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

Abstract

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.

Improved transformation efficiency of group A Streptococcus by inactivation of a type I restriction modification system

“…A previous study reported that deletion of the hsdRSM locus in an M28 GAS strain resulted in reduced transcription of mga , which encodes a positive regulator of genes encoding several virulence factors including M protein ( emm28 ) and C5a peptidase ( scpA ) [ 2 ]. To determine the impact of hsdM deletion on the transcript abundance of key virulence factors in the M1 strain 854, we used qRT-PCR to compare relative expression of genes encoding nine virulence factors in 854 wild type and 854Δ hsdM mutant cells.…”

“…A global analysis of transcript levels in the hsdM deletion strain revealed no significant changes in expression of other genes compared to wild type. A previous study in an M28 strain found that deletion of the entire hsdRSM locus was associated with reduced expression of mga and Mga-regulated genes [ 2 ]. That we did not detect changes in transcription of these or other genes may reflect strain- or serotype-specific differences or effects of deleting hsdM alone in contrast to the entire hsd locus.…”

“…Previous studies have reported that inactivation of the Hsd system in GAS is associated with increased transformation efficiency [ 2 , 3 ]. One study done in an M28 strain found that deletion of the three components of the Hsd system was associated with increased transformation efficiency [ 2 ]. Another study characterized a group of emm1 clinical isolates associated with invasive infections.…”

Methylation by multiple type I restriction modification systems avoids influencing gene regulation in uropathogenicEscherichia coli

“…The general observation that DNA methylation, particularly that mediated by orphan methyltransferases (such as Dam or CcrM, which may themselves have evolved from RMSs), could alter transcription (44,48,84), led to the discovery of a broad suite of associated roles in DNA metabolism, cell physiology, and virulence (12,51,55,(85)(86)(87). An active research community continues to describe and characterize the additional roles that DNA methylation (from both restriction and orphan systems) play in bacteria (4,39,88), some of which can be very specific (67,69). We now show that, in contrast with expectations from this literature, the archetypal EcoKI Type I RMS has nearly zero impact on gene regulation or any phenotype among more than 1000 growth conditions tested.…”

Improved transformation efficiency of group AStreptococcusby inactivation of a type I restriction modification system

“…Previous studies have reported that inactivation of the Hsd system in GAS is associated with increased transformation efficiency [2,3]. One study done in an M28 strain found that deletion of the three components of the Hsd system was associated with increased transformation efficiency [2]. Another study characterized a group of emm1 clinical isolates associated with invasive infections.…”