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The intracellular sRNA transcriptome of Listeria monocytogenes during growth in macrophages.

Mraheil MA, Billion A, Mohamed W, Mukherjee K, Kuenne C, Pischimarov J, Krawitz C, Retey J, Hartsch T, Chakraborty T, Hain T - Nucleic Acids Res. (2011)

Bottom Line: Currently extensive information exists on the sRNAs of Listeria monocytogenes expressed during growth in extracellular environments.A total of 29 regulatory RNAs, including small non-coding antisense RNAs, are specifically expressed intracellularly.Our analyses reveal extensive sRNA expression as an important feature of bacterial regulation during intracellular growth.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medical Microbiology, Justus-Liebig-University, Frankfurter Strasse 107, 35392 Giessen, Germany.

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 expressed during growth in extracellular environments. We used deep sequencing of cDNAs obtained from fractioned RNA (<500 nt) isolated from extracellularly growing bacteria and from L. monocytogenes infected macrophages to catalog the sRNA repertoire during intracellular bacterial growth. Here, we report on the discovery of 150 putative regulatory RNAs of which 71 have not 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 sRNA candidates 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.

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Survival of L. monocytogenes sRNA mutants in P388D1 murine macrophage cells. The macrophages were infected with the wild-type L. monocytogenes EGD-e and its isogenic deletion mutants, rli31, rli33-1 and rli50*, with an MOI of 10 in 24-well plates and bacterial CFU counts were measured on agar plated following lysis of the P388D1 cells after 4 h post-infection. n = 5; error bars indicate standard deviations (*P ≤ 0.005, **P ≤ 0.05).
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Figure 3: Survival of L. monocytogenes sRNA mutants in P388D1 murine macrophage cells. The macrophages were infected with the wild-type L. monocytogenes EGD-e and its isogenic deletion mutants, rli31, rli33-1 and rli50*, with an MOI of 10 in 24-well plates and bacterial CFU counts were measured on agar plated following lysis of the P388D1 cells after 4 h post-infection. n = 5; error bars indicate standard deviations (*P ≤ 0.005, **P ≤ 0.05).

Mentions: We assessed the mutant strains’ capability to grow in P388D1 murine macrophages together with the wild-type strain. Whereas the mutants Δrli31, Δrli33-1 and Δrli50* were significantly impaired in their abilities to proliferate intracellularly in macrophages as compared with the wild-type strain (Figure 3), no differences were observable in their in vitro growth in BHI (Supplementary Figure S6).Figure 3.


The intracellular sRNA transcriptome of Listeria monocytogenes during growth in macrophages.

Mraheil MA, Billion A, Mohamed W, Mukherjee K, Kuenne C, Pischimarov J, Krawitz C, Retey J, Hartsch T, Chakraborty T, Hain T - Nucleic Acids Res. (2011)

Survival of L. monocytogenes sRNA mutants in P388D1 murine macrophage cells. The macrophages were infected with the wild-type L. monocytogenes EGD-e and its isogenic deletion mutants, rli31, rli33-1 and rli50*, with an MOI of 10 in 24-well plates and bacterial CFU counts were measured on agar plated following lysis of the P388D1 cells after 4 h post-infection. n = 5; error bars indicate standard deviations (*P ≤ 0.005, **P ≤ 0.05).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3105390&req=5

Figure 3: Survival of L. monocytogenes sRNA mutants in P388D1 murine macrophage cells. The macrophages were infected with the wild-type L. monocytogenes EGD-e and its isogenic deletion mutants, rli31, rli33-1 and rli50*, with an MOI of 10 in 24-well plates and bacterial CFU counts were measured on agar plated following lysis of the P388D1 cells after 4 h post-infection. n = 5; error bars indicate standard deviations (*P ≤ 0.005, **P ≤ 0.05).
Mentions: We assessed the mutant strains’ capability to grow in P388D1 murine macrophages together with the wild-type strain. Whereas the mutants Δrli31, Δrli33-1 and Δrli50* were significantly impaired in their abilities to proliferate intracellularly in macrophages as compared with the wild-type strain (Figure 3), no differences were observable in their in vitro growth in BHI (Supplementary Figure S6).Figure 3.

Bottom Line: Currently extensive information exists on the sRNAs of Listeria monocytogenes expressed during growth in extracellular environments.A total of 29 regulatory RNAs, including small non-coding antisense RNAs, are specifically expressed intracellularly.Our analyses reveal extensive sRNA expression as an important feature of bacterial regulation during intracellular growth.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medical Microbiology, Justus-Liebig-University, Frankfurter Strasse 107, 35392 Giessen, Germany.

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 expressed during growth in extracellular environments. We used deep sequencing of cDNAs obtained from fractioned RNA (<500 nt) isolated from extracellularly growing bacteria and from L. monocytogenes infected macrophages to catalog the sRNA repertoire during intracellular bacterial growth. Here, we report on the discovery of 150 putative regulatory RNAs of which 71 have not 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 sRNA candidates 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.

Show MeSH
Related in: MedlinePlus