To Örebro University

Introduction: A significant increase in invasive meningococcal disease (IMD) due to serogroup Y Neisseria meningitidis (MenY) ST-23 clonal complex (cc23) emerged in the United States during the 1990s, spreading to Europe shortly thereafter. The largest increase was observed in Sweden with incidence proportions up to 53%. Genome analysis of all MenY isolates causing IMD between 1995 to 2012 in Sweden revealed that a distinct strain (YI) and more specifically a subtype (1) of this strain was found to be responsible for the increase of MenY IMD in Sweden [1]. In this study, we compared the methylomes of subtype 1 to the less successful subtype 2, using Single Molecule Real-Time (SMRT) sequencing technology.

Methods: Ten genomes belonging to subtype 1 (n=7) and 2 (n=3) and one MenY genome without connection to a specific lineage were sequenced using SMRT sequencing on a PacBio®RS II. The analysis platform SMRT Portal v2 was used to identify modified positions and for the genome-wide analysis of modified motifs. DNA methyltransferase genes associated with the different methyltransferase recognition motifs identified were searched using SEQWARE. The modification-dependent restriction endonucleases MspJI and FspEI were used to determine the m5C recognition sites of the active m5C methylases in the strains.

Results: The genome-wide analysis of the methylomes identified two m6A modified motifs: GATC and CACNNNNNTAC, but the latter was only found in isolates belonging to subtype 2 due to a transposase inserted in the candidate gene in subtype 1 strains: a Type I restriction system specificity protein (NEIS2535). The motif CACNNNNNTAC was only found in one other meningococcal isolate in REBASE, belonging to cc23, suggesting that this is a cc23 specific motif. Eleven putative restriction modification (RM) systems were found when comparing the sequences of all 11 genomes to DNA methyltransferase genes in REBASE. Five m5C genes were predicted, however, only three of these corresponding to the motifs: GCRYGC, GGNNCC and CCAGR were confirmed as active using MspJI and FspEI cleavage. The apparent CCAGR motif may be the result of two methylases, one recognizing CCWGG and the other CCAGA, but this will have to be verified.

Conclusion: These results are consistent with previous studies [2] that have shown that the composition of different RM systems are clade specific suggesting that the unique RM system of cc23 isolates will most likely result in a specific DNA methylation pattern unique to this particular cc. However, although the majority of methyltransferases were shared between the two subtypes, there was one difference in a m6A modified motif between these two highly similar cc23 subtypes, which may lead to an altered gene expression pattern.