Epigenetic variability in the facultative human pathogen "Neisseria meningitidis"

Abstract

Meningococcal disease occurs worldwide as endemic infections. The gram-negative bacteria Neisseria meningitidis is a predominant cause of septicemia and meningitis, at the same time it is also a frequent commensal of the human nasopharynx. Meningococci displays a high genetic diversity, yet to present the results from multilocus sequence typing (MLST) and whole genome sequencing have not defined a strict core patho-genome enabling the prediction of virulence on a genetic basis. Towards a better understanding of meningococcal complexity and genomic plasticity, we address in this thesis the epigenetic diversity and the consequences of DNA methylation as well as developing tools to assess phase-variation events within a population of Neisseria meningitidis isolates. We applied single-molecule real-time (SMRT) sequencing technology to establish genome wide DNA modification profiles of two closely related N. meningitidis strains. DNA modifications, and in particular DNA methylation as the most common DNA modification, are thereby detected based on delay in the kinetics of the DNA synthesis in vitro. Our approach revealed a high diversity in DNA methylation between closely related Neisseria strains. The methylated sequences as defined by the SMRT sequencing results largely correspond to putative target sequence of DNA methyltransferases identified in N. meningitidis and to the observed protection from digestion by methylation-sensitive cognate restriction enzymes. Association of epigenetic modifications with phenotypes is still poorly characterized in bacteria. Our analysis of the methylation patterns along the genome showed a biased distribution evident by a clear depletion of 5-methylcytosine motifs relative to gene start positions. These results suggest a more complex role for DNA methylation in terms of the hypothesized regulatory functions. In addition, we identified a striking co-localization of mutations at methylated bases indicating additional un-known consequences of DNA methylation in relation to bacterial evolution and adaptation. We also developed a novel bioinformatic tool to infer the precise number of repeat units at specific tandem repeat loci exploiting increasing read length resulting from recent versions of large scale sequencing assays. Our probabilistic approach detects divergent repeat length configurations and therefore functional states of ORFs directly from raw sequencing data, offering an enhanced detection power and accuracy over conventional tools. We integrated our tool into a fast and efficient computational pipeline to detect genome wide phase-variation events by comparing a large number of sequenced meningococcal genomes. Our comprehensive approach identified a high number of hyper-variable repeat regions mostly associated with genes encoding outer membrane components and other virulence determinants.