Functional decoding reveals a hidden regulatory layer of the Salmonella transcriptome during infection

Bacterial transcriptomes contain extensive, largely unexplored regulatory information beyond annotated genes, including small regulatory RNAs (sRNAs), yet distinguishing functionally active transcripts from the broader non-coding transcriptome remains a fundamental challenge, particularly in the context of host-pathogen interactions. Here, we develop an unbiased highthroughput functional screening strategy to decode the regulatory potential of the Salmonella enterica transcriptome during macrophage infection. A pooled expression library comprising 875 RNA fragments derived from infection-relevant conditions was screened for effects on bacterial invasion and intracellular survival, revealing distinct and stage-specific regulatory activities. Functional characterization uncovered non-canonical sRNAs originating from 5' untranslated regions that differentially modulate virulence-associated programs spanning the SPI-1, SPI-4 and SPI-2 pathogenicity islands. One attenuates the SPI-1 and SPI-4 secretion systems, limiting bacterial adhesion and invasion, whereas the other promotes invasion-associated programs while repressing pathways supporting intracellular survival. Rather than acting solely on individual virulence determinants, these regulatory activities reveal a broader layer of posttranscriptional control that coordinates bacterial adaptation across the dynamic host environment. Our study establishes a scalable strategy for extracting functional regulatory information from condition-specific bacterial transcriptomes and provides a framework for uncovering context-dependent RNA-mediated control of complex host-associated phenotypes.