Variation and molecular evolution of HmbR, the Neisseria meningitidis haemoglobin receptor

Abstract

Meningococcal disease caused by serogroup B Neisseria meningitidis remains an important health problem in many parts of the world, and there are currently no comprehensive vaccines. Poor immunogenicity, combined with immunological identity to human sialic acids, have hindered the development of a serogroup B conjugate vaccine, resulting in the development of alternative vaccine candidates, including many outer-membrane protein (OMP)-based formulations. However, the design of protein-based meningococcal vaccines is complicated by the high level of genetic and antigenic diversity of the meningococcus. Knowledge of the extent and structuring of this diversity can have implications for the use of particular proteins as potential vaccine candidates. With this in mind, the diversity of the meningococcal OMP HmbR was investigated among N. meningitidis isolates representative of major hyper-invasive lineages. In common with other meningococcal antigens, the genetic diversity of hmbR resulted from a combination of intraspecies horizontal genetic exchange and de novo mutation. Furthermore, genealogical analysis showed an association of hmbR genes with clonal complexes and the occurrence of two hmbR families, A and B. Three variable regions (VR1-VR3), located in loops 2, 3 and 4, were observed with clonal complex structuring of VR types. A minority of codons (3.9 %), located within putative surface-exposed loop regions of a 2D model, were under diversifying selection, indicating regions of the protein likely to be subject to immune attack.

Fig. 1.

clonalframe analysis of the hmbR sequences. Phylogenetic trees were constructed using clonalframe version 1.1 available at http://www2.warwick.ac.uk/fac/sci/statistics/staff/research/didelot/clonalframe/ (Didelot & Falush, 2007). In the present study, over 100 000 iterations and 100 000 burn-ins were performed with every hundredth tree sampled, after which a 75 % majority-rule consensus tree was derived. Annotation was then undertaken by importing the tree into the Molecular Evolutionary Genetics Analysis software package (mega v4.0) (Tamura et al., 2007).

Fig. 2.

clonalframe representation of hmbR recombination events. The nucleotide sequence of hmbR genes is represented on the x axis, with the grey line indicating at each locus the probability for an import on a scale from 0 (bottom of the y axis) to 1 (top of the y axis). Each inferred substitution in the graph is represented by a cross, the intensity of which indicates the posterior probability for that substitution. In (a), a single recombination event occurring at node A among family A hmbR sequences is represented (Fig. 1). In (b), horizontal genetic exchange at node B and among family B sequences is depicted occurring approximately from bases 300 to 500, 500 to 750 and 750 to 1000. The amino acid location of each loop is represented with additional functional information (Perkins-Balding et al., 2003): loop 1, 484–501; *loop 2, 589–681 haem binding; *loop 3, 775–831 haem binding; *loop 4, 928–1020; *loop 5, 1147–1218; loop 6, 1315–1377 haem utilization; loop 7, 1465–1545 haem utilization; *loop 8, 1627–1764; loop 9, 1870–1905; *loop 10, 1993–2067; and *loop 11, 2161–2274. Asterisks indicate loops containing positively selected sites.

Fig. 3.

Location of positively selected residues on a structural topology model of HmbR. Positively selected codon sites (black background with white lettering) inferred using the maximum-likelihood method with BEB and the M8 model are identified on a structural topology model of HmbR. Amino acids that correspond to inserted/deleted codons are indicated by asterisks. The glycines encoded by the polyG tract are indicated by hashes.