Identification of two major families of transferrin receptors among Neisseria meningitidis strains based on antigenic and genomic features

Abstract The transferrin receptor or transferrin-binding proteins (Tbps) of 50 strains of Neisseria meningitidis belonging to different serogroups were examined by Western blotting using two rabbit antisera raised against Tbp purified from N. meningitidis strains B16B6 and M982. On the basis of the reactivity of Tbp2 with the antisera two patterns were observed and allowed the classification of 74% of the strains in group I (M982-like strains) and 26% in group II (B16B6-like strains). Southern blot analysis was performed on the genomic DNA of 16 meningococcal strains and showed that under stringent conditions, the tbp2 probes were specific for each group identified. Both immunological and genomic analyses have led to the identification within N. meningitidis strains of two major families distinguished on the basis of the characteristics of Tbp2 molecules, independently of serogroup, type or subtype.     

Identification of variable region differences in Neisseria meningitidis class 3 protein sequences among five group B serotypes

Strains of Neisseria meningitidis express one of two porin proteins. These porins have been identified as the class 2 and class 3 proteins, and express serotype-specific epitopes. The gene for the class 3 protein was amplified by the polymerase chain reaction from the DNA of a serotype 4 strain as a 1025 bp fragment. The nucleotide sequence of this gene was determined and compared with two recently published sequences. On the basis of this comparison we have identified two major variable regions in the translated protein sequence, VR1 and VR2, that may be associated with serotype specificity. Three other variable regions were also identified. The sequences in the VR1 and VR2 regions from five additional group B N. meningitidis strains of serotypes 1, 4, 8, 12, and 15, all expressing class 3 proteins, were determined. The VR1 and VR2 regions were variable and were flanked by highly conserved regions among eight different class 3 sequences. These two variable regions of 15 and 9 amino acids are predicted to be in surface-exposed loops.     

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.     

Transformational exchanges in the dihydropteroate synthase gene of Neisseria meningitidis: a novel mechanism for acquisition of sulfonamide resistance.

The nucleotide sequences of the chromosomal dihydropteroate synthase (dhps) genes in sulfonamide-susceptible and sulfonamide-resistant strains of Neisseria meningitidis of serogroups A, B and C were determined. The molecular weights and the amino acid sequences showed similarity to those of all other known dihydropteroate synthase polypeptides. Sequence comparison of the N. meningitidis dhps genes indicated horizontal transfer of DNA segments rather than point mutations as the cause for resistance in meningococci. The dhps genes in three of four sulfonamide-resistant meningococci contained identical central regions of 424 bp. Compared with the corresponding genes in susceptible strains, each central region included an insert of 6 bp. In one of the sulfonamide-resistant strains, the dhps gene was similar to the corresponding genes in the sensitive strains in its NH2-terminal and C-terminal parts. Its central region, however, was identical to the corresponding regions of two of the other resistant genes, and thus it could be seen as a hybrid dhps gene. Transformation experiments and mapping of transformed dhps genes indicated the existence of a novel mechanism for the dissemination of sulfonamide resistance in N. meningitidis. The origin of the resistance-mediating segment of the gene is unknown, but hybridization results showed the presence of homologous dhps genes in Neisseria gonorrhoeae and N. lactamica but not in N. subflava or Branhamella catarrhalis.     

A novel interpretation of structural dot plots of genomes derived from the analysis of two strains of Neisseria meningitidis

Neisseria meningitidis is the agent of invasive meningococcal disease, including cerebral meningitis and septicemia. Because the diseases caused by different clonal groups (sequence types) have their own epidemiological characteristics, it is important to understand the differences among the genomes of the N. meningitidis clonal groups. To this end, a novel interpretation of a structural dot plot of genomes was devised and applied; exact nucleotide matches between the genomes of N. meningitidis serogroup A strain Z2491 and serogroup B strain MC58 were identified, leading to the specification of various structural regions. Known and putative virulence genes for each N. meningitidis strain were then classified into these regions. We found that virulence genes of MC58 tend more to the translocated regions (chromosomal segments in new sequence contexts) than do those of Z2491, notably tending towards the interface between one of the translocated regions and the collinear region. Within the col-linear region, virulence genes tend to occur within 16 kb of gaps in the exact matches. Verification of these tendencies using genes clustered in the cps locus was sufficiently supportive to suggest that these tendencies can be used to focus the search for and understanding of virulence genes and mechanisms of pathogenicity in these two organisms.     

Cloning and partial sequence of transferrin-binding protein 2 of Neisseria meningitidis using a novel method: Twin N-terminal PCR

Abstract The genes encoding transferrin-binding proteins (TBPs) 1 and 2 of Neisseria meningitidis and N. gonorrhoeae were used as model loci in a novel method of cloning (twin N-terminal polymerase chain reaction; TNT-PCR) involving amplification between the 5' ends of two genes. Primers were based on N-terminal amino-acid sequences. A 2.1-kb product amplified from N. meningitidis strain SD (B15 P1.16) was cloned into a plasmid vector and partially sequenced. Translated sequence immediately downstream of the primer at both ends of this product correlated to the additional known N-terminal amino acids of TBP-1 and 2. The protein encoded by the cloned sequence reacted with TBP-2-specific antiserum. The size of products generated in TNT-PCR correlated exactly with the different sized TBP-2 produced by 10 strains of the Neisseria spp. examined, indicating successful cloning of the gene for TBP-2 and showing it to be adjacent to and preceding TBP-1 on the chromosome for both N. meningitidis and N. gonorrhoeae .     

Sequence diversity within the argF, fbp and recA genes of natural isolates of Neisseria meningitidis: interspecies recombination within the argF gene

Studies of natural populations of Neisseria meningitidis using multilocus enzyme electrophoresis have shown extensive genetic variation within this species, which, it has been proposed, implies a level of sequence diversity within meningococci that is greater than that normally considered as the criterion for species limits in bacteria. To obtain a direct measure of the sequence diversity among meningococci, we obtained the nucleotide sequences of most of the argF, recA and fbp genes of eight meningococci of widely differing electrophoretic type (from the reference collection of Caugant). Sequence variation between the meningococcal strains ranged from 0-0.6% for fbp, 0-1.3% for argF, and 0-3.3% for recA. These levels of diversity are no greater than those found within Escherichia coli 'housekeeping' genes and suggest that multilocus enzyme electrophoresis may overestimate the extent of nucleotide sequence diversity within meningococci. The average sequence divergence between the Neisseria meningitidis strains and N. gonorrhoeae strain FA19 was 1.0% for fbp and 1.6% for recA. The argF gene, although very uniform among the eight meningococcal isolates, had a striking mosaic structure when compared with the gonococcal argF gene: two regions of the gene differed by greater than 13% in nucleotide sequence between meningococci and gonococci, whereas the rest of the gene differed by less than 1.7%. One of the diverged regions was shown to have been introduced from the argF gene of a commensal Neisseria species that is closely related to Neisseria cinerea. The source of the other region was unclear.     

Genes associated with meningococcal capsule complex are also found in Neisseria gonorrhoeae.

A homolog of the meningococcal cps locus region E has been identified in Neisseria gonorrhoeae immediately upstream of the gonococcal region D locus. Region E has no detectable function in capsule biosynthesis in Neisseria meningitidis or in lipopolysaccharide biosynthesis in either organism. The open reading frame is homologous to proteins of unknown function in Escherichia coli and Haemophilus influenzae. Further analysis of the N. meningitidis cps cluster has identified a second copy of region D encoding three additional open reading frames, including homologs of DNA methyltransferases. The organization of the region D and E genes in N. gonorrhoeae and N. meningitidis in relation to the cps genes provides some insight into the evolutionary origin of encapsulation in N. meningitidis.     

Structure of the C-terminal domain of Neisseria heparin binding antigen (NHBA), one of the main antigens of a novel vaccine against Neisseria meningitidis

Neisseria heparin binding antigen (NHBA), also known as GNA2132 (genome-derived Neisseria antigen 2132), is a surface-exposed lipoprotein from Neisseria meningitidis that was originally identified by reverse vaccinology. It is one the three main antigens of a multicomponent vaccine against serogroup B meningitis (4CMenB), which has just completed phase III clinical trials in infants. In contrast to the other two main vaccine components, little is known about the origin of the immunogenicity of this antigen, and about its ability to induce a strong cross-bactericidal response in animals and humans. To characterize NHBA in terms of its structural/immunogenic properties, we have analyzed its sequence and identified a C-terminal region that is highly conserved in all strains. We demonstrate experimentally that this region is independently folded, and solved its three-dimensional structure by nuclear magnetic resonance. Notably, we need detergents to observe a single species in solution. The NHBA domain fold consists of an 8-strand β-barrel that closely resembles the C-terminal domains of N. meningitidis factor H-binding protein and transferrin-binding protein B. This common fold together with more subtle structural similarities suggest a common ancestor for these important antigens and a role of the β-barrel fold in inducing immunogenicity against N. meningitidis. Our data represent the first step toward understanding the relationship between structural, functional, and immunological properties of this important vaccine component.     

PilC of pathogenic Neisseria is associated with the bacterial cell surface

Adherence of pathogenic Neisseria to target host cells is mediated by pili. PilC1 and PilC2 are two high-molecular-weight proteins involved in pilus assembly and cellular adherence functions of the pili. Inactivation of pilC1 or pilC2 in N. meningitidis resulted in clones that expressed the same number of pili as the parent, contained no alterations in pilE and showed no detectable differences in PilE glycosylation. However, the PilC2+ pilC1- mutant showed much reduced adherence to target cells, indicating that production of PilC1 is essential for pilus-mediated adherence. To study further the functional differences between the meningococcal pilC genes, we determined the complete nucleotide sequence of pilC1 and pilC2 of N. meningitidis. Alignment of six PilC sequences demonstrated that PilC is composed of both conserved and variable regions. By immunogold labelling of bacterial sections we showed that PilC is present in the membranes of both piliated and non-piliated bacteria. Further, we demonstrated that PilC is associated with the bacterial cell surface.     

Genotypic characterization of Neisseria meningitidis using pyrosequencing™

Pyrosequencing involves the synthesis of single-stranded deoxyribonucleic acid leading to rapid and accurate analysis of nucleotide sequences. This article describes the development of typing assays for the characterization of Neisseria meningitidis using Pyrosequencing. This involved developing methods for the nucleotide sequence analysis of important variable regions contained on a major capsule gene and on outer membrane protein genes that are used for grouping, typing, and subtyping meningococci. To achieve this, primers were designed for amplification of three genes, siaD, porB, and porA from the four main serogroups B, C, Y, and W135. To facilitate throughput and reproducibility, the method was also automated. Data from 717 isolates have shown that Pyrosequencing can be used for the single nucleotide polymorphism and sequence-analysis characterization of meningococci.     

Characterization of the opa (class 5) gene family of Neisseria meningitidis

Class 5 outer membrane proteins of Neisseria meningitidis show both phase- and antigenic variation of expression. The proteins are encoded by a family of opa genes that share a conserved framework interspersed with three variable regions, designated the semivariable (SV) region and hypervariable regions 1 (HV1) and 2 (HV2). In this study, we determined the number and DNA sequence of all of the opa genes of meningococcal strain FAM18, to assess the structural and antigenic variability in the family of proteins made by one strain. Pulsed field electrophoresis and Southern blotting showed that there are four opa genes in the FAM18 chromosome, and that they are not tightly clustered. DNA sequence analysis of the four cloned genes showed a modest degree of diversity in the SV region and more extensive differences in the HV1 and HV2 regions. There were four versions of HV1 and three versions of HV2 among the four genes. Each of the FAM18 opa loci contained a gene with a unique combination of SV, HV1, and HV2 sequences. We used lambda gt11 cloning and synthetic peptides to demonstrate that HV2 sequences completely encode the epitopes for two monoclonal antibodies specific for different class 5 proteins of FAM18.     

Antigenic and molecular heterogeneity of the transferrin-binding protein of Neisseria meningitidis

The transferrin-binding protein in 35 Neisseria meningitidis isolates was examined using a binding assay involving 125I-transferrin. The results show that most strains have a binding protein with a Mr between 78 kDa and 83 kDa; only 4 strains had a binding protein with a Mr of about 68 kDa. The side of the protein appears unrelated to the serogroup or serotype of the organism. Using antibodies raised to whole cells of N. meningitidis grown under iron restriction we show also that considerable antigenic heterogeneity exists amongst the transferrin-binding proteins. This makes it a less than promising vaccine candidate antigen, although conserved antigenic domains are now being sought.     

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.     

Characterization of a highly structured domain in Tbp2 from Neisseria meningitidis involved in binding to human transferrin.

The binding of iron-loaded human transferrin at the surface of Neisseria meningitidis is mediated by two polypeptides, Tbp1 and Tbp2. Predicted Tbp amino acid sequences from N. meningitidis strains are highly divergent. This variability is particularly pronounced throughout the Tbp2 polypeptide. In this study, a highly structured and extremely stable Tbp2 domain of about 270 to 290 amino acids which is involved in the binding to transferrin and whose position is well conserved has been characterized. The conservation of such a remarkable structure in a very divergent protein domain (there is only 43% amino acid identity within this region) suggests that is plays an essential biological role and raises a number of questions regarding tbp2 evolution.     

Identification of sequences in human transferrin that bind to the bacterial receptor protein, transferrin-binding protein B

Alignment of amino-acid sequences from the N-terminal and C-terminal halves of transferrin-binding protein B revealed an underlying bilobed nature with several regions of identity. Based on this analysis, purified recombinant fusion proteins of maltose-binding protein (Mbp) with intact TbpB, its N-terminal half or C-terminal half from the human pathogens Neisseria meningitidis and Moraxella catarrhalis were produced. Solid-phase binding assays and affinity isolation assays demonstrated that the N-terminal and C-terminal halves of TbpB could bind independently to human transferrin (hTf). A solid-phase overlapping synthetic peptide library representing the amino-acid sequence of hTf was probed with soluble, labelled Mbp-TbpB fusions to localize TbpB-binding regions on hTf. An essentially identical series of peptides from domains within both lobes of hTf was recognized by intact TbpB from both organisms, demonstrating a conserved TbpB-hTf interaction. Both halves of TbpB from N. meningitidis bound the same series of peptides, which included peptides from equivalent regions on the two hTf lobes, indicating that TbpB interacts with each lobe of hTf in a similar manner. Mapping of the peptide-binding regions on a molecular model of hTf revealed a series of nearly adjacent surface regions that nearly encircled each lobe. Binding studies with chimeric hTf/bTf transferrins demonstrated that regions in the C-lobe of hTf were preferentially recognized by the N-terminal half of TbpB. Collectively, these results provide evidence that TbpB consists of two lobes, each with distinct yet homologous Tf-binding regions.     

Iron piracy: acquisition of transferrin-bound iron by bacterial pathogens

The mechanism of iron utilization from transferrin has been most extensively characterized in the pathogenic Neisseria species and Haemophilus species. Two transferrin-binding proteins, Tbp1 and Tbp2, have been identified in these pathogens and are thought to be components of the transferrin receptor. Tbp1 appears to be an integral, TonB-dependent outer membrane protein while Tbp2, a lipoprotein, may be peripherally associated with the outer membrane. The relative contribution of each of these proteins to transferrin binding and utilization is discussed and a model of iron uptake from transferrin is presented. Sequence comparisons of the genes encoding neisserial transferrin-binding proteins suggest that they are probably under positive selection for variation and may have resulted from inter-species genetic exchange.     

Sulfonamide resistance in Neisseria meningitidis as defined by site-directed mutagenesis could have its origin in other species.

Sulfonamide resistance in Neisseria meningitidis is mediated by altered forms of the chromosomal gene for the drug target enzyme dihydropteroate synthase. Sulfonamides have been used for decades both for prophylaxis and the treatment of meningococcal disease, and resistance is common. Two types of resistance determinants have been identified, and regions important for drug insusceptibility to the corresponding enzyme have been defined by site-directed mutagenesis. Both types of resistance traits have spread among strains of N. meningitidis of different serogroups and serotypes, and the large differences at the nucleotide level in a comparison of the resistance genes with the dhps genes of susceptible meningococci indicate the origin of one or maybe both types in other Neisseria species. One sulfonamide-sensitive strain of N. meningitidis was found to have a mosaic dhps gene with a central part identical to the corresponding part of a gonococcal strain. This observation supports the idea of an interspecies transfer of genetic material in Neisseria species as a mechanism for the development of chromosomally mediated resistance.     

Molecular analysis of region 1 of the Escherichia coli K5 antigen gene cluster: a region encoding proteins involved in cell surface expression of capsular polysaccharide.

The nucleotide sequence of region 1 of the K5 antigen gene cluster of Escherichia coli was determined. This region is postulated to encode functions which, at least in part, participate in translocation of polysaccharide across the periplasmic space and onto the cell surface. Analysis of the nucleotide sequence revealed five genes that encode proteins with predicted molecular masses of 75.7, 60.5, 44, 43, and 27 kDa. The 27-kDa protein was 70.7% homologous to the CMP-2-keto-3-deoxyoctulosonic acid synthetase enzyme encoded by the E. coli kdsB gene, indicating the presence of a structural gene for a similar enzyme within the region 1 operon. The 43-kDa protein was homologous to both the Ctrb and BexC proteins encoded by the Neisseria meningitidis and Haemophilus influenzae capsule gene clusters, respectively, indicating common stages in the expression of capsules in these gram-negative bacteria. However, no homology was detected between the 75.7, 60.5-, and 44-kDa proteins and any of the proteins so far described for the H. influenzae and N. meningitidis capsule gene clusters.     

In vivo human immune response to transferrin-binding protein 2 and other iron-regulated proteins of Neisseria meningitidis

Abstract When grown under iron restriction, Neisseria meningitidis expresses new outer-membrane proteins, some of which are antigenic and potentially useful as vaccine components. This is particularly relevant to N. meningitidis serogroup B, against which neither polysaccharide nor conjugate vaccines are effective. We investigated recognition of N. meningitidis serogroup B outer-membrane antigens by three sera from patients recovered from meningitis. Recognition of antigens from the homologous strain provided information on in vivo expression during infection and immunogenicity, while cross-reactivity with outer membrane proteins from the other two strains and from another five strains in our collection allowed evaluation of antigenic heterogeneity. Our results demonstrate that transferrin-binding protein 2 (TBP2) is immunogenic in humans, to varying degrees depending on the strain, and that TBP2s (like the equivalent proteins of Haemophilus influenzae type b) are among the most important iron-regulated outer membrane antigens expressed during infection. Other immunogenic outer membrane proteins (some iron-regulated) are also expressed during infection; in a previous study in mouse, three of these proteins (with M _r of 50, 70 and 77 kDa) did not induce an immune response. Our cross-reactivity data provide some support for Robki et al.'s two-group classification of N. meningitidis strains, and provide evidence against the possibility that the antigenic domains shared by the TBP2s of all N . meningitidis strains induce immune responses in vivo.