A novel antisense RNA from the Salmonella virulence plasmid pSLT expressed by non-growing bacteria inside eukaryotic cells

PLoS One. 2013 Oct 31;8(10):e77939. doi: 10.1371/journal.pone.0077939. eCollection 2013.

Abstract

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 'Intracellular-expressed-sRNA-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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Biomarkers / metabolism
  • Blotting, Northern
  • Blotting, Western
  • Eukaryotic Cells / metabolism*
  • Eukaryotic Cells / microbiology
  • Fibroblasts / microbiology*
  • Gene Expression Profiling
  • Gene Expression Regulation, Bacterial*
  • Host-Pathogen Interactions
  • Mice
  • Oligonucleotide Array Sequence Analysis
  • Plasmids / genetics*
  • RNA, Antisense / genetics
  • RNA, Antisense / metabolism*
  • RNA, Bacterial / genetics*
  • RNA, Messenger / genetics
  • Rats
  • Real-Time Polymerase Chain Reaction
  • Reverse Transcriptase Polymerase Chain Reaction
  • Salmonella Infections / genetics
  • Salmonella Infections / microbiology
  • Salmonella typhimurium / genetics*
  • Salmonella typhimurium / growth & development
  • Salmonella typhimurium / metabolism
  • Virulence / genetics*
  • Virulence Factors / genetics
  • Virulence Factors / metabolism

Substances

  • Bacterial Proteins
  • Biomarkers
  • RNA, Antisense
  • RNA, Bacterial
  • RNA, Messenger
  • Virulence Factors

Grants and funding

This work was supported by grants BIO2010-18885 and Consolider-CSD2008-00013-INTERMODS from the Spanish Ministry of Economy and Competitiveness; and, PIE-201320E020 from the ‘Agencia Estatal CSIC’ (to F.G.-P.). J.G.A. held a ‘Juan de la Cierva’ contract from the Spanish Ministry of Economy and Competitiveness. G.R.P. holds a FPU (Formación de Profesorado Universitario) fellowship from the Spanish Ministry of Education and Culture. A.D.O. is contracted under the CSD2008-00013-INTERMODS grant. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.