*The Peter Medawar Building for Pathogen Research and Department of Zoology, University of Oxford;
Department of Zoology, University of Oxford;
Meningococcus Reference Unit, Public Health Laboratory, Withington Hospital, Manchester;
Department of Biomolecular Sciences, University of Manchester Institute of Science and Technology
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Abstract |
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Introduction |
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The meningococcus is an appropriate model system to evaluate this approach because it is a pathogen of global significance which is genetically and antigenically diverse and for which no comprehensive vaccine exists (Pollard and Frasch 2001
). Further, large genetically defined isolate collections have been assembled and models of the population biology of this organism are available (Caugant et al. 1987
; Maiden et al. 1998
). Amongst the candidate vaccine components proposed are the variable outer membrane proteins, the trimeric porins, which act as pores for the passage of solutes into the cell (Tommassen et al. 1990
). These molecules are targeted by the host immune response and have been used in meningococcal typing schemes (Bjune et al. 1991a
, 1991b
; Sierra et al. 1991
; van der Ley and Poolman 1992
; van der Ley, van der Biezen, and Poolman 1995
). Unlike most other Neisseria species, the meningococcus expresses two porins, PorA and PorB. Expression of PorA is regulated at transcription and exhibits three levels depending on the length of the polyguanidine stretch in the promoter region of the porA gene (van der Ende et al. 1995
), whereas there is no evidence to suggest that PorB proteins are subject to phase variation. Mutant meningococcal strains that lack PorB do not grow well (Tommassen et al. 1990
), suggesting that PorB has a function essential for growth. PorB is also capable of translocating vectorially into the membranes of mammalian cells (Blake and Gotschlich 1987
), and of binding ATP and GTP, which down regulates pore size and alters voltage dependence and ion selectivity (Rudel et al. 1996
). These functional and structural characteristics are thought to influence the early stages of neutrophil activation and therefore implicate the PorB protein in meningococcal pathogenesis (Rudel et al. 1996
).
A PorB topology model has been constructed on the basis of nucleotide sequence data (Maiden et al. 1991
; van der Ley et al. 1991
) and, more recently, the structural similarity between the Neisseria porins and the Escherichia coli porins OmpF and PhoE has been exploited to generate a three-dimensional homology model for Neisseria porins (Derrick et al. 1999
). These models predicted eight surface exposed "loops" interspersed with highly conserved outer membrane-spanning sequences that formed a "ß-barrel" (Kleffel et al. 1985
). The antigenically variable epitopes targeted in the host immune response (Saukkonen et al. 1989
) were proposed to reside in the surface-exposed loops (McGuinness et al. 1990
; Maiden et al. 1991
). Serological and molecular characterization of the meningococcal porins have been used for epidemiological analyses of meningococcal carriage and disease (Frasch, Zollinger, and Poolman 1985
; Poolman et al. 1986
; Maiden et al. 1991
), although the variability of these proteins means that they are not always reliable epidemiological markers (Achtman 1995
; Urwin et al. 1998a
, 1998b
).
The meningococcus possesses one of two PorB protein classes, PorB2 or PorB3, which are encoded by alternate allele classes present at the porB locus. Phylogenetic analyses show that the porB3 gene is most closely related to one of the gonococcal porin genes, porB1a (Smith, Maynard Smith, and Spratt 1995
) and that these gene sequences form a clade together with gonococcal porB1b, N. lactamica por, and N. polysaccharea por gene sequences (Derrick et al. 1999
). The meningococcal porB2 gene shares sequence similarity both with members of this clade (specifically, the sequence encoding the putative ATP and GTP binding site) and with the porin genes of most human commensal and animal Neisseria species, suggesting that porB2 may have arisen because of interspecies recombination (Derrick et al. 1999
). Indeed, the incongruence between phylogenetic trees drawn for individual loop-encoding regions of porB3 genes (Bash et al. 1995
) suggests that inter- and intraspecies recombination is a mechanism that increases genetic variation among porB alleles.
Previous comparisons of the rates and distribution of synonymous (dS) and nonsynonymous (dN) substitutions among meningococcal porin genes have concluded that, unlike the gonococcal PIA and PIB porins which were under positive selection in the surface loop regions, meningococcal porB genes were subject to only weak positive selection and purifying selection (Smith, Maynard Smith, and Spratt 1995
). This observation supports the hypothesis that PorB is a less important vaccine constituent than the PorA protein. This conclusion was also drawn from studies of the bactericidal activity of monoclonal (Saukkonen et al. 1987
) and polyclonal (van der Ley and Poolman 1992
) antibodies in mice and immunological results from human vaccine trials (Rosenqvist et al. 1995
; Perkins et al. 1998
). In addition, some PorB3 proteins expressed on the surfaces of live meningococci have been reported to be poorly accessible for antibody binding (Michaelsen et al. 2001
). Consequently, PorB has been deliberately excluded from some vaccine formulations (van der Ley, van der Biezen, and Poolman 1995
). But the previous selection analysis was conducted on very small data sets, and dS and dN were estimated as mean values across whole or partial gene sequences, making it possible that strongly selected sites were missed in this broadscale comparison (Smith, Maynard Smith, and Spratt 1995
). More recently, phylogenetic analyses carried out on a larger set of gonococcal porin gene sequences concluded that there were differences in the evolution of PIA and PIB homology groups, with positive selection driving evolution of the PIA proteins and both positive and purifying selection acting on PIB protein sequences (Posada et al. 2000
).
In this work we undertook rigorous maximum likelihood analyses of selection pressures acting on a large set of meningococcal PorB sequences. A likelihood-based approach was also used to determine with more accuracy the extent of recombination in porB2 and porB3. Our study reveals that both genes are subject to exceptionally high rates of positive selection, as well as frequent recombination, which has important implications for the use of PorB as a potential vaccine candidate.
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Materials and Methods |
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For de novo sequencing, the propagation of isolates, preparation of DNA, porB gene amplification, and nucleotide sequence determination were as described previously (Feavers et al. 1999
; Urwin et al. 1998a
). The sequences were assembled with the Staden sequence analysis package (Staden 1996
) and all sequences aligned manually in the Seqlab alignment program (Genetics Computer Group, Madison, Wis.) (Devereux, Haeberli, and Smithies 1984
). The sequence alignment was trimmed at 5'- and 3'-ends so that all sequences began at the 5'-end with the thirteenth codon of the sequence encoding the mature protein (GAA in all sequences) and ended seven codons from the 3'-end of this sequence (ATG in porB2; GGT in porB3) because this corresponded to the length of the shortest sequences in the data set. Calculation of the number of nucleotide differences between pairs of alleles was determined using MEGA version 1.01 (Kumar, Tamura, and Nei 1994
). The porB allele sequences and alignments can be viewed at http://neisseria.org/typing/porb. There were 125 unique porB allele sequences, 46 of which were porB2 sequences (named porB2-1 to porB2-46, according to a previously defined nomenclature [Feavers and Maiden 1998
]) and 79, which were porB3 sequences (porB3-1 to porB3-79). Previously unpublished sequences have been submitted to GenBank, accession numbers AF520356AF520416.
Analysis of Selection Pressures
A maximum likelihood (ML) approach was used to examine selection pressures acting on the meningococcal porB genes. Here, dN and dS were examined codon-by-codon, using different models of codon substitution that differed in how dN/dS ratios (parameter ) varied along sequences, as well as incorporating information about the phylogenetic relationships of the sequences in question so that comparisons are independent (Yang et al. 2000
). Model M0 estimated a single
parameter for all sites, whereas the M1 model divided codons into conserved sites (p0), with
0 fixed at 0 and neutral sites (p1) with
1 set to 1. The M2 model could account for positive selection through a third category of sites (p2) with
2 estimated from the data. M3 provided a more sensitive test by estimating, from the data,
values for three classes of site all of which could be >1. The M7 and M8 models both used a discrete beta distribution (with 10 categories and described by parameters p and q) to model
ratios among sites, although M8, unlike M7, considered an extra class of sites for which
could be >1. Nested models could be compared using a likelihood ratio test (LRT) in which twice the difference in log likelihood between models was compared with the value obtained under a
2 distribution (degrees of freedom equal to the difference in the number of parameters between models). Finally, Bayesian methods were used to determine the probability that a particular codon site fell into the positively selected class. All these analyses used the CODEML program from the PAML package (Yang 1997
).
Phylogenetic trees for the two data sets were constructed using the maximum likelihood method available in the PAUP* package (Swofford 1998
). The HKY85 model of nucleotide substitution was used, with values for both the transition-transversion ratio and the shape parameter (
) of a gamma distribution of rate variation among sites (with eight categories) estimated during tree reconstruction (trees and parameter values available on request).
Recombination Analysis
The extent of recombination in the porB sequence data was analyzed by assessing the degree of phylogenetic congruence. Because of the large numbers of sequences available, this analysis was performed on a set of 35 randomly sampled porB2 and porB3 alleles. The porB2 and porB3 alignments were first split into two equal-sized fragments. ML phylogenetic trees were then estimated for both halves of the alignments using the procedures described above. To establish whether the trees constructed on each half of the alignment were significantly different in topology, as might be expected given frequent recombination, the difference in log likelihood () between the ML tree for the first half of the gene and the ML tree topology for the second half of the gene fitted to the data from the first half, but with branch lengths reoptimized, were compared. The significance of the likelihood differences was assessed using two randomization tests. The first test used Monte Carlo simulation on 100 replicate data sets simulated under the ML model parameters using the program Seq-Gen (Rambaut and Grassly 1997
). Maximum likelihood trees were then constructed for each of these simulated data sets, using the procedures described above, and their likelihoods compared with those of ML topology on each data set. If the
values for the real data fell within this null distribution of
values, then the trees constructed for each half of the gene were not significantly different in topology. In the second test, 200 random trees were created using PAUP*. The likelihoods of these trees were then estimated on the data from the first half of the porB2 and porB3 alignments, again with the reoptimization of branch lengths, and the
values between these random trees and the two ML trees were then compared. If the
values for the two ML trees fall within the 99th percentile of this null distribution then we may say that they are no more similar than two random trees inferred from these data (Holmes, Urwin, and Maiden 1999
).
Structural Models
Structural models for the PorB2 and PorB3 proteins were generated using the software package Modeller (Sali et al. 1995
), using the crystal structure of the porin Omp32 from Comamonas acidovorans as a template (Zeth et al. 2000
; PDB accession 1E54). The sequence alignment was based on that given by Zeth et al. for PorB, with minor modifications.
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Results |
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Analysis of Selection Pressures Acting on the porB Genes of N. meningitidis
The maximum likelihood analysis of the selection pressures acting on the porB2 and porB3 alleles provided strong evidence for positive selection (tables 2
and 3
). For both porB2 and porB3, the best-supported M3 model estimated (dN/dS) parameters >>1, indicative of strong positive selection. Similarly, the M8 model, which could incorporate positive selection, was significantly favored over the M7 model which did not, and estimated
>> 1 for both porB2 and porB3. The strength of the inferred selection pressures acting on both porB2 and porB3 was also striking, as was the similarity in selection pressures between these allele classes. For the M3 model, two classes of positively selected sites were apparent. In the case of porB2
4.5% of sites fell into a relatively weakly positively selected class, where
0 = 4.163, whereas
1.1% of sites are seemingly subject to very strong positive selection with
2 = 18.553. The remaining 94% of sites were highly conserved (
1 = 0.067). A similar distribution of sites was apparent in porB3. Here,
4.6% of sites had an
value of 3.229, whereas 0.7% of sites were subject to much stronger positive selection pressures with
2 = 13.923. In the case of porB3, 95% of sites were subject to strong selective constraints (
1 = 0.033).
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Discussion |
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In addition, the analysis also revealed that although the PorB2 and PorB3 proteins were subject to very similar selection pressures they exhibited different distributions of positively selected sites. These findings were supported by flow cytometry analysis of live meningococci which demonstrated that some PorB3 variants were not easily accessible for antibody binding (Michaelsen et al. 2001
), possibly because of shielding or due to PorB3 extracellular loops being shorter than those of PorB2, so that fewer residues were subject to intense immune selection. In contrast, the gonococcal porins PIA and PIB showed no significant difference in the distribution of selected sites despite reported differences in selection intensities between the two homology groups (Posada et al. 2000
). Posada et al. therefore concluded that epidemiological differences between gonococci expressing PIA or PIB proteins were responsible for differences in selection rather than structural differences in the proteins. In meningococci, however, it is possible that the differences in the lengths of the surface loops of PorB2 and PorB3 porins are sufficient to affect the conformation and structure of epitopes presented by these proteins and that this will determine which sites are exposed to selective pressure from the host responses. Furthermore, although there may be some epidemiological differences between meningococci expressing PorB2 and PorB3 proteins, there is no evidence from these data that this has led to concomitant selective differences, although additional studies are required to test this hypothesis further.
The conformational effects of particular amino acid substitutions on PorB structure remain difficult to determine. The homology models used were useful for examining the approximate disposition of residues in space and could provide insights not readily apparent from a sequence alignment. The crystal structure of Omp32 from C. acidovorans was used as the basis for the homology models of PorB2 and PorB3; this is a closer homolog to the Neisseria porins than the E. coli porin crystal structures that were used previously (Derrick et al. 1999
). But the size of most of the external loop regions precluded an accurate estimation of their conformations by standard homology modeling techniques, and the location of selected residues shown in external loop regions in figure 2
were approximate.
Figures 1
and 2
illustrate that the strongly and weakly selected sites are not distributed evenly across the loop regions. The presence of conserved residues at the apices of the PorB2 variable loops I, IV, and V suggested that these amino acids were not exposed to the host immune response because of protein folding, or perhaps they fulfill an important role retaining the surface loop structure. For example, the L2 loop in the OmpF protein of E. coli contributes to the stabilization of the porin trimer (Phale et al. 1998
) and residues in other loop regions could play analogous roles in stabilizing the protein, using loop-loop interactions. Indeed, although the portions of polypeptide chain joining the ends of the ß-barrel strands are frequently referred to as loops, they are likely to contain regions of regular secondary structure, as is seen in other porin structures (Koebnik, Locher, and Van Gelder 2000
), and this would place constraints on the sequence variation within these regions in PorB2 and PorB3 proteins. These observations are borne out by the difficulty in mimicking PorB epitopes with linear peptides (Zapata et al. 1992
) and by sequence variation identified among serologically similar PorB antigens (Urwin et al. 1998a
, 1998b
).
Although most residues within the transmembrane ß-strands of PorB2 were highly conserved, five positively selected sites were located among these structural regions. According to the structural model, two of these sites (positions 39 and 43 in fig. 1a
) were located close to one another on opposite sides of a ß-turn and were identified as positively selected with >99% probability (although falling into the weakly rather than the strongly positively selected class). A leucine residue at position 39 was invariably accompanied by a methionine residue at position 43, whereas the phenylalanine residue at position 39 was accompanied by the smaller hydrophobic residues valine or isoleucine at position 43. Within the limitations of the homology model, the side chains of the two residues may be in contact, suggesting that these are compensatory mutations. Elsewhere, the reasons for strong positive selection within the ß-barrel region were more difficult to discern. The PorB2 protein contained a number of mutations within the L3 loop, which folds back into the center of the barrel creating a constricted channel for the passage of low molecular weight solutes (Zeth et al. 2000
). Residues within the L3 loop were unlikely to be subject to immune selection but are probably involved in ion selectivity: the presence of a number of mutations which resulted in changes in charge within this region of the protein are consistent with this idea (Bauer et al. 1989
; Benz et al. 1989
; Saint et al. 1996
; Schirmer and Phale 1999
).
Despite the extensive evidence for recombinational reassortment in PorB proteins, (Vázquez et al. 1995
; Cooke et al. 1998
; Derrick et al. 1999
), this has not been so frequent as to remove all phylogenetic signals. In contrast, congruence tests between different housekeeping genes from N. meningitidis, subject to strong purifying selection, revealed that most gene trees were no more similar to one another than expected by chance (Holmes, Urwin, and Maiden 1999
). Overall, the diversity of meningococcal protein antigens explored here exhibits features which phylogenetic and structural models can exploit in the elucidation of the function and vaccine potential of these molecules. The insights obtained can complement data from both genomic studies and experimental studies of immunogenicity in man and animals to provide more complete information on the interactions of pathogen proteins with the hosts and its immune system.
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Acknowledgements |
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Footnotes |
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Keywords: Neisseria meningitidis
porB
evolution
recombination
selection
Address for correspondence and reprints: Martin C. J. Maiden, Department of Zoology, University of Oxford, South Parks Road, Oxford OX1 3PS, U.K. martin.maiden{at}zoo.ox.ac.uk
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