Unknown,Transcriptomics,Genomics,Proteomics

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Non-coding RNA-Seq of Listeria monocytogenes from bacteria growing extracellularly and in macrophages

ABSTRACT: Small non-coding RNAs (sRNAs) are widespread effectors of post-transcriptional gene regulation in bacteria. Currently extensive information exists on the sRNAs of Listeria monocytogenes (Lm) expressed during growth in extracellular environments. We used deep sequencing of cDNAs obtained from fractioned RNA (<500nt) isolated from extracellularly growing bacteria and from Lm-infected macrophages to catalog the sRNA repertoire during intracellular bacterial growth. Here we report on the discovery of 150 regulatory RNAs of which 71 have never been previously described. A total of 29 regulatory RNAs, including small non-coding antisense RNAs, are specifically expressed intracellularly. We validated highly expressed sRNAs by Northern blotting and demonstrated by the construction and characterization of isogenic mutants of rli31, rli33-1 and rli50 for intracellular expressed sRNA candidates, that their expression is required for efficient growth of bacteria in macrophages. All three mutants were attenuated when assessed for growth in mouse and insect models of infection. Comparative genomic analysis revealed the presence of lineage specific sRNAs and the absence of sRNA loci in genomes of naturally-occurring infection-attenuated bacteria, with additional loss in non-pathogenic listerial genomes. Our analyses reveal extensive sRNA expression as an important feature of bacterial regulation during intracellular growth.

ORGANISM(S): Listeria monocytogenes EGD-e

SUBMITTER: Andre Billion

PROVIDER: E-MTAB-329 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Objective: Reg3g has been proposed to have a protective role against infection due to its bactericidal effect on Gram-positive bacteria, but evidence from in vivo studies is lacking. Therefore we generated a Reg3g-/- mouse, to determine its role in intestinal homeostasis and protection against experimental infection. Methods: Reg3g-/- mice were phenotyped using histological methods and a range of innate and immune markers. To investigate the antimicrobial role of Reg3g we experimentally infected mice with Gram-positive Listeria monocytogenes and Gram-negative Salmonella entertitidis and measured translocated bacteria, mucosal and systemic markers of infection. Results: Reg3g-/- mice display altered ileal mucus distribution and increased bacterial contact with the epithelium. , concomitant with This increased the inflammatory status in of the ileal mucosa and increased expression of Il-22, myeloperoxidase (MPO) and serum chemokines in serum. In response to infection, Reg3g-/- mice showed transcriptome changes and elevated levels of mucosal MPO in the ileum, but no increased bacterial translocation to the organs. Conclusions: Reg3g is equally distributed throughout the mucus of wild type (wt) mice and its absence results in an altered distribution of the ileal mucus. Reg3g deficiency also results resulted in increased bacterial contact with the epithelium and heightened inflammatory responses in the ileal mucosa. We propose that Reg3g binds pathogens suggesting it and contributes to mucus barrier function by ensnaring bacteria. Compared to wt mice, Reg3g-/- mice infected with S. enteritidis and L. monocytogenes show an increase of mucosal inflammatory markers indicating the protective, anti-microbial roles of Reg3g in defence against both Gram-positive and -negative bacteria. This study was set up according to a one-treatment, one-control design; treatments were inoculation with either Listeria monocytogenes or Salmonella enteritidis bacterial pathogens. The study results contain transcriptional profiles from infected and sham-infected control C57Bl/6 mice. In total, this study includes data from 2 treatments and 1 control of (pooled) wild-type C57Bl/6 mice and Reg3g-/- KO mutant C57Bl/6 mice = 6 arrays.

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Non-coding RNA-Seq of Listeria monocytogenes from bacteria growing extracellularly and in macrophages

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