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.     

Identification of epitopes recognized by monoclonal antibodies SM1 and SM2 which react with all pili of Neisseria gonorrhoeae but which differentiate between two structural classes of pili expressed by Neisseria meningitidis and the distribution of their encoding sequences in the genomes of Neisseria spp

The pili expressed by all isolates of Neisseria gonorrhoeae react with two monoclonal antibodies, SM1 and SM2. In contrast, although many isolates of Neisseria meningitidis also express pili (class I) which react with antibodies SM1 and SM2, a proportion express pili (class II) which fail to react. In order to define the epitopes recognized by these antibodies, a series of overlapping peptides corresponding to the amino acid sequence of conserved regions of gonococcal pili have been synthesized. The minimum epitope recognized by antibody SM1 was found to comprise a linear peptide EYYLN, corresponding to residues 49-53 of mature pilin. In contrast, antibody SM2 reacted with a number of peptides from around the cysteine residue (Cys 1) at position 120, suggesting that an extended region may contribute to a conformational epitope recognized by this antibody in the native protein. The identification of the two epitopes defines structural differences between the classes of pili expressed by meningococci. In order to determine the distribution of pilin gene sequences in Neisseria we used as hybridization probes an oligonucleotide (PS1) with the sequence 5'-GAGTATTACCTGAATCA-3' which spans the coding region for the SM1 epitope, and a fragment of the 3' end of the gonococcal pilE gene which contains conserved sequences flanking the two Cys codons and encodes the SM2 epitope. All strains of N. gonorrhoeae and N. meningitidis tested, regardless of piliation phenotype, harboured DNA sequences homologous to those encoding the carboxy-terminus of meningococcal class I pilin. Furthermore, all gonococci and all meningococci producing class I pili hybridized with oligonucleotide probe PS1. Non-reverting non-piliated derivatives of previously class I pilus-producing strains showed reduced hybridization signals with this probe, but nevertheless retained sequences homologous to the coding sequence for the SM1 epitope. However, meningococci producing class II pili could be divided into two groups on the basis of their reaction with the PS1 probe: half the strains tested failed to react, which is consistent with our previous analysis of silent class I pilin sequences; the remainder reacted (relatively weakly) with the probe, suggesting that the silent pil sequences in these strains extend further towards the 5' end of the pilin gene than in strains studied previously. Some strains of Neisseria lactamica reacted weakly with both types of probe but failed to produce SM1-reactive pili. In contrast, isolates of Neisseria flava, Neisseria pharyngis, Neisseria sicca and a series of unrelated bacteria failed to react with both SM1 antibody and the DNA probes. This confirms that possession of 'gonococcal' pilin sequences is limited to the pathogenic neisseriae.     

PilC of Neisseria meningitidis is involved in class II pilus formation and restores pilus assembly, natural transformation competence and adherence to epithelial cells in PilC-deficient gonococci

Type 4 pili produced by the pathogenic Neisseria species constitute primary determinants for the adherence to host tissues. In addition to the major pilin subunit (PilE), neisserial pili contain the variable PilC proteins represented by two variant gene copies in most pathogenic Neisseria isolates. Based upon structural differences in the conserved regions of PilE, two pilus classes can be distinguished in Neisseria meningitidis . For class I pili found in both Neisseria gonorrhoeae and N. meningitidis , PilC proteins have been implicated in pilus assembly, natural transformation competence and adherence to epithelial cells. In this study, we used primers specific for the pilC2 gene of N. gonorrhoeae strain MS11 to amplify, by the polymerase chain reaction, and clone a homologous pilC gene from N. meningitidis strain A1493 which produces class II pili. This gene was sequenced and the deduced amino acid sequence showed 75.4% and 73.8% identity with the gonococcal PilC1 and PilC2, respectively. These values match the identity value of 74.1% calculated for the two N. gonorrhoeae MS11 PilC proteins, indicating a horizontal relationship between the N. gonorrhoeae and N. meningitidis pilC genes. We provide evidence that PilC functions in meningococcal class II pilus assembly and adherence. Furthermore, expression of the cloned N. meningitidis pilC gene in a gonococcal pilC1,2 mutant restores pilus assembly, adherence to ME-180 epithelial cells, and transformation competence to the wild-type level. Thus, PilC proteins exhibit indistinguishable functions in the context of class I and class II pili.     

Silent pilin genes of Neisseria gonorrhoeae MS11 and the occurrence of related hypervariant sequences among other gonococcal isolates

Pilin variation in Neisseria gonorrhoeae depends on a family of variant genes that undergo homologous, intragenic recombination. This work focuses on the repertoire of silent variant pilin genes in strain MS11, which contribute to the extensive variation of the expressed gene copy. A total of 17 silent copies were identified, which are, to varying degrees, truncated at their 5' coding region and grouped in seven distinct pil loci. Most silent copies belong to loci pilS1, pilS2 and pilS6, which contain six, two and three silent copies, respectively, tandemly arranged. The pilS5 and pilS7 loci each contain only a single copy. In addition, two silent copies are associated with each of the two pilE loci. By comparison with sequences present in the expressed gene of other variants of the same strain, it is suggested that each silent locus is capable of donating variant sequences into the expression locus and, thus, each silent copy can contribute to the variability of pilin expression. Often, concomitant with changes in the expressed copy, the silent copies of the pilE1 locus undergo recombinations as well. Analyses of unrelated clinical isolates of N. gonorrhoeae reveal homologies of hypervariant pilin sequences with those present in strain MS11, suggesting a limited diversity of such sequences within the gonococcal population and the existence of substantial functional constraints on the variability of pilin and pili. The data further indicate that hypervariant pilin sequences are subject to horizontal exchange and interstrain recombination.     

Analysis of the Piv recombinase-related gene family of Neisseria gonorrhoeae

Neisseria gonorrhoeae (the gonococcus) is an obligate human pathogen and the causative agent of the disease gonorrhea. The gonococcal pilus undergoes antigenic variation through high-frequency recombination events between unexpressed pilS silent copies and the pilin expression locus pilE. The machinery involved in pilin antigenic variation identified to date is composed primarily of genes involved in homologous recombination. However, a number of characteristics of antigenic variation suggest that one or more recombinases, in addition to the homologous recombination machinery, may be involved in mediating sequence changes at pilE. Previous work has identified several genes in the gonococcus with significant identity to the pilin inversion gene (piv) from Moraxella species and transposases of the IS110 family of insertion elements. These genes were candidates for a recombinase system involved in pilin antigenic variation. We have named these genes irg for invertase-related gene family. In this work, we characterize these genes and demonstrate that the irg genes do not complement for Moraxella lacunata Piv invertase or IS492 MooV transposase activities. Moreover, by inactivation of all eight gene copies and overexpression of one gene copy, we conclusively show that these recombinases are not involved in gonococcal pilin variation, DNA transformation, or DNA repair. We propose that the irg genes encode transposases for two different IS110-related elements given the names ISNgo2 and ISNgo3. ISNgo2 is located at multiple loci on the chromosome of N. gonorrhoeae, and ISNgo3 is found in single and duplicate copies in the N. gonorrhoeae and Neisseria meningitidis genomes, respectively.     

Production of Neisseria gonorrhoeae pili (fimbriae) in Pseudomonas aeruginosa.

Pseudomonas aeruginosa K/2PfS, when transformed with an expression plasmid harboring the pilin gene (pilE1) of Neisseria gonorrhoeae MS11, was able to express and assemble gonococcal pilin monomers into surface-associated pili, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, and immunoelectron microscopy. Concomitant with the expression of gonococcal pili in P. aeruginosa was the virtual loss of production of P. aeruginosa K/2PfS pili normally associated with the host cell.     

Inter-strain homology of pilin gene sequences in Neisseria meningitidis isolates that express markedly different antigenic pilus types

Neisseria meningitidis isolates examined in this study elaborated one of two pilus types that were antigenically markedly different. Each pilus type reacted either with SM1, a monoclonal antibody that recognizes an epitope common to all gonococcal pili, or with a polyclonal antiserum raised against meningococcal pili that did not react with SM1, but not both. Total genomic DNA from all N. meningitidis isolates analysed, irrespective of pilus type, contained at least one region with extensive homology to a gonococcal pilE probe. Different N. meningitidis strains possessed one of several configurations of genomic pilE-homologous segments. Chromosomal rearrangement of pilE-homologous sequences was associated with P+ to P- pilus phase transition in the strains examined. The arrangement of pilE-homologous segments in total genomic DNA from N. meningitidis isolated from the blood and cerebro-spinal fluid of the same patient was apparently identical.     

L-pilin variants of Neisseria gonorrhoeae MS11

Phase- and antigenic variation of pilin expression in Neisseria gonorrhoeae is based on the genetic exchange between silent pilin genes (pilS) and the pilin expression locus (pilE). Similarly, the non-piliated L-variants of strain MS11, which show an increased resistance to certain antibiotics, are the result of recombination with the pilE locus. However, this recombination is atypical in that pilE(L) carries a tandem arrangement of a complete pilin gene and additional partial pilin genes under the control of the same pilE promoter. Since the two pilin gene copies are tandemly arranged and are often in the same translational frame, oversized pilin molecules are produced, which do not assemble into pili. The tandem gene copies introduced in a pilE(L) locus originate from silent loci where they are already joint. Upon reversion to the P+ phenotype the L-variants lose one pilin gene copy from the pilE(L) in a process reminiscent of the deletion events that otherwise lead to the formation of the non-revertible and non-piliated Pn mutants of MS11 gonococci. Thus deletion of pilin genes from pilE can be regarded as a third mechanism of pilin variation in gonococci.     

Structural characterization of outer membrane components of the type IV pili system in pathogenic Neisseria

Structures of the type IV pili secretin complexes from Neisseria gonorrhoeae and Neisseria meningitidis, embedded in outer membranes were investigated by transmission electron microscopy. Single particle averaging revealed additional domains not observed previously. Secretin complexes of N. gonorrhoeae showed a double ring structure with a 14-15-fold symmetry in the central ring, and a 14-fold symmetry of the peripheral ring with 7 spikes protruding. In secretin complexes of N. meningitidis, the spikes were absent and the peripheral ring was partly or completely lacking. When present, it had a 19-fold symmetry. The structures of the complexes in several pil mutants were determined. Structures obtained from the pilC1/C2 adhesin and the pilW minor pilin deletion strains were similar to wild-type, whereas deletion of the homologue of N. meningitidis PilW resulted in the absence of secretin structures. Remarkably, the pilE pilin subunit and pilP lipoprotein deletion mutants showed a change in the symmetry of the peripheral ring from 14 to 19 and loss of spikes. The pilF ATPase mutant also lost the spikes, but maintained 14-fold symmetry. These results show that secretin complexes contain previously unidentified large and flexible extra domains with a probable role in stabilization or assembly of type IV pili.     

Structural analysis of the pilE region of Neisseria gonorrhoeae P9

We have determined the nucleotide sequence of an expressed structural pilus gene (pilE) derived from Neisseria gonorrhoeae strain P9-2. Detailed analysis of nucleotide sequences upstream from pilE revealed a silent, truncated pilin gene segment that was linked to families of DNA elements (RS1 and RS3) that have previously been identified at the major silent pilin gene locus (pilS1) and at pilE of the independently isolated N. gonorrhoeae strain MS11ms. A nucleotide sequence downstream from pilE was reminiscent of the recognition sequences of several recombinases, including Tn3 tnpR product (resolvase), suggesting a possible role for site-specific events in the recombinational modulation of pilus expression.     

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.     

Sequence changes in the pilus subunit lead to tropism variation of Neisseria gonorrhoeae to human tissue

Summary
Pili of Neisseria gonorrhoeae are correlated with Increased bacterial attachment to epithelial cells and undergo both phase and antigenic variation. Phase variation of gonococcal pili can be brought about by recombination events in the pilin structural gene, pilE , or by the on/off switch in expression of PilC, a pilus biogenesis protein for which two loci exist. We have studied the binding to epithelial cell lines and to fixed tissue sections of N. gonorrhoeae MS11 derivatives and mutants carrying structurally defined PilE and PilC proteins, in situ binding studies of N. gonorrhoeae to formalin-fixed tissue sections resulted in a binding pattern similar to that obtained using viable epithelial cell lines of different origin. Piliated gonococcal clones, containing different pilE sequences, varied dramatically from one another in their efficiencies at binding to corneal and conjunctival tissue, but bound equally well to cervical and endometrial tissues. Further, the binding data suggested that PJIC expression by itself, i.e. without pili, cannot confer bacterial binding and that expression of either PilC1 or PiiC2 does not confer different binding properties to the bacterial cells. Possible receptors for piliated gonococci were expressed in human tissues, such as cervix, endometrium, cornea, intestine, stomach, mid-brain and meninges, but not in human kidney. Pretreatment of the target tissues with Proteinase K decreased the gonococcal binding dramatically, whereas pretreatment with neuraminidase and meta-periodate, which cleave carbon-carbon linkages between vicinal hydroxyl groups in carbohydrates, did not affect attachment of gonococci. These data argue that pilus-dependent attachment of N. gonorrhoeae to human tissue may be mediated by a eukaryotic receptor having protein characteristics, and that the pilus subunit sequence may play an important role in the interaction with human cornea.     

Identification and characterization of specific sequences encoding pathogenicity associated proteins in the genome of commensal Neisseria species

Abstract The distribution of distinct sequences in pathogenic and commensal Neisseria species was investigated systematically by dot blot analysis. Probes representing the genes of Rmp, pilin and IgA1 protease were found to hybridize exclusively to the chromosomal DNA of the pathogenic species, Neisseria gonorrhoeae and/or Neisseria meningitidis . In contrast, specific sequences for the genes of the porin protein Por and the opacity protein (Opa) were also detected in a panel of commensal Neisseria species such as N. lactamica, N. subflava, N, flava, N. mucosa and N. sicca . Using opa -specific oligonucleotides as probes in chromosomal blots, the genomes of the commensal Neisseria species show a totally reduced repertoire of cross-hybridizing loci compared to the complex opa gene family of N. gonorrhoeae . DNA sequence analysis of one opa -related gene derived from N. flava and N. sicca , respectively, revealed a large degree of homology with previously described gonococcal and meningococcal genes e.g., a typical repetitive sequence in the leader peptide and the distribution of the hypervariable and conserved regions. This observation, together with the finding, that the gene is constitutively transcribed, leads to the assumption that some of the commensal Neisseria species may have the potential for the expression of a protein harboring similar functions as the Opa proteins in pathogenic Neisseriae .     

Phase variation of gonococcal pili by frameshift mutation in pilC, a novel gene for pilus assembly.

Pili prepared from Neisseria gonorrhoeae contain minor amounts of a 110 kd outer membrane protein denoted PilC. The corresponding gene exists in two copies, pilC1 and pilC2, in most strains of N.gonorrhoeae. In the piliated strain MS11(P+), only one of the genes, pilC2, was expressed. Inactivation of pilC2 by a mTnCm insertion resulted in a nonpiliated phenotype, while a mTnCm insertion in pilC1 had no effect on piliation. Expression of pilC was found to be controlled at the translational level by frameshift mutations in a run of G residues positioned in the region encoding the signal peptide. Nonpilated (P-), pilin expressing colony variants that did not express detectable levels of PilC were selected; all P+ backswitchers from these P-, PilC- clones were found to be PilC+. The structural gene for pilin, pilE, was sequenced and found to be identical in one P-, PilC- and P+, PilC+ pair. Most PilC- cells were completely bald whereas the PilC+ backswitcher had 10-40 pili per cell. Thus, a turn ON and turn OFF in the expression of PilC results in gonococcal pili phase variation. These results suggest that PilC is required for pilus assembly and/or translocation across the gonococcal outer membrane.     

The Use of High-Throughput DNA Sequencing in the Investigation of Antigenic Variation: Application to Neisseria Species

Antigenic variation occurs in a broad range of species. This process resembles gene conversion in that variant DNA is unidirectionally transferred from partial gene copies (or silent loci) into an expression locus. Previous studies of antigenic variation have involved the amplification and sequencing of individual genes from hundreds of colonies. Using the pilE gene from Neisseria gonorrhoeae we have demonstrated that it is possible to use PCR amplification, followed by high-throughput DNA sequencing and a novel assembly process, to detect individual antigenic variation events. The ability to detect these events was much greater than has previously been possible. In N. gonorrhoeae most silent loci contain multiple partial gene copies. Here we show that there is a bias towards using the copy at the 3′ end of the silent loci (copy 1) as the donor sequence. The pilE gene of N. gonorrhoeae and some strains of Neisseria meningitidis encode class I pilin, but strains of N. meningitidis from clonal complexes 8 and 11 encode a class II pilin. We have confirmed that the class II pili of meningococcal strain FAM18 (clonal complex 11) are non-variable, and this is also true for the class II pili of strain NMB from clonal complex 8. In addition when a gene encoding class I pilin was moved into the meningococcal strain NMB background there was no evidence of antigenic variation. Finally we investigated several members of the opa gene family of N. gonorrhoeae, where it has been suggested that limited variation occurs. Variation was detected in the opaK gene that is located close to pilE, but not at the opaJ gene located elsewhere on the genome. The approach described here promises to dramatically improve studies of the extent and nature of antigenic variation systems in a variety of species.     

Genetic analysis of variant pilin genes from Neisseria gonorrhoeae P9 cloned in Escherichia coli: physical and immunological properties of encoded pilins

A series of genomic DNA fragments that encode gonococcal pilins from four well-characterized pilus variants of Neisseria gonorrhoeae strain P9 have been cloned in Escherichia coli K12. At least nine classes of cloned P9 pilin genes have been identified on the basis of restriction mapping of cloned pilin-encoding DNA and physical and immunological analysis of expressed pilin proteins. Each antigenic variant of strain P9 possesses many genomic segments of pilin gene information, although our results suggest that strain P9 contains only a single pilin-expressing (pilE) locus.     

Sequence analysis and relationships between meningococcal class 3 serotype proteins and other porins from pathogenic and non-pathogenic Neisseria species

The presence of highly conserved regions within previously determined porin gene sequences from Neisseria meningitidis and Neisseria gonorrhoeae permitted the construction of oligonucleotide primers for PCR amplification of other neisserial porin genes. Although two separate porin genes (porA and porB) are present in N. meningitidis only a single fragment, corresponding to porB, could be amplified from this species. The amplified porB genes from four different meningococcal serotypes, which express the class 3 outer membrane protein, were sequenced. Amplified fragments corresponding to porin genes from N. lactamica and N. sicca were also sequenced. In common with the known neisserial porins, models of the organisation of the predicted proteins indicated trans-membrane structures with eight surface exposed loops. In the meningococcal class 3 proteins the main regions of sequence variation, which must be responsible for serotype specificity, were located on loops 5 and 7. A phylogenetic analysis of the family of porins from the Neisseria confirmed the close relationship of the meningococcal class 3 protein with the gonococcal PIA protein, while the gonococcal PIB protein was shown to be closely related to the N. lactamica porin. The close relationship seen between porins of the pathogenic and non-pathogenic Neisseriae identified no obvious virulence-associated regions in the proteins, but did suggest that the current nomenclature for neisserial porin genes may need reviewing.