DNA methylation from a Type I restriction modification system influences gene expression and virulence in Streptococcus pyogenes.

DNA methylation is pervasive across all domains of life. In bacteria, the presence of N6-methyladenosine (m6A) has been detected among diverse species, yet the contribution of m6A to the regulation of gene expression is unclear in many organisms. Here we investigated the impact of DNA methylation on gene expression and virulence within the human pathogen Streptococcus pyogenes, or Group A Streptococcus. Single Molecule Real-Time sequencing and subsequent methylation analysis identified 412 putative m6A sites throughout the 1.8 Mb genome. Deletion of the Restriction, Specificity, and Methylation gene subunits (Delta RSM strain) of a putative Type I restriction modification system lost all detectable m6A at the recognition sites and failed to prevent transformation with foreign-methylated DNA. RNA-sequencing identified 20 genes out of 1,895 predicted coding regions with significantly different gene expression. All of the differentially expressed genes were down regulated in the Delta RSM strain relative to the parent strain. Importantly, we found that the presence of m6A DNA modifications affected expression of Mga, a master transcriptional regulator for multiple virulence genes, surface adhesins, and immune-evasion factors in S. pyogenes. Using a murine subcutaneous infection model, mice infected with the Delta RSM strain exhibited an enhanced host immune response with larger skin lesions and increased levels of pro-inflammatory cytokines compared to mice infected with the parent or complemented mutant strains, suggesting alterations in m6A methylation influence virulence. Further, we found that the Delta RSM strain showed poor survival within human neutrophils and reduced adherence to human epithelial cells. These results demonstrate that, in addition to restriction of foreign DNA, gram-positive bacteria also use restriction modification systems to regulate the expression of gene networks important for virulence. Author summary DNA methylation is common among many bacterial species, yet the contribution of DNA methylation to the regulation of gene expression is unclear outside of a limited number of gram-negative species. We characterized sites of DNA methylation throughout the genome of the gram-positive pathogen Streptococcus pyogenes or Group A Streptococcus. We determined that the gene products of a functional restriction modification system are responsible for genome-wide m6A. The mutant strain lacking DNA methylation showed altered gene expression compared to the parent strain, with several genes important for causing human disease down regulated. Furthermore, we showed that the mutant strain lacking DNA methylation exhibited altered virulence properties compared to the parent strain using various models of pathogenesis. The mutant strain was attenuated for both survival within human neutrophils and adherence to human epithelial cells, and was unable to suppress the host immune response in a murine subcutaneous infection model. Together, these results show that bacterial m6A contributes to differential gene expression and influences the ability of Group A Streptococcus to cause disease. DNA methylation is a conserved feature among bacteria and may represent a potential target for intervention in effort to interfere with the ability of bacteria to cause human disease.