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Bacterial small RNAs (sRNAs) are regulatory molecules playing relevant roles in response to environmental changes, stressful conditions and pathogenesis. The intracellular bacterial pathogen  Salmonella enterica  serovar Typhimurium ( S.  Typhimurium) is known to regulate expression of some sRNAs during colonization of fibroblasts. Here, we characterize a previously unknown sRNA encoded in the  S.  Typhimurium pSLT virulence plasmid that is specifically up-regulated by non-growing dormant bacteria persisting inside fibroblasts. This sRNA was inferred in microarray expression analyses, which unraveled enhanced transcriptional activity in the  PSLT047- PSLT046  ( mig5 ) intergenic region. The sRNA transcript was further identified as a 597-nucleotide molecule, which we named IesR-1, for ‘ I ntracellular- e xpressed- sR NA-1′. IesR-1 expression is low in bacteria growing in axenic cultures across a variety of experimental conditions but displays a marked increase (∼200–300 fold) following bacterial entry into fibroblasts. Remarkably, induction of IesR-1 expression is not prominent in bacteria proliferating within epithelial cells. IesR-1 deletion affects the control of bacterial growth in defined fibroblast cell lines and impairs virulence in a mouse infection model. Expression analyses performed in the  PSLT047-iesR-1 - PSLT046  ( mig5 ) region support a  cis -acting regulatory mechanism of IesR-1 as antisense RNA over the  PSLT047  transcript involving interaction at their respective 3′ ends and modulation of PSLT047 protein levels. This model is sustained by the scarce production of PSLT047 protein observed in non-growing intracellular bacteria and the high amount of PSLT047 protein produced by bacteria carrying a truncated IesR-1 version with separated 5′ and 3′ regions. Taken together, these data reveal that  S.  Typhimurium sRNAs encoded in the pSLT virulence plasmid respond to a state of persistence inside the host cell. As exemplified by IesR-1, some of these sRNAs may contribute to diminish the relative levels of proteins, such as PSLT047, which are probably dispensable for the intracellular lifestyle.

A Novel Antisense RNA from the Salmonella Virulence Plasmid pSLT Expressed by Non-Growing Bacteria inside Eukaryotic Cells