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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 Galleria mellonella larvae after inoculation with different L. monocytogenes sRNA mutants and L. innocua. Time course of survival of the larvae varies with the type of sRNA mutants employed for inoculation. Inoculation with 106 CFU/larvae EGD-e resulted in significantly higher killing rate of larvae in comparison to (A) rli31, (B) rli33-1 and (C) rli50*. The non-pathogenic L. innocua showed no mortality. Values represent means of at least three independent experiments ± standard deviations for 20 larvae per treatment (*P ≤ 0.005).
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Figure 4: Survival of Galleria mellonella larvae after inoculation with different L. monocytogenes sRNA mutants and L. innocua. Time course of survival of the larvae varies with the type of sRNA mutants employed for inoculation. Inoculation with 106 CFU/larvae EGD-e resulted in significantly higher killing rate of larvae in comparison to (A) rli31, (B) rli33-1 and (C) rli50*. The non-pathogenic L. innocua showed no mortality. Values represent means of at least three independent experiments ± standard deviations for 20 larvae per treatment (*P ≤ 0.005).

Mentions: We investigated the survival of larvae from Galleria following injection with different sRNA mutants of L. monocytogenes. Bacterial cultures grown to exponential phase were injected dorsolaterally into the hemocoel at 106 CFU/larva. We observed significant attenuation in the mortality rates of larvae when injected with Δrli31, Δrli33-1 and Δrli50* in comparison to wild-type EGD-e (Figure 4A–C).Figure 4.


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 Galleria mellonella larvae after inoculation with different L. monocytogenes sRNA mutants and L. innocua. Time course of survival of the larvae varies with the type of sRNA mutants employed for inoculation. Inoculation with 106 CFU/larvae EGD-e resulted in significantly higher killing rate of larvae in comparison to (A) rli31, (B) rli33-1 and (C) rli50*. The non-pathogenic L. innocua showed no mortality. Values represent means of at least three independent experiments ± standard deviations for 20 larvae per treatment (*P ≤ 0.005).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 4: Survival of Galleria mellonella larvae after inoculation with different L. monocytogenes sRNA mutants and L. innocua. Time course of survival of the larvae varies with the type of sRNA mutants employed for inoculation. Inoculation with 106 CFU/larvae EGD-e resulted in significantly higher killing rate of larvae in comparison to (A) rli31, (B) rli33-1 and (C) rli50*. The non-pathogenic L. innocua showed no mortality. Values represent means of at least three independent experiments ± standard deviations for 20 larvae per treatment (*P ≤ 0.005).
Mentions: We investigated the survival of larvae from Galleria following injection with different sRNA mutants of L. monocytogenes. Bacterial cultures grown to exponential phase were injected dorsolaterally into the hemocoel at 106 CFU/larva. We observed significant attenuation in the mortality rates of larvae when injected with Δrli31, Δrli33-1 and Δrli50* in comparison to wild-type EGD-e (Figure 4A–C).Figure 4.

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