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

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