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 R estriction, S pecificity, and M ethylation gene subunits ( Δ 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 Δ 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 Δ 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 Δ 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.

Background DNA methylation has been extensively studied as a regulator of gene expression among eukaryotes, but the regulatory role for DNA methylation has been far less studied in bacterial pathogens. Streptococcus pyogenes, or Group A Streptococcus, is an important bacterial pathogen of children. Our group has recently shown that S. pyogenes utilizes DNA methylation at N6-methyladenine (m6A) as a regulatory mechanism, modulating gene transcription and influencing the expression of several genes recognized as potential virulence factors. Our goal was to further explore how DNA methylation impacts virulence properties of S. pyogenes through adherence to epithelial cells and persistence on a mucosal surface. Methods S. pyogenes strains HSC12 (M14), MEW123 (M28), and MEW431 (M4) were modified by in-frame genetic deletion of a 3-gene operon encoding the only Type-I Restriction-Modification locus, resulting in mutant strains lacking the majority of m6A base modifications (ΔRSM). S. pyogenes parent and ΔRSM mutant strains were subjected to transcriptional profiling by RT–PCR and RNA-Seq analysis. Adherence rates of streptococci to D562 human pharyngeal epithelial cells and VKE6E7 human vaginal epithelial cells were assessed. A murine vaginal mucosa colonization model was used to monitor streptococcal mucosal persistence. Results The ΔRSM mutants of all three strains lacked essentially all m6A DNA base modifications by dot-blot with anti-m6A antibody and PacBio™ sequencing with methylation analysis. Transcriptional profiling demonstrated that a limited subset of ~20 genes was strongly down-regulated in all of the ΔRSM mutant strains, most notably genes in the core Mga regulon involved in tissue adherence and evasion of the host immune response, including the M protein (emm gene). The ΔRSM mutants of all 3 strains were attenuated for adherence to human respiratory and vaginal epithelial cells compared with parent strains or complemented mutant strains. The HSC12 and MEW431 ΔRSM mutant strains exhibited significantly decreased bacterial burdens over time compared with parent strains in the murine mucosal carriage model. The bacterial burdens of strain MEW123 and its ΔRSM mutant were not significantly different in the murine mucosal carriage model. Expression of R28, an adhesin specifically promoting adherence to vaginal epithelial cells, was not altered in the MEW123 ΔRSM mutant, which may explain the continued persistence of this strain in the murine model. Conclusions DNA methylation influences gene expression at the transcriptional level in S. pyogenes and affects virulence properties in both in vitro and in vivo models of infection. We report that methylation promotes activation of several important virulence factors, including the M protein and other members of the Mga regulon, and influences epithelial cell adherence and streptococcal persistence on a mucosal surface. DNA methylation appears to be an important contributor to bacterial physiology and pathogenesis. Future work will identify the interaction of m6A base modifications and transcriptional regulatory proteins.