DOI: 10.1128/mbio.00637-26 ISSN: 2150-7511
Discovery of
Staphylococcus aureus
small RNAs highly expressed during human chronic infection
Paul Briaud, Julia Schumacher, Marvin Whiteley ABSTRACT
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 (
ne
twork transcriptomics of
m
icrobes in native envir
o
nments), 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.
IMPORTANCE
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.