Institut für Hygiene und Mikrobiologie

    Evolutionary Pathogenomics of N. meningitidis

    The analysis of meningococcal population structure by multilocus sequence typing (MLST) showed that disease-causing meningococci belong to particular groups of related sequence types (STs), termed clonal complexes (CCs), that are overrepresented in disease isolates relative to their carriage prevalences and that only few so called hyperinvasive lineages are responsible for most disease.

    We recently analyzed a national, well characterized collection of strains that was sampled  predominantly in Germany in the years 1999–2000 using a combination of comparative genome hybridization using microarrays (mCGH) and computational approaches to systematically analyze possible associations between gene content and hyperinvasive lineages. The sample population covered over 98% of the observed genetic diversity in a population of carriage strains with assigned CCs and included all major hyperinvasive lineages associated with invasive disease as determined via MLST. The panel comprised 29 strains isolated from patients (n= 13) as well as from healthy carriers (n= 16) which belonged to six different serogroups including also capsule null locus (cnl) strains and 22 different CCs, respectively. Fifteen strains were from CCs more frequently associated with invasive meningococcal disease (IMD) than carriage, and 14 in turn belonged to CCs that are mostly associated with asymptomatic carriage in healthy individuals. Twenty-six strains (90%) were isolated in Germany either in the course of the Bavarian carriage study [1] or were taken from the strain collection of the German Reference Laboratory for Meningococci (NRZM, Würzburg, Germany) spanning the same time period.
    Extensive gene content comparison revealed novel associations of virulence with genetic elements besides the recently discovered meningococcal disease associated (MDA) island. In particular, we identified an association of virulence with a recently described canonical genomic island termed IHT-E and a differential distribution of genes encoding RTX toxin- and two-partner secretion systems among hyperinvasive and non-hyperinvasive lineages (Figure 1).

    Figure 1. Clustering of strains based on the accessory genome
    A maximum parsimony (MP) tree is shown with bootstrap values at nodes used for grouping of strains into eight genome groups (GGs) from their gene content comprising 470 parsimony informative genes as revealed by mCGH. Strains with an asterisk next to their name have further been used for the in silico screening for intragenomic recombination in 1092 genes from the core genome as estimated via mCGH of the entire sample population. Next to the MP tree, the CCs and serogroups (Sg) of the respective strains are given with hypervirulent CCs in black boxes, and right to the Sgs a virtual array image displaying the presence and absence of 1679 genes is shown. Strains from the same serogroup have in general highly similar gene content, and strains from the same CC always belong to the same GG. In turn, a GG can comprise strains from different CCs, and with the exception of the two serogroup W-135 strains split between GG-II and GG-III and the serogroup B strain DE8638, GGs always comprise strains from the same serogroup. However, no two strains have exactly the same gene content. Right below the virtual array, the spotted genes are color coded according to the source genome (representing the genomes of strain Z2491, MC58, FAM18 and α14), and the presence of putatively mobile DNA is depicted below with IHT-B, IHT-C, IHT-E as well as the λ prophage denoted as B, C, E and λ, respectively, in the respective lanes (Abbreviations: IHT, island of horizontal transfer; Φ: prophage; MME, minimal mobile element). At the lower margin, the FDR for the association with hyperinvasive lineages is color coded for each gene with genes having a FDR < 0.05 depicted in blue.

    How the proteins encoded on these genetic elements might contribute to meningococcal disease and whether they affect, e. g., the interaction with human host cells is currently a matter of ongoing in vitro experimental investigations in the lab.

    In addition to the aforementioned virulence-associated genetic factors, meningococci often produce a polysaccharide capsule which mediates resistance against complement-mediated lysis and opsonophagocytosis. Based on the chemical composition and the immunological characteristics of the capsular polysaccharide, meningococci are divided into 13 serogroups with serogroups A, B, C, W-135 and Y being most frequently associated with human disease.
    Although several carriage isolates can also express polysaccharide capsules and is therefore likely not sufficient to confer full virulence in N. meningitidis, epidemiological observations over the last decades have clearly shown that the a capsule is essential for causing invasive meningococcal disease. The capsule is therefore one of the best established virulence-associated factors in N. meningitidis and understanding the evolution of the capsule genes in N. meningitidis is paramount to an understanding of virulence evolution in this basically commensal species.
    A whole genome-based reconstruction of neisserial phylogeny suggests that, among the five meningococcal strains compared, the unencapsulated strain alpha14 is the strain most similar to N. gonorrhoeae and N. lactamica. Therefore, alpha14 might be close to the branching point of N. meningitidis and N. gonorrhoeae from N. lactamica, and since N. lactamica and N. gonorrhoeae both lack a capsule this provides evidence that N. meningitidis may have originated as an un-encapsulated species (Figure 2).

    Figure 2: Genome-based inference of meningococcal phylogeny.
    Hypothetical scenario of the evolution of encapsulated meningococcal strains from an un-encapsulated common ancestor with N. gonorrhoeae and N. lactamica as suggested by whole-genome comparisons (neighbour net reconstruction based on genome rearrangement distances).

    A closer bioinformatic analysis of the cps locus required for the synthesis of the polysaccharide capsule in N. meningitidis suggest that large parts of this locus might have been acquired by un-encapsulated ancestor via horizontal gene transfer (HGT) from another bacterial species. In particular, the cps locus consists of five regions, A to E. While regions E and D might belong to the neisserial core genome, as they can be found in many other Neisseria spp., regions A and C reside on an island of horizontal transfer called IHT-A1, which has a lower G + C content when compared to the rest of the meningococcal genome. Also the ctrABCD genes of region C and the lipAB genes of region B are highly similar in sequence and operon organization to the hexABCD (PMO0778-0781) and phyAB (PMO0772-0773) genes, respectively, in the Pasteurella multocida genome (GenBank AE004439). These results are in line with previous observations of HGT from Haemophilus influenzae being also a member of the Pasteurellaceae and inhabitant of the human airways to N. meningitdis. Therefore, the encapsulated and thus potentially pathogenic strains of N. meningitidis might have evolved from an un-encapsulated ancestor by horizontal acquisition of the cps locus from other bacteria residing in the human nasopharynx.
    What this donor species might be, at what time in the evolutionary past of this species this event might have taken place and under what circumstances associated with human population history are all questions of ongoing research activities in our group.

    Relevant recent publications:

    Joseph, B. R. F. Schwarz, B. Linke, J. Blom, A. Becker, H. Claus, A. Goesmann, M. Frosch, T. Müller, U. Vogel and C. Schoen (2011) Virulence evolution of the human pathogen Neisseria meningitidis by recombination in the core and accessory genome. PLoS ONE 6:e18441

    Schoen, C., H. Tettelin, J. Parkhill and M. Frosch (2009) Genome flexibility in Neisseria meningitidis. Vaccine 27S: B103-B111

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