Conservation of meningococcal antigens in the genus Neisseria

Abstract

Neisseria meningitidis, one of the major causes of bacterial meningitis and sepsis, is a member of the genus Neisseria, which includes species that colonize the mucosae of many animals. Three meningococcal proteins, factor H-binding protein (fHbp), neisserial heparin-binding antigen (NHBA), and N. meningitidis adhesin A (NadA), have been described as antigens protective against N. meningitidis of serogroup B, and they have been employed as vaccine components in preclinical and clinical studies. In the vaccine formulation, fHbp and NHBA were fused to the GNA2091 and GNA1030 proteins, respectively, to enhance protein stability and immunogenicity. To determine the possible impact of vaccination on commensal neisseriae, we determined the presence, distribution, and conservation of these antigens in the available genome sequences of the genus Neisseria, finding that fHbp, NHBA, and NadA were conserved only in species colonizing humans, while GNA1030 and GNA2091 were conserved in many human and nonhuman neisseriae. Sequence analysis showed that homologous recombination contributed to shape the evolution and distribution of both NHBA and fHbp, three major variants of which have been defined. fHbp variant 3 was probably the ancestral form of meningococcal fHbp, while fHbp variant 1 from N. cinerea was introduced into N. meningitidis by a recombination event. fHbp variant 2 was the result of a recombination event inserting a stretch of 483 bp from variant 1 into the variant 3 background. These data indicate that a high rate of exchange of genetic material between neisseriae that colonize the human upper respiratory tract exists. IMPORTANCE The upper respiratory tract of healthy individuals is a complex ecosystem colonized by many bacterial species. Among these, there are representatives of the genus Neisseria, including Neisseria meningitidis, a major cause of bacterial meningitis and sepsis. Given the close relationship between commensal and pathogenic species, a protein-based vaccine against N. meningitidis has the potential to impact the other commensal species of Neisseria. For this reason, we have studied the distribution and evolutionary history of the antigen components of a recombinant vaccine, 4CMenB, that recently received approval in Europe under the commercial name of Bexsero®. We found that fHbp, NHBA, and NadA can be found in some of the human commensal species and that the evolution of these antigens has been essentially shaped by the high rate of genetic exchange that occurs between strains of neisseriae that cocolonize the same environment.

FIG 1

Maximum-likelihood phylogenetic tree obtained with 16S rRNA sequences. Bootstrap values are shown with red labels. In this tree, strains of the same species do not form monophyletic branches. A branch containing sequences from N. meningitidis, N. gonorrhoeae, N. polysaccharea, and N. cinerea can be identified. Colored circles and labels indicate species whose whole-genome sequences were available. In particular, purple indicates those sequences where none of the three antigens was conserved, while the other sequences are colored according to the species. A capital T indicates the type strain of the species.

FIG 2

Neighbor-joining phylogenetic tree with evolutionary distances calculated by the maximum composite likelihood method obtained from the SNPs in the portion of the multiple-sequence alignment shared by all of the strains studied. The alignment includes 64 strains of N. meningitidis, N. gonorrhoeae, N. polysaccharea, N. cinerea, and N. lactamica. The presence of fHbp, nhba, and nadA is indicated. The colors indicate the major allelic variants of fHbp. One of the three major fHbp variants is present in all of the strains of N. meningitidis. nadA is present in N. meningitidis in 7 out of 27 strains with the correct translation frame. nhba is ubiquitous, with the exception of N. cinerea.

FIG 3

Maximum-likelihood phylogenetic tree of the fHbp gene. The tree was obtained with the Kimura two-parameter model with gamma correction. Bootstrap values are shown with red labels. The average gene variability (Pi) was 0.131 (SE, 0.011). Also, including sequences from other species, the tree showed the typical structuring in three branches, variants 1, 2, and 3, as already described in N. meningitidis. In particular, N. gonorrhoeae strains harbored variant 3-like genes, as did the two N. polysaccharea strains. N. cinerea harbored a variant 1 fHbp gene very similar to that of N. meningitidis. Colored circles and labels indicate the different Neisseria species. The § symbol indicates sequences that were downloaded from the BigsDB database; the others came from GenBank. The # symbol indicates genes that were at the border of a contig. Asterisks indicate genes that were FS.

FIG 4

(a) Alignment of the fHbp locus in N. lactamica strain 020-06, N. meningitidis strain M01-240355, N. gonorrhoeae strain FA1090, and N. polysaccharea strain ATCC 43768. The fHbp gene is indicated in green. In three cases (N. meningitidis M01-240149, WUE_2594, and Z2491), the intergenic region between the homologues of NMB1869 and the fHbp gene showed the insertion of a highly conserved, AT-rich DNA fragment of 186 or 187 bp (49) that corresponds to the direct-repeat portion of IS1106 (GenBank accession no. Z11857.1) and is responsible for the separation of the fHbp promoter from its start codon. The entire locus is conserved in the reverse strand in all strains of N. gonorrhoeae and N. polysaccharea, where fHbp is always variant 3, while in both strains of N. polysaccharea the homologue of the fHbp gene is FS. The fHbp gene is absent from all strains of N. lactamica, where it is replaced by a protein annotated as a “putative opacity protein” on the opposite strand (in yellow). (b) fHbp loci in N. meningitidis MC58 and N. cinerea strain ATCC 1468. In both cases, the fHbp gene is variant 1.

FIG 5

Schema of the recombination events spanning the fHbp gene. The positions of the genes in the MC58 sequence are shown. The black box indicates the position of the fHbp gene. A single representative of each group of sequences showing a similar pattern of recombination events is reported. Each horizontal bar represents the locus in the genome indicated on the left. Light boxes drawn within each bar represent putative segments transferred by recombination. The imported fragments are represented by specific boxes that are drawn below each bar. The name of a putative donor strain is indicated on the right. fHbp variant 1 (strain MC58) appears to have been imported into N. meningitidis by a recombination event also including the upstream cbbA gene, while fHbp variant 2 was the result of the recombination of a small fragment of a variant 1 sequence in a variant 3 background.

FIG 6

Alignment of the nhba loci of N. lactamica strain 020-06, N. meningitidis strain MC58, N. gonorrhoeae strain FA1090, and N. polysaccharea strain ATCC 43768. The nhba gene is indicated in green.

FIG 7

Maximum-likelihood phylogenetic tree of the nhba gene. The tree was obtained with the Kimura two-parameter model with gamma correction. Bootstrap values are shown with red lebels. The average gene variability (Pi) was 0.084 (SE, 0.004). Gonococci formed a monophyletic branch closely related to the rest of the isolates. The other species, N. polysaccharea, N. lactamica, and N. flavescens, were scattered throughout the entire tree. Colored circles and labels indicate the different Neisseria species. The § symbol indicates sequences that were downloaded from the BigsDB database; the others were from GenBank. The # symbol indicates genes that were at the border of a contig. Asterisks indicate genes that were FS.

FIG 8

Alignment of the nadA loci of N. meningitidis serogroup A strain Z2491, N. meningitidis serogroup B strain MC58, and N. lactamica strain 020-06. The nadA gene is indicated in green. Although the locus is well conserved, the nadA gene is missing from both N. meningitidis serogroup A strain Z2491 and N. lactamica strain 020-06. In N. cinerea ATCC 14685, the nadA gene is interrupted by a contig edge; in N. cinerea CCUG346T, the gene is intact. In both strains, the gene is placed in the same locus as in N. meningitidis serogroup B strain MC58.

FIG 9

Maximum-likelihood phylogenetic tree of the nadA gene. The tree was obtained with the Kimura two-parameter model with gamma correction. Bootstrap values are shown with red labels. The sequence of the nadA gene from N. cinerea strain ATCC 14685 was obtained by joining two fragments at the border of two distinct contigs, and it was therefore not possible to assess the integrity of the gene. Colored circles and labels indicate the different Neisseria species. The § symbol indicates sequences that were downloaded from the BigsDB database; the others were from GenBank. The # symbol indicates genes that were at the border of a contig. The plus sign indicates genes that were interrupted by IS4. Asterisks indicate genes that were FS.