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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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Mice infection studies with sRNA deletion mutants and of L. monocytogenes. Bacterial load in mice organs were also determined following in vitro infection with 2000 CFU of L. monocytogenes EGD-e wild-type strain as well as its isogenic sRNA mutants rli31, rli33-1 and rli50*. On day 3 after infection, the numbers of viable bacteria in spleens (A) and livers (B) of three animals per group were determined of wild-type EGD-e versus rli31, rli33-1 and rli50* in spleen and liver, respectively (n = 4). Error bars indicate standard deviations (*P ≤ 0.005, **P < 0.05).
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Figure 5: Mice infection studies with sRNA deletion mutants and of L. monocytogenes. Bacterial load in mice organs were also determined following in vitro infection with 2000 CFU of L. monocytogenes EGD-e wild-type strain as well as its isogenic sRNA mutants rli31, rli33-1 and rli50*. On day 3 after infection, the numbers of viable bacteria in spleens (A) and livers (B) of three animals per group were determined of wild-type EGD-e versus rli31, rli33-1 and rli50* in spleen and liver, respectively (n = 4). Error bars indicate standard deviations (*P ≤ 0.005, **P < 0.05).

Mentions: We used the mouse infection model to assess the virulence properties of the sRNA mutants Δrli31, Δrli33-1 and Δrli50* to that of wild-type L. monocytogenes EGD-e. For all three mutants, survival and/or growth in the spleen, and in particular in the liver, were significantly reduced at day 3 post-infection when compared with wild-type (Figure 5A and B).Figure 5.


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)

Mice infection studies with sRNA deletion mutants and of L. monocytogenes. Bacterial load in mice organs were also determined following in vitro infection with 2000 CFU of L. monocytogenes EGD-e wild-type strain as well as its isogenic sRNA mutants rli31, rli33-1 and rli50*. On day 3 after infection, the numbers of viable bacteria in spleens (A) and livers (B) of three animals per group were determined of wild-type EGD-e versus rli31, rli33-1 and rli50* in spleen and liver, respectively (n = 4). 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 5: Mice infection studies with sRNA deletion mutants and of L. monocytogenes. Bacterial load in mice organs were also determined following in vitro infection with 2000 CFU of L. monocytogenes EGD-e wild-type strain as well as its isogenic sRNA mutants rli31, rli33-1 and rli50*. On day 3 after infection, the numbers of viable bacteria in spleens (A) and livers (B) of three animals per group were determined of wild-type EGD-e versus rli31, rli33-1 and rli50* in spleen and liver, respectively (n = 4). Error bars indicate standard deviations (*P ≤ 0.005, **P < 0.05).
Mentions: We used the mouse infection model to assess the virulence properties of the sRNA mutants Δrli31, Δrli33-1 and Δrli50* to that of wild-type L. monocytogenes EGD-e. For all three mutants, survival and/or growth in the spleen, and in particular in the liver, were significantly reduced at day 3 post-infection when compared with wild-type (Figure 5A and B).Figure 5.

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