Intramacrophage RIL-seq uncovers an RNA antagonist of the <i>Salmonella</i> virulence-associated small RNA PinT

Salmonella virulence chiefly relies upon two major pathogenicity islands, SPI-1 and SPI-2, which enable host cell invasion and intracellular survival, respectively. Growing evidence suggests post-transcriptional control of SPI gene expression by Hfq-dependent small regulatory RNAs (sRNAs) such as PinT. This 80-nucleotide sRNA is highly expressed after Salmonella enters host cells and modulates the transition from the SPI-1 to SPI-2 program by targeting the mRNAs of different virulence factors. However, it remains unclear how PinT activity can be counteracted when the suppression of virulence genes needs to be relieved. Here, we mapped the RNA interactome of Salmonella recovered from infected macrophages, using an optimized version of RIL-seq. In addition to offering an unprecedented view of Hfq-mediated RNA interactions during Salmonella's intracellular infection stage, RIL-seq uncovered the previously described 3' end-derived sRNA InvS as a direct negative regulator of PinT. Biochemical and genetic experiments suggest a decoy mechanism by which InvS lifts PinT-mediated target repression. Moreover, InvS acts as an mRNA repressor of the adhesion protein MipA and PinT interaction with InvS relieves mipA repression. Together, our work identifies a pair of antagonistic sRNAs in a growing post-transcriptional network of virulence gene regulation.