstrating the considerable variation in that part of the antigen which is exposed on the surface of the gonococcus and is closely involved in pathogenic mechanisms.     

Limited variation and maintenance of tight genetic linkage characterize heteroallelic pilE recombination following DNA transformation of Neisseria gonorrhoeae

Genetic recombination impacts on neisserial biology in two ways: (i) specific loci undergo rearrangement at high frequency leading to the formation of many different alleles; and (ii) Neisseria , being naturally competent for DNA transformation, provide a means to disseminate the novel alleles throughout a population. In this study pilE was used as a model system to examine heteroallelic recombination following DNA transformation. When gonococci were transformed with chromosomal donor DNA containing different pilE alleles, the majority of pilE recombinants arose through allelic replacement. Co-conversion analysis across pilE showed that in ∼ 85–90% of recombination events encompassing pilE and an adjacent opa locus, linkage was maintained (i.e. ∼ 10–15% of recombination events terminated within the ∼ 1000 base pair pilE/opaE interval). In addition to those recombinants that arose through allelic replacement, a large pilus-minus subpopulation was also observed (∼ 10% of all recombinants), indicating that many recombination events did not yield recombinant pilE s that could be assembled into functional pili. PilE mosaics increased following transformation with plasmid donor DNAs carrying pilE with limited flanking-sequence homology, suggesting a potential role for flanking-sequence homologies in mosaic formation. Overall, the data support the view that horizontal transmission of chromosomal DNA between gonococci will favour the spread of intact alleles, as opposed to expanding the allelic repertoire through the formation of gene mosaics.     

Structural comparison of Neisseria gonorrhoeae outer membrane proteins.

Outer membranes from opaque colonia variants of Neisseria gonorrhoeae P9 contain a major outer membrane protein (protein I) together with one or more of a series of heat-modifiable proteins (proteins II). Proteins I. II, and IIa have been isolated by detergent extraction of outer membranes. Amino acid analysis showed proteins II and IIa to have a very similar composition. Cyanogen bromide cleavage of proteins II and IIa produced a pair of fragments with identical molecular weight and a pari which differed by an amount (0.5K) equivalent to the difference between the intact proteins. Tryptic peptide maps of 125I-labeled proteins II, IIa, and IIb showed many similarities, with only a few peptides unique to any one protein. Peptide maps of protein IIa from cells which had been surface labeled showed that the unique peptides were exposed on the surface. The heat-modifiable proteins thus appear to form a family of proteins with closely related structure probably differing in that part which is exposed on the bacterial surface.     

Recombinational error and deletion formation in Neisseria gonorrhoeae: a role for RecJ in the production of pilE L deletions

Genetic linkage within Neisseria gonorrhoeae populations is in equilibrium, yet the physical linkage map indicates a relatively stable chromosome structure, despite an apparently vast potential for mispairing between repeated sequences (e.g. between the multiple pil or opa alleles, or through mispairing of any of the numerous small repeated sequences that are liberally scattered throughout the chromosome). Therefore, the stability of the physical linkage map suggests that aberrant recombination between repeated sequences is a rare event. This study was undertaken to explore some of the parameters that may govern deletion events between short direct oligonucleotide repeats, using a chromosomal locus that appears to be especially prone to deletions (the pilin expression locus; pilE). In this report, we demonstrate that deletion formation at pilE occurs primarily through recombinational error following a pilE/pilS interaction; illegitimate (i.e. RecA-independent) events can occur, but they are infrequent. In contrast, when genetically engineered opa deletion substrates were constructed and placed in the chromosome, deletions at the opa loci were infrequent even under rec(+) conditions. A model is presented in which the gonococcal RecA and RecJ proteins promote pilE deletions through a recombination event that is templated or stabilised by a pilE/pilS interaction.     

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

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.     

Biological properties of two distinct pilus types produced by isogenic variants of Neisseria gonorrhoeae P9.

Isogenic variants from a single strain of Neisseria gonorrhoeae were shown to produce two distinct types of pili. These pili, designated alpha and beta, differed in both subunit molecular weight and in ability to attach to buccal epithelial cells.     

The cohesive properties of variants of Neisseria gonorrhoeae strain P9: specific pilus-mediated and non-specific interactions

The cohesive properties of virulent pilated Neisseria gonorrhoeae strain P9 (P++) have been compared with those of a non-pilated isogenic variant (P-) possessing the same outer membrane components. The binding of P++ gonococci to buccal epithelial cells was dependent on pH, with an optimum at pH 6.5 to 7.0 . This adhesion was markedly inhibited by treatment of the buccal epithelial cells with a neuraminidase/exoglycosidase mixture. In contrast, the binding of P++ gonococci to erythrocytes was unaffected by pH. A possible explanation is that pili bind to a carbohydrate receptor present on buccal epithelial cells but lacking on erythrocytes. The adhesion of P- gonococci to erythrocytes and to buccal epithelial cells was unaffected by pH but enhanced by treatment of the cells with neuaminidase or periodate. Presumably, neuraminic acid residues on host cell surface carbohydrates inhibit adhesion. The finding that P- gonococci bind to amphipathic gels suggests hydrophobic interactions as a possible non-specific mechanism attaching P- gonococci to host cell surfaces.     

Ultrastructural analysis of the pathogenesis of Neisseria gonorrhoeae endometrial infection

We have studied gonococcal infection in human endometrium organ culture and in human primary endometrial epithelial cells using various microscopic techniques including scanning electron microscopy, transmission electron microscopy, bright field light microscopy and laser scanning confocal microscopy. Here we describe the interactions between Neisseria gonorrhoeae and human endometrial luminal epithelial cells at the ultrastructural levels. N. gonorrhoeae attached to cilia but were not observed associated with the plasma membrane of ciliated epithelial cells or internalized into ciliated epithelial cells. N. gonorrhoeae could be found in intracellular vacuoles in secretory epithelial cells. N. gonorrhoeae have diverse interactions with endometrial epithelium. These include intimate association and colocalization with asialoglycoprotein receptor (ASGP-R) and CEACAM, lamellipodia and ruffle formation and colocalization with CR3, and microvillus engagement. These studies indicate that N. gonorrhoeae utilize multiple mechanisms to associate with endometrial epithelial cells and can associate with both ciliated and secretory cells. This diversity is consistent with a role of the endometrium as a transition zone between frequently asymptomatic cervical gonorrhoea and symptomatic pelvic inflammatory disease.