Meningococcal genetic variation mechanisms viewed through comparative analysis of serogroup C strain FAM18

Abstract

The bacterium Neisseria meningitidis is commonly found harmlessly colonising the mucosal surfaces of the human nasopharynx. Occasionally strains can invade host tissues causing septicaemia and meningitis, making the bacterium a major cause of morbidity and mortality in both the developed and developing world. The species is known to be diverse in many ways, as a product of its natural transformability and of a range of recombination and mutation-based systems. Previous work on pathogenic Neisseria has identified several mechanisms for the generation of diversity of surface structures, including phase variation based on slippage-like mechanisms and sequence conversion of expressed genes using information from silent loci. Comparison of the genome sequences of two N. meningitidis strains, serogroup B MC58 and serogroup A Z2491, suggested further mechanisms of variation, including C-terminal exchange in specific genes and enhanced localised recombination and variation related to repeat arrays. We have sequenced the genome of N. meningitidis strain FAM18, a representative of the ST-11/ET-37 complex, providing the first genome sequence for the disease-causing serogroup C meningococci; it has 1,976 predicted genes, of which 60 do not have orthologues in the previously sequenced serogroup A or B strains. Through genome comparison with Z2491 and MC58 we have further characterised specific mechanisms of genetic variation in N. meningitidis, describing specialised loci for generation of cell surface protein variants and measuring the association between noncoding repeat arrays and sequence variation in flanking genes. Here we provide a detailed view of novel genetic diversification mechanisms in N. meningitidis. Our analysis provides evidence for the hypothesis that the noncoding repeat arrays in neisserial genomes (neisserial intergenic mosaic elements) provide a crucial mechanism for the generation of surface antigen variants. Such variation will have an impact on the interaction with the host tissues, and understanding these mechanisms is important to aid our understanding of the intimate and complex relationship between the human nasopharynx and the meningococcus.

Figure 1. Rearrangements between the Meningococcal Genomes

The dotplots were generated using MUMmer version 3.15 (http://mummer.sourceforge.net) and indicate matching sequences with codirectional and reversed regions of synteny shown in red and green, respectively. Genome sequences are aligned to start/finish at the origin of replication with the approximate position of the terminus of replication indicated (Ter) (note this required rotation of the publicly available sequences for Z2491 and MC58, see Materials and Methods). Also shown are the positions of the foci of the three major inversion events (IE1, IE2, and IE3, see text for detail).

Figure 2. Syntenic Repeat Arrays Showing Variation in Repeat Number and Array Length and tbpAB Divergence

CDS are shown as arrowed boxes with colours common for orthologues. dRS3 repeat sequences are shown as red lines. Green blocks indicate percentage identity of amino acid sequence between CDS.

Figure 3. Sequence Divergence in Orthologues Flanking Repeat Arrays

(A) Plot of repeat array length against flanking orthologue sequence identity for FAM18 versus Z2491 (blue diamond), Z2491 versus MC58 (red square), and FAM18 versus MC58 (green triangle). (B) Plot of distance from array versus orthologue identity for FAM18 versus Z2491, Z2491 versus MC58, and FAM18 versus MC58. (C) The same as (B), ignoring the first CDS.

Figure 4. Pie Charts Showing Association between Repeat Arrays and Surface Proteins in FAM18

Chart sectors are coloured according to gene function (see colour key).

Figure 5. NIME Array Percentage G + C Content Profile for an Array Located at 1283600–1284640 bp in the FAM18 Genome

The % G + C window size is 24 bases. Maximum, minimum, and median are also shown. Red blocks represent dRS3 with inverted repeats indicated by grey triangles. Blue blocks represent RS elements.

Figure 6. Silent Gene Cassette–Mediated Variation

(A) maf1 locus (NMA0305–0286, NMB2104–2127, NMC2083–2102). (B) fha loci (NMA0687–0698, NMB0496–0521, NMC0443–0459). Purple and red boxes indicate the coding sequences and corresponding downstream alternative C termini-encoding silent sequences, respectively. Pale blue boxes represent intervening annotated coding sequences. Pink and orange boxes represent transposase and hypothetical protein encoding, flanking CDS, respectively. Coloured blocks between gene clusters represent regions of sequence similarity; green blocks indicate large syntenic regions of similarity, and blue blocks represent shorter internal repeat sequences, which may facilitate recombination between loci.