DNA methylation from a Type I restriction modification system influences gene expression and virulence in Streptococcus pyogenes
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ABSTRACT:
ORGANISM(S): Streptococcus pyogenes
PROVIDER: GSE130429 | GEO | 2019/04/30
REPOSITORIES: GEO
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Project description: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 (ΔRSM 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 ΔRSM 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 ΔRSM 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 ΔRSM 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.
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Project description:We used Pacific Biosciences Single Molecule Real-Time sequencing platform to identify modified motifs in an RM deficient strain of Streptococcus pyogenes
2019-04-30 | GSE130428 | GEO
Project description:We used Illumina NGS to measure mRNA levels and perform subsequent differential expression analysis between wild type and RM system deficient strains of Streptococcus pyogenes
2019-04-30 | GSE130427 | GEO
Project description:DNA methylation from a Type I restriction modification system influences gene expression and virulence in Streptococcus pyogenes
| PRJNA540241 | ENA
Project description:DNA methylation from a Type I restriction modification system influences gene expression and virulence in Streptococcus pyogenes [PacBio]
| PRJNA540244 | ENA
Project description:DNA methylation from a Type I restriction modification system influences gene expression and virulence in Streptococcus pyogenes [RNA-seq]
| PRJNA540245 | ENA
Project description:Bacterial defense barriers, such as DNA methylation-associated restriction-modification (R-M) and the CRISPR-Cas system, play an important role in bacterial antimicrobial resistance (AMR). Recently, a novel R-M system based on DNA phosphorothioate (PT) modification has been shown to be widespread in the kingdom of Bacteria as well as Archaea. However, the potential role of the PT R-M system in bacterial AMR remains unclear. In this study, we explored the role of PT R-Ms in AMR with a series of common clinical pathogenic bacteria. By analyzing the distribution of AMR genes related to mobile genetic elements (MGEs), it was shown that the presence of PT R-M effectively reduced the distribution of horizontal gene transfer (HGT)-derived AMR genes in the genome, even in the bacteria that did not tend to acquire AMR genes by HGT. In addition, unique gene variation analysis based on pangenome analysis and MGE prediction revealed that the presence of PT R-M could suppress HGT frequency. Thus, this is the first report showing that the PT R-M system has the potential to repress HGT-derived AMR gene acquisition by reducing the HGT frequency. IMPORTANCE In this study, we demonstrated the effect of DNA PT modification-based R-M systems on horizontal gene transfer of AMR genes in pathogenic bacteria. We show that there is no apparent association between the genetic background of the strains harboring PT R-Ms and the number of AMR genes or the kinds of gene families. The strains equipped with PT R-M harbor fewer plasmid-derived, prophage-derived, or integrating mobile genetic element (iMGE)-related AMR genes and have a lower HGT frequency, but the degree of inhibition varies among different bacteria. In addition, compared with Salmonella enterica and Escherichia coli, Klebsiella pneumoniae prefers to acquire MGE-derived AMR genes, and there is no coevolution between PT R-M clusters and bacterial core genes.
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Project description:Streptococcus pyogenes or group A Streptococcus (GAS) is a leading cause of bacterial pharyngitis, skin and soft tissue infections, life-threatening invasive infections, and the post-infectious autoimmune syndromes of acute rheumatic fever and post-streptococcal glomerulonephritis. Genetic manipulation of this important pathogen is complicated by resistance of the organism to genetic transformation. Very low transformation efficiency is attributed to recognition and degradation of introduced foreign DNA by a type I restriction-modification system encoded by the hsdRSM locus. DNA sequence analysis of this locus in ten GAS strains that had been previously transformed with an unrelated plasmid revealed that six of the ten harbored a spontaneous mutation in hsdR, S, or M. The mutations were all different, and at least five of the six were predicted to result in loss of function of the respective hsd gene product. The unexpected occurrence of such mutations in previously transformed isolates suggested that the process of transformation selects for spontaneous inactivating mutations in the Hsd system. We investigated the possibility of exploiting the increased transformability of hsd mutants by constructing a deletion mutation in hsdM in GAS strain 854, a clinical isolate representative of the globally dominant M1T1 clonal group. Mutant strain 854ΔhsdM exhibited a 5-fold increase in electrotransformation efficiency compared to the wild type parent strain and no obvious change in growth or off-target gene expression. We conclude that genetic transformation of GAS selects for spontaneous mutants in the hsdRSM restriction modification system. We propose that use of a defined hsdM mutant as a parent strain for genetic manipulation of GAS will enhance transformation efficiency and reduce the likelihood of selecting spontaneous hsd mutants with uncharacterized genotypes.
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Project description:The CovR/S system in Streptococcus pyogenes (Group A Streptococcus, or GAS), a two-component signal transduction/transcription regulation system, controls the expression of major virulence factors. The presence of a negative feedback loop distinguishes the CovR/S system from the majority of bacterial two-component systems. We developed a deterministic model of the CovR/S system consisting of eight delay differential equations. Computational experiments showed that the system possessed a unique stable steady state. The dynamical behavior of the system showed a tendency for oscillations becoming more pronounced for longer but still biochemically realistic delays resulting from reductions in the rates of translation elongation. We have devised an efficient procedure for computing the system's steady state. Further, we have shown that the signal-response curves are hyperbolic for the default parameter values. However, in experiments with randomized parameters we demonstrated that sigmoidality of signal-response curves, implying a response threshold, is not only possible, but seems to be rather typical for CovR/S-like systems even when binding of the CovR response regulator protein to a promoter is non-cooperative. We used sensitivity analysis to simplify the model in order to make it analytically tractable. The existence and uniqueness of the steady state and hyperbolicity of signal-response curves for the majority of the variables was proved for the simplified model. Also, we found that provided CovS was active, the system was insensitive to changes in the concentration of any other phosphoryl donor such as acetyl phosphate.
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Project description:The human pathogen Streptococcus pyogenes secretes many proteins to the cell wall and extracellular environment that contribute to virulence. Rgg regulates the expression of several exoproteins including a cysteine protease (SPE B), a nuclease (MF-1), a putative nuclease (MF-3), and autolysin. The functional heterogeneity of Rgg-regulated exoproteins and the lack of a conserved regulatory motif in the promoter regions of the genes suggested that Rgg interacts with additional regulatory networks to influence gene expression. DNA microarrays were used to test this hypothesis by comparing genomewide transcript profiles of S. pyogenes NZ131 and isogenic derivative NZ131 rgg during the exponential phase of growth. Transcripts of known and putative virulence-associated genes were more abundant in the rgg mutant, including emm, scpA, orfX, scl1, hasAB, slo, sagA, ska, speH, grab, mac, mf-1, and mf-3. Increased transcription of emm, scpA, and orfX in the rgg mutant was associated with increased production of the corresponding proteins. Differences in the expression of virulence-associated genes were associated with changes in the expression of several regulatory genes, including mga, sagA, csrRS, and fasBCA. The results show that Rgg influences the expression of multiple regulatory networks to coregulate virulence factor expression in S. pyogenes.
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