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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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IesR-1 regulates production of the PSLT047 protein by a mechanism involving interaction at the 3′ ends of the respective RNA molecules.(A) expression pattern of iesR-1 and PSLT047 in extracellular bacteria grown in LB broth and in intracellular bacteria at 24 h post-infection of NRK-49F fibroblasts. Transcript levels were determined by reverse transcription and RT-qPCR. Expression data were calculated relative to the levels in bacteria cultured to early-exponential phase. 5S ribosomal RNA and the ompA transcript were used as endogenous controls for iesR-1 and PSLT047 transcripts, respectively. Bars indicate the mean ± standard deviation of three independent experiments. (B) Non-growing intracellular bacteria persisting in fibroblasts down-regulate the levels of the PSLT047 protein. The relative levels of PSLT047 were determined using a PSLT047::3×FLAG-tagged variant. Samples were prepared from bacteria grown in LB broth overnight in no-shaking conditions (inoculum) and from non-growing intracellular bacteria collected from NRK-49F fibroblasts at 24 h post-infection. The results from two independent experiments are shown. (C) Increased production of the PSLT047::3×FLAG-tagged variant in bacteria in which interaction between the 3′ ends of iesR-1 and PSLT047 is impeded by the presence of an antibiotic resistance cassette. The genetic configuration of the strains used is indicated. Results from two independent clones are shown. DnaK and OmpA were used as loading controls.
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pone-0077939-g005: IesR-1 regulates production of the PSLT047 protein by a mechanism involving interaction at the 3′ ends of the respective RNA molecules.(A) expression pattern of iesR-1 and PSLT047 in extracellular bacteria grown in LB broth and in intracellular bacteria at 24 h post-infection of NRK-49F fibroblasts. Transcript levels were determined by reverse transcription and RT-qPCR. Expression data were calculated relative to the levels in bacteria cultured to early-exponential phase. 5S ribosomal RNA and the ompA transcript were used as endogenous controls for iesR-1 and PSLT047 transcripts, respectively. Bars indicate the mean ± standard deviation of three independent experiments. (B) Non-growing intracellular bacteria persisting in fibroblasts down-regulate the levels of the PSLT047 protein. The relative levels of PSLT047 were determined using a PSLT047::3×FLAG-tagged variant. Samples were prepared from bacteria grown in LB broth overnight in no-shaking conditions (inoculum) and from non-growing intracellular bacteria collected from NRK-49F fibroblasts at 24 h post-infection. The results from two independent experiments are shown. (C) Increased production of the PSLT047::3×FLAG-tagged variant in bacteria in which interaction between the 3′ ends of iesR-1 and PSLT047 is impeded by the presence of an antibiotic resistance cassette. The genetic configuration of the strains used is indicated. Results from two independent clones are shown. DnaK and OmpA were used as loading controls.

Mentions: sRNAs that partially overlap with transcripts originated from flanking genes usually exert a cis-regulatory mechanism by antisense pairing [26]. Since IesR-1 partially overlaps with the 3′UTR of the PSLT047 transcript, we hypothesized on a possible antisense regulatory mechanism. Our previous results indicated that IesR-1 is selectively induced in non-growing dormant intracellular bacteria (Figure 2B). An inverse correlation between IesR-1 levels and its putative cis-regulated target would suggest a negative post-transcriptional regulation at the level of target mRNA stability. To test this, we monitored the expression pattern of PSLT047 and iesR-1 transcripts in different growth conditions (Figure 5A). Unexpectedly, PSLT047 mRNA expression followed essentially the same induction pattern as its putative sRNA cis-acting regulator, IesR-1 (Figure 5A). These results indicated that IesR-1 could have either no influence or, alternatively, a positive effect on PSLT047 mRNA stability. In this scenario, post-transcriptional control at the level of PSLT047 translation could also occur. When analyzed in non-growing intracellular bacteria, we observed that PSLT047 protein levels dropped significantly compared to extracellular bacteria in the inoculum (Figure 5B). Experiments with two strains carrying the PSLT047-3xFLAG tagged allele but differing in the integrity of the IesR-1 molecule provided additional support to the model involving interaction between 3′ regions of IesR-1 and the PSLT047 transcript with effects in PSLT047 protein levels. Thus, the amount of PSLT047 increased significantly in extracellular bacteria having an IesR-1 molecule interrupted with a Km resistance cassette, therefore unable to interact at its 3′ end with the PSLT047 transcript (Figure 5C). Altogether, these results are consistent with a cis-acting regulatory mechanism involving interaction of the antisense IesR-1 molecule with the PSLT047 mRNA, which could ultimately modulate the translation rate of this protein.


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)

IesR-1 regulates production of the PSLT047 protein by a mechanism involving interaction at the 3′ ends of the respective RNA molecules.(A) expression pattern of iesR-1 and PSLT047 in extracellular bacteria grown in LB broth and in intracellular bacteria at 24 h post-infection of NRK-49F fibroblasts. Transcript levels were determined by reverse transcription and RT-qPCR. Expression data were calculated relative to the levels in bacteria cultured to early-exponential phase. 5S ribosomal RNA and the ompA transcript were used as endogenous controls for iesR-1 and PSLT047 transcripts, respectively. Bars indicate the mean ± standard deviation of three independent experiments. (B) Non-growing intracellular bacteria persisting in fibroblasts down-regulate the levels of the PSLT047 protein. The relative levels of PSLT047 were determined using a PSLT047::3×FLAG-tagged variant. Samples were prepared from bacteria grown in LB broth overnight in no-shaking conditions (inoculum) and from non-growing intracellular bacteria collected from NRK-49F fibroblasts at 24 h post-infection. The results from two independent experiments are shown. (C) Increased production of the PSLT047::3×FLAG-tagged variant in bacteria in which interaction between the 3′ ends of iesR-1 and PSLT047 is impeded by the presence of an antibiotic resistance cassette. The genetic configuration of the strains used is indicated. Results from two independent clones are shown. DnaK and OmpA were used as loading controls.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3815029&req=5

pone-0077939-g005: IesR-1 regulates production of the PSLT047 protein by a mechanism involving interaction at the 3′ ends of the respective RNA molecules.(A) expression pattern of iesR-1 and PSLT047 in extracellular bacteria grown in LB broth and in intracellular bacteria at 24 h post-infection of NRK-49F fibroblasts. Transcript levels were determined by reverse transcription and RT-qPCR. Expression data were calculated relative to the levels in bacteria cultured to early-exponential phase. 5S ribosomal RNA and the ompA transcript were used as endogenous controls for iesR-1 and PSLT047 transcripts, respectively. Bars indicate the mean ± standard deviation of three independent experiments. (B) Non-growing intracellular bacteria persisting in fibroblasts down-regulate the levels of the PSLT047 protein. The relative levels of PSLT047 were determined using a PSLT047::3×FLAG-tagged variant. Samples were prepared from bacteria grown in LB broth overnight in no-shaking conditions (inoculum) and from non-growing intracellular bacteria collected from NRK-49F fibroblasts at 24 h post-infection. The results from two independent experiments are shown. (C) Increased production of the PSLT047::3×FLAG-tagged variant in bacteria in which interaction between the 3′ ends of iesR-1 and PSLT047 is impeded by the presence of an antibiotic resistance cassette. The genetic configuration of the strains used is indicated. Results from two independent clones are shown. DnaK and OmpA were used as loading controls.
Mentions: sRNAs that partially overlap with transcripts originated from flanking genes usually exert a cis-regulatory mechanism by antisense pairing [26]. Since IesR-1 partially overlaps with the 3′UTR of the PSLT047 transcript, we hypothesized on a possible antisense regulatory mechanism. Our previous results indicated that IesR-1 is selectively induced in non-growing dormant intracellular bacteria (Figure 2B). An inverse correlation between IesR-1 levels and its putative cis-regulated target would suggest a negative post-transcriptional regulation at the level of target mRNA stability. To test this, we monitored the expression pattern of PSLT047 and iesR-1 transcripts in different growth conditions (Figure 5A). Unexpectedly, PSLT047 mRNA expression followed essentially the same induction pattern as its putative sRNA cis-acting regulator, IesR-1 (Figure 5A). These results indicated that IesR-1 could have either no influence or, alternatively, a positive effect on PSLT047 mRNA stability. In this scenario, post-transcriptional control at the level of PSLT047 translation could also occur. When analyzed in non-growing intracellular bacteria, we observed that PSLT047 protein levels dropped significantly compared to extracellular bacteria in the inoculum (Figure 5B). Experiments with two strains carrying the PSLT047-3xFLAG tagged allele but differing in the integrity of the IesR-1 molecule provided additional support to the model involving interaction between 3′ regions of IesR-1 and the PSLT047 transcript with effects in PSLT047 protein levels. Thus, the amount of PSLT047 increased significantly in extracellular bacteria having an IesR-1 molecule interrupted with a Km resistance cassette, therefore unable to interact at its 3′ end with the PSLT047 transcript (Figure 5C). Altogether, these results are consistent with a cis-acting regulatory mechanism involving interaction of the antisense IesR-1 molecule with the PSLT047 mRNA, which could ultimately modulate the translation rate of this protein.

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