Stress-induced DNA methylome plasticity and transcriptional re-programming in Staphylococcus aureus

Abstract

Staphylococcus aureus, a major human and livestock pathogen, is the second biggest cause of antimicrobial resistance-associated mortality. Although S. aureus transcriptional regulation has been extensively characterised, the potential role of DNA methylation in S. aureus transcriptional regulation and stress response remains largely undefined. We tackled this gap by combining genome-wide nanopore sequencing-derived DNA methylation data and transcriptomic data, acquired before and during exposure of methicillin-resistant Staphylococcus aureus strain USA300 to clinically relevant oxidative, antibiotic and nitrosative stresses. Stress-induced significant DNA methylation changes were rare, with <0.1% of cytosines/adenines undergoing [≥]20% change in methylation; these changes were enriched within genomic features (protein-coding genes, predicted promoter regions, ncRNAs). Transcription changes reflected metabolic, regulatory and stress-specific pathway adjustments. Many of the stress-induced DNA methylation changes occurred alongside transcription changes, although there was no obvious overarching relationship between the directions of changes in DNA methylation and transcription. Pre-stress treatment methylation entropy tended to be elevated at sites containing a base that underwent stress-induced change in methylation level, identifying focal sites of pre-existing methylome heterogeneity relative to both local and genome-wide backgrounds; the magnitude of the site-specific methylation entropy peak, moreover, correlated with the magnitude of subsequent methylation change. Genome-wide entropy levels were consistent with a finite number of methylation patterns, distinguished by key bases, that could correspond to clinically important S. aureus subpopulations exhibiting persistence, dormancy and immune evasion. These findings support the principle that DNA methylation is an important component of the regulatory machinery underpinning S. aureus adaptability and persistence. ImportanceStaphylococcus aureus survives antibiotic treatment and host immune attack partly through phenotypic diversity, yet the regulatory processes that support this adaptability remain incompletely understood. Here, we integrate genome-wide DNA methylation and transcriptomic profiling to examine how epidemic methicillin-resistant S. aureus USA300 responds to oxidative, antibiotic, and nitrosative stresses. Stress-induced methylation changes were rare but non-random, concentrated in coding and regulatory regions linked to stress defence, metabolism, persistence, virulence, biofilm formation, and host interaction. Sites that underwent stress-induced methylation changes tended to have unusually high pre-stress methylation pattern diversity relative to local and genome-wide pattern diversity. These findings support a model in which an S. aureus population contains a pre-stress repertoire of epigenetic states that stress selectively redistributes via bases that differentiate stable methylation patterns. By revealing a layer of heterogeneity that may support pathogen resilience, this work provides a framework for investigating whether bacterial methylation dynamics can be exploited clinically.