4methylcytosine DNA modification is critical for global epigenetic regulation and virulence in the human pathogen Leptospira interrogans

In bacteria, DNA methylation is an important epigenetic marker, as it can regulate DNA repair, replication timing, and transcription. This modification can be facilitated by orphan DNA methyltransferases lacking cognate restriction endonucleases, but the mechanisms by which these enzymes control key cellular processes of these organisms is poorly understood. A specific modification, 4-methylcytosine (4mC), is even less understood, as this epigenetic marker is unique to bacteria and archaea, whereas the bulk of epigenetic research is currently performed on eukaryotes. Here, we characterize a 4mC methyltransferase found in pathogenic species from the genus Leptospira, a grievous human pathogen responsible for over one million severe annual infections, but absent from saprophytic members of the genus. Inactivating this enzyme resulted in complete abrogation of the methylation of CTAG motifs in the genome, leading to genome-wide dysregulation of gene expression, particularly affecting genes responsible for motility and envelope biogenesis. Consistently, mutants exhibited growth defects and showed decreased adhesion to host cells, as well as higher susceptibility to LPS-targeting antibiotics. Further investigation resulted in the discovery of at least one gene, that of an ECF sigma factor, whose transcription was altered in the methylase mutant and, subsequently, by mutation of the CTAG motifs in the promoter and coding sequence of the gene in the WT strain. The genes that comprise regulon of this sigma factor were, accordingly, highly dysregulated in the methylase mutant and in strain engineered to overexpress the sigma factor. Finally, we also demonstrate the methylase mutant and the sigma factor overexpressing strain were less virulent in an acute model of infection. These findings indicate that 4mC epigenetic modification is involved in global gene regulation and virulence in this spirochete, and, likely, other bacteria.