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

Gonzalo-Asensio J, Ortega AD, Rico-Pérez G, Pucciarelli MG, García-Del Portillo F - PLoS ONE (2013)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas (CNB-CSIC), Madrid, Spain.

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.

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Identification of a novel sRNA in the virulence plasmid pSLT of S. Typhimurium.(A) Venn diagrams showing the number of oligonucleotide probes corresponding to intergenic regions (IGR) induced in three experimental conditions (intracellular wild-type, intracellular phoP, late stationary phase wild-type) as compared to wild-type bacteria growing in exponential phase of growth (see text and ref. [23] for details). Significance threshold was established at log2-expression ratios ≥2.0. The table refers to microarray data of the CNB1344-0995 probe expressed as log2 changes in relative levels. The probe was spotted as duplicate in different locations of the microarray slide; (B) Schematic representation of the pSLT plasmid region encompassing the PSLT048 (tlpA)-pSLT047-PSLT046 (mig5) genes. Bended arrows indicate predicted transcriptional start sites of RNAs expressed in this region. Hairpins indicate predicted Rho-independent terminators. Relative positions of the primers used in circular RACE are indicated as colored arrows; (C) Amplification products obtained from circular RACE in pyrophosphatase-treated (TAP+) and control (TAP−) RNA samples. On the left, approximate electrophoretic mobility of molecular weight standards corresponding to 0.7, 0.6, 0.5, 0.4, 0.3 and 0.2 Kb. Results for IGR-0995 correspond to the amplification products obtained using the two primer pairs indicated in the panel B as pairs “a” and “b”; (D) Sequence coverage of clones obtained by circular RACE. Y-axis represents the number of times that a nucleotide in a certain position was found in the sequenced clones. X-axis represents the sequence of the pSLT virulence plasmid, and the coordinates in Kb are in accordance to the NCBI reference sequence NC_017720. Arrows indicate the position of the different coding sequences.
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pone-0077939-g001: Identification of a novel sRNA in the virulence plasmid pSLT of S. Typhimurium.(A) Venn diagrams showing the number of oligonucleotide probes corresponding to intergenic regions (IGR) induced in three experimental conditions (intracellular wild-type, intracellular phoP, late stationary phase wild-type) as compared to wild-type bacteria growing in exponential phase of growth (see text and ref. [23] for details). Significance threshold was established at log2-expression ratios ≥2.0. The table refers to microarray data of the CNB1344-0995 probe expressed as log2 changes in relative levels. The probe was spotted as duplicate in different locations of the microarray slide; (B) Schematic representation of the pSLT plasmid region encompassing the PSLT048 (tlpA)-pSLT047-PSLT046 (mig5) genes. Bended arrows indicate predicted transcriptional start sites of RNAs expressed in this region. Hairpins indicate predicted Rho-independent terminators. Relative positions of the primers used in circular RACE are indicated as colored arrows; (C) Amplification products obtained from circular RACE in pyrophosphatase-treated (TAP+) and control (TAP−) RNA samples. On the left, approximate electrophoretic mobility of molecular weight standards corresponding to 0.7, 0.6, 0.5, 0.4, 0.3 and 0.2 Kb. Results for IGR-0995 correspond to the amplification products obtained using the two primer pairs indicated in the panel B as pairs “a” and “b”; (D) Sequence coverage of clones obtained by circular RACE. Y-axis represents the number of times that a nucleotide in a certain position was found in the sequenced clones. X-axis represents the sequence of the pSLT virulence plasmid, and the coordinates in Kb are in accordance to the NCBI reference sequence NC_017720. Arrows indicate the position of the different coding sequences.

Mentions: The transcriptomic analyses in non-growing intracellular wild-type bacteria revealed a significant induction (>4-fold, p<0.05) in probes mapping to 73 IGRs (Figure 1A). Ten of these 73 probes were also registered with increased signal in intracellular phoP overgrowing bacteria and 32 were in common with enhanced signals obtained in bacteria grown to late-stationary phase in LB broth (Figure 1A). Interestingly, 39 transcripts encoded in IGRs were found specifically induced in non-growing intracellular dormant bacteria, which suggests the occurrence of presumptive sRNAs with a putative role in pathogen persistence within fibroblasts (Figure 1A). Among these 39 probes, two of them (CNB1344-0963 and CNB1344-0995) lie to the Salmonella virulence plasmid pSLT. The sequence of these two probes in shown as supporting information (Figure S1). At the time of designing the ‘Salgenomics’ microarray, the CNB1344-0963 probe was located within an “empty” IGR non-containing any protein-encoding open reading frame. However, the subsequent comparison to other S. Typhimurium plasmids (pSTU288-1, p798_93, pYT2, pSal8934b, pSal6919a, pSTUK-100, TY474p1, pSTMDT12_L, pYT1) indicated that trbE gene maps to this region. Since the CNB1344-0963 probe is located between traN and traF and they are likely to be transcribed as an operon, the transcriptional signal detected from this probe could most probably correspond to the polycistronic mRNA rather than to a novel sRNA. We therefore focused in the transcript detected by the CNB1344-0995 probe (Figure 1A).


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

Gonzalo-Asensio J, Ortega AD, Rico-Pérez G, Pucciarelli MG, García-Del Portillo F - PLoS ONE (2013)

Identification of a novel sRNA in the virulence plasmid pSLT of S. Typhimurium.(A) Venn diagrams showing the number of oligonucleotide probes corresponding to intergenic regions (IGR) induced in three experimental conditions (intracellular wild-type, intracellular phoP, late stationary phase wild-type) as compared to wild-type bacteria growing in exponential phase of growth (see text and ref. [23] for details). Significance threshold was established at log2-expression ratios ≥2.0. The table refers to microarray data of the CNB1344-0995 probe expressed as log2 changes in relative levels. The probe was spotted as duplicate in different locations of the microarray slide; (B) Schematic representation of the pSLT plasmid region encompassing the PSLT048 (tlpA)-pSLT047-PSLT046 (mig5) genes. Bended arrows indicate predicted transcriptional start sites of RNAs expressed in this region. Hairpins indicate predicted Rho-independent terminators. Relative positions of the primers used in circular RACE are indicated as colored arrows; (C) Amplification products obtained from circular RACE in pyrophosphatase-treated (TAP+) and control (TAP−) RNA samples. On the left, approximate electrophoretic mobility of molecular weight standards corresponding to 0.7, 0.6, 0.5, 0.4, 0.3 and 0.2 Kb. Results for IGR-0995 correspond to the amplification products obtained using the two primer pairs indicated in the panel B as pairs “a” and “b”; (D) Sequence coverage of clones obtained by circular RACE. Y-axis represents the number of times that a nucleotide in a certain position was found in the sequenced clones. X-axis represents the sequence of the pSLT virulence plasmid, and the coordinates in Kb are in accordance to the NCBI reference sequence NC_017720. Arrows indicate the position of the different coding sequences.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3815029&req=5

pone-0077939-g001: Identification of a novel sRNA in the virulence plasmid pSLT of S. Typhimurium.(A) Venn diagrams showing the number of oligonucleotide probes corresponding to intergenic regions (IGR) induced in three experimental conditions (intracellular wild-type, intracellular phoP, late stationary phase wild-type) as compared to wild-type bacteria growing in exponential phase of growth (see text and ref. [23] for details). Significance threshold was established at log2-expression ratios ≥2.0. The table refers to microarray data of the CNB1344-0995 probe expressed as log2 changes in relative levels. The probe was spotted as duplicate in different locations of the microarray slide; (B) Schematic representation of the pSLT plasmid region encompassing the PSLT048 (tlpA)-pSLT047-PSLT046 (mig5) genes. Bended arrows indicate predicted transcriptional start sites of RNAs expressed in this region. Hairpins indicate predicted Rho-independent terminators. Relative positions of the primers used in circular RACE are indicated as colored arrows; (C) Amplification products obtained from circular RACE in pyrophosphatase-treated (TAP+) and control (TAP−) RNA samples. On the left, approximate electrophoretic mobility of molecular weight standards corresponding to 0.7, 0.6, 0.5, 0.4, 0.3 and 0.2 Kb. Results for IGR-0995 correspond to the amplification products obtained using the two primer pairs indicated in the panel B as pairs “a” and “b”; (D) Sequence coverage of clones obtained by circular RACE. Y-axis represents the number of times that a nucleotide in a certain position was found in the sequenced clones. X-axis represents the sequence of the pSLT virulence plasmid, and the coordinates in Kb are in accordance to the NCBI reference sequence NC_017720. Arrows indicate the position of the different coding sequences.
Mentions: The transcriptomic analyses in non-growing intracellular wild-type bacteria revealed a significant induction (>4-fold, p<0.05) in probes mapping to 73 IGRs (Figure 1A). Ten of these 73 probes were also registered with increased signal in intracellular phoP overgrowing bacteria and 32 were in common with enhanced signals obtained in bacteria grown to late-stationary phase in LB broth (Figure 1A). Interestingly, 39 transcripts encoded in IGRs were found specifically induced in non-growing intracellular dormant bacteria, which suggests the occurrence of presumptive sRNAs with a putative role in pathogen persistence within fibroblasts (Figure 1A). Among these 39 probes, two of them (CNB1344-0963 and CNB1344-0995) lie to the Salmonella virulence plasmid pSLT. The sequence of these two probes in shown as supporting information (Figure S1). At the time of designing the ‘Salgenomics’ microarray, the CNB1344-0963 probe was located within an “empty” IGR non-containing any protein-encoding open reading frame. However, the subsequent comparison to other S. Typhimurium plasmids (pSTU288-1, p798_93, pYT2, pSal8934b, pSal6919a, pSTUK-100, TY474p1, pSTMDT12_L, pYT1) indicated that trbE gene maps to this region. Since the CNB1344-0963 probe is located between traN and traF and they are likely to be transcribed as an operon, the transcriptional signal detected from this probe could most probably correspond to the polycistronic mRNA rather than to a novel sRNA. We therefore focused in the transcript detected by the CNB1344-0995 probe (Figure 1A).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas (CNB-CSIC), Madrid, Spain.

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.

Show MeSH
Related in: MedlinePlus