In vivo metatranscriptomics reveals sRNA-driven zinc-sparing responses in chronic Staphylococcus aureus infection

ABSTRACT: Understanding bacterial gene regulation during human infection remains challenging, as most regulatory networks are inferred from in vitro models that poorly recapitulate host environments. Here, we present INTENSE (In-human Transcriptional Expression Networks in Site-specific Environments), a pangenome-based metatranscriptomic pipeline designed to profile Staphylococcus aureus transcription directly from human infection samples. Using a curated S. aureus pangenome and stringent decoy-based filtering, we analyzed RNA-seq data from chronic wound and cystic fibrosis sputum samples and uncovered infection-specific transcriptional signatures that clearly diverge from in vitro growth conditions. Small RNAs (sRNAs) emerged as major contributors to transcriptomic variability during chronic infection, with nine sRNAs consistently induced in human samples. Among these, we identify rsaX20 as a previously uncharacterized, zinc-responsive sRNA that also encodes a small peptide. Meta-analysis of over 2,000 publicly available S. aureus RNA-seq datasets revealed that rsaX20 is recurrently induced under low-zinc and host-associated stress conditions and displays co-regulation patterns with known metal-responsive sRNAs. Genetic, transcriptomic, and proteomic analyses demonstrate that rsaX20 is repressed by the zinc uptake regulator Zur, induced upon zinc limitation, and functions as a dual-purpose sRNA/sORF. Functional dissection revealed that the sRNA and peptide exert distinct and often opposing regulatory effects on protein expression, collectively targeting a network of metal-binding proteins. Notably, rsaX20-mediated repression of zinc-dependent enzymes supports a zinc-sparing adaptive response during infection. Together, these findings establish pangenome-based metatranscriptomics as a powerful approach to interrogate bacterial regulation in vivo and uncover rsaX20 as a key dual-function regulator shaping zinc homeostasis during chronic S. aureus infection

ORGANISM(S): Homo sapiens

PROVIDER: GSE319561 | GEO | 2026/07/20

REPOSITORIES: GEO