Discovery of Staphylococcus aureus small RNAs highly expressed during human chronic infection

Understanding bacterial gene expression in natural environments remains challenging because most regulatory networks are inferred from in vitro models that poorly capture in situ environmental conditions. Here, we present NEMO (network transcriptomics of microbes in native environments), a generalizable pangenome-based metatranscriptomic framework for extracting microbial transcriptional programs from metatranscriptomes generated from natural samples. We applied NEMO to Staphylococcus aureus metatranscriptomes from human chronic wounds and cystic fibrosis sputum, revealing infection-associated transcriptional states that diverged markedly from in vitro growth conditions. Notably, small RNAs (sRNAs) were key drivers of the transcriptional divergence between human infection-derived and in vitro transcriptomes. Among these, we identify rsaX20 as a previously uncharacterized, zinc-responsive sRNA that also encodes a small peptide. Meta-analysis of more than 2,000 publicly available S. aureus RNA-seq data sets showed that rsaX20 is induced under zinc limitation and host-associated stress and is co-regulated with known metal-responsive sRNAs. Genetic, transcriptomic, and proteomic analyses demonstrated that rsaX20 is repressed by the zinc regulator Zur and functions as a dual-purpose sRNA/small open reading frame, with the RNA and peptide exerting distinct, sometimes opposing, effects on target proteins. Together, these findings establish NEMO as a broadly applicable framework for interrogating microbial gene regulation directly from native metatranscriptomes and identify rsaX20 as a key regulator of zinc homeostasis during chronic human S. aureus infection.

Most knowledge of bacterial gene regulation comes from laboratory cultures, yet pathogens behave very differently inside the human body. We introduce NEMO, a pangenome-based framework that enables direct analysis of microbial gene expression from natural infection samples. Applying NEMO to human Staphylococcus aureus infections revealed transcriptional programs that differ substantially from standard in vitro models and highlighted small regulatory RNAs as major drivers of this divergence. Using this approach, we discovered rsaX20, a previously uncharacterized zinc-responsive regulatory RNA that also encodes a small peptide with distinct biological functions. These findings demonstrate the power of studying microbes in their native environments and uncover a key regulator of zinc homeostasis during chronic human infection.