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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 affects the capacity of S. Typhimurium to invade and control growth within the human fibroblast cell line BJ-5ta.S. Typhimurium wild type (white bars) and its isogenic ΔiesR-1/5′ mutant (black bars) were used to infect rat and human fibroblasts (NRK-49F and BJ-5ta, respectively). HeLa epithelial cells were also infected for comparison. Viable intracellular bacteria were counted at 2 h, 6 h (HeLa cells) and 2 h, 24 h (fibroblasts) post-infection. (A) Invasion rates. Bars represent the percentage of bacteria from the initial inoculum that was internalized by the cells upon 30 min of incubation. (B) Intracellular proliferation rates. Bars represent the ratio between the number of viable intracellular bacteria counted at 24 h (fibroblasts) or 6 h (HeLa cells) relative to that determined at 2 h post-infection. Values are the mean ± standard deviation from three independent experiments. (*) P<0.05 in student’s t test.
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pone-0077939-g003: IesR-1 affects the capacity of S. Typhimurium to invade and control growth within the human fibroblast cell line BJ-5ta.S. Typhimurium wild type (white bars) and its isogenic ΔiesR-1/5′ mutant (black bars) were used to infect rat and human fibroblasts (NRK-49F and BJ-5ta, respectively). HeLa epithelial cells were also infected for comparison. Viable intracellular bacteria were counted at 2 h, 6 h (HeLa cells) and 2 h, 24 h (fibroblasts) post-infection. (A) Invasion rates. Bars represent the percentage of bacteria from the initial inoculum that was internalized by the cells upon 30 min of incubation. (B) Intracellular proliferation rates. Bars represent the ratio between the number of viable intracellular bacteria counted at 24 h (fibroblasts) or 6 h (HeLa cells) relative to that determined at 2 h post-infection. Values are the mean ± standard deviation from three independent experiments. (*) P<0.05 in student’s t test.

Mentions: Wild-type and ΔiesR-1/5′ strains were used in in vitro infection assays with rat and human fibroblasts (NRK-49F and BJ-5ta cell lines, respectively) and human HeLa epithelial cells. Except for BJ-5ta fibroblasts, in which the lack of IesR-1 was linked to a decrease of ∼2-fold in the invasion rate, no significant differences were observed in the rest of cell lines used (Figure 3A). Concerning intracellular phenotypes, the only observed change was also observed in BJ-5ta human fibroblasts, in which the lack of IesR-1 correlated to an increase of ∼3-fold in the intracellular proliferation rate (Figure 3B). Since only minor alterations were observed in the in vitro infection models, we tested these strains in the mouse typhoid model. Previous studies reported a strong correlation between S. Typhimurium functions required for maintaining a persistent state within fibroblasts and their relative contribution to virulence [43]–[46]. So, we hypothesized a probable role in virulence of IesR-1, strongly up-regulated by non-growing intracellular bacteria (Figure 2B). We then examined the fitness of ΔiesR-1/5′::cat and ΔiesR-1/5′ strains in BALB/c mice using competitive infections upon intraperitoneal challenge. The ΔiesR-1/5′::cat mutant displayed a decreased fitness compared to wild-type bacteria (competitive index, CI ∼ 0.33) (Figure 4A). This result suggested that either the overexpression of the 3′ region, the lack of the 5′ region, or a combination of both, might attenuate virulence. Similar results were obtained when infecting mice by the oral route (results not shown). Interestingly, the ΔiesR-1/5′::cat mutant also displayed decreased fitness when mixed with ΔiesR-1/5′ mutant bacteria (Figure 4A). Since both strains are devoid of an identical 5′ region of IesR-1, this finding indicates that an increased expression of the 3′ region of IesR-1 might impair virulence. Competition experiments were also performed with the ΔiesR-1/5′ strain expressing in trans two versions of IesR-1: one encompassing the entire 597 nt molecule, MD2277 (ΔiesR-1/5′, cat::PLtetO iesR ); and, the other carrying only the first 208 nt of the 5′ region, MD2276 (ΔiesR-1/5′, cat::PLtetO iesR-5′). Expression of these IesR-1 molecules was verified by RT-PCR (data not shown). When these two complemented strains were confronted against the ΔiesR-1/5′ mutant no changes in virulence were observed (Figure 4B). Altogether, these results indicate that deregulation of IesR-1 expression in S. Typhimurium impacts of the progression of the infection and, simultaneously, suggest that the regulation that IesR-1 exerts over the PSLT047 transcript may require interaction of both RNA molecules while they are co-expressed in a specific location of the virulence plasmid.


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 affects the capacity of S. Typhimurium to invade and control growth within the human fibroblast cell line BJ-5ta.S. Typhimurium wild type (white bars) and its isogenic ΔiesR-1/5′ mutant (black bars) were used to infect rat and human fibroblasts (NRK-49F and BJ-5ta, respectively). HeLa epithelial cells were also infected for comparison. Viable intracellular bacteria were counted at 2 h, 6 h (HeLa cells) and 2 h, 24 h (fibroblasts) post-infection. (A) Invasion rates. Bars represent the percentage of bacteria from the initial inoculum that was internalized by the cells upon 30 min of incubation. (B) Intracellular proliferation rates. Bars represent the ratio between the number of viable intracellular bacteria counted at 24 h (fibroblasts) or 6 h (HeLa cells) relative to that determined at 2 h post-infection. Values are the mean ± standard deviation from three independent experiments. (*) P<0.05 in student’s t test.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3815029&req=5

pone-0077939-g003: IesR-1 affects the capacity of S. Typhimurium to invade and control growth within the human fibroblast cell line BJ-5ta.S. Typhimurium wild type (white bars) and its isogenic ΔiesR-1/5′ mutant (black bars) were used to infect rat and human fibroblasts (NRK-49F and BJ-5ta, respectively). HeLa epithelial cells were also infected for comparison. Viable intracellular bacteria were counted at 2 h, 6 h (HeLa cells) and 2 h, 24 h (fibroblasts) post-infection. (A) Invasion rates. Bars represent the percentage of bacteria from the initial inoculum that was internalized by the cells upon 30 min of incubation. (B) Intracellular proliferation rates. Bars represent the ratio between the number of viable intracellular bacteria counted at 24 h (fibroblasts) or 6 h (HeLa cells) relative to that determined at 2 h post-infection. Values are the mean ± standard deviation from three independent experiments. (*) P<0.05 in student’s t test.
Mentions: Wild-type and ΔiesR-1/5′ strains were used in in vitro infection assays with rat and human fibroblasts (NRK-49F and BJ-5ta cell lines, respectively) and human HeLa epithelial cells. Except for BJ-5ta fibroblasts, in which the lack of IesR-1 was linked to a decrease of ∼2-fold in the invasion rate, no significant differences were observed in the rest of cell lines used (Figure 3A). Concerning intracellular phenotypes, the only observed change was also observed in BJ-5ta human fibroblasts, in which the lack of IesR-1 correlated to an increase of ∼3-fold in the intracellular proliferation rate (Figure 3B). Since only minor alterations were observed in the in vitro infection models, we tested these strains in the mouse typhoid model. Previous studies reported a strong correlation between S. Typhimurium functions required for maintaining a persistent state within fibroblasts and their relative contribution to virulence [43]–[46]. So, we hypothesized a probable role in virulence of IesR-1, strongly up-regulated by non-growing intracellular bacteria (Figure 2B). We then examined the fitness of ΔiesR-1/5′::cat and ΔiesR-1/5′ strains in BALB/c mice using competitive infections upon intraperitoneal challenge. The ΔiesR-1/5′::cat mutant displayed a decreased fitness compared to wild-type bacteria (competitive index, CI ∼ 0.33) (Figure 4A). This result suggested that either the overexpression of the 3′ region, the lack of the 5′ region, or a combination of both, might attenuate virulence. Similar results were obtained when infecting mice by the oral route (results not shown). Interestingly, the ΔiesR-1/5′::cat mutant also displayed decreased fitness when mixed with ΔiesR-1/5′ mutant bacteria (Figure 4A). Since both strains are devoid of an identical 5′ region of IesR-1, this finding indicates that an increased expression of the 3′ region of IesR-1 might impair virulence. Competition experiments were also performed with the ΔiesR-1/5′ strain expressing in trans two versions of IesR-1: one encompassing the entire 597 nt molecule, MD2277 (ΔiesR-1/5′, cat::PLtetO iesR ); and, the other carrying only the first 208 nt of the 5′ region, MD2276 (ΔiesR-1/5′, cat::PLtetO iesR-5′). Expression of these IesR-1 molecules was verified by RT-PCR (data not shown). When these two complemented strains were confronted against the ΔiesR-1/5′ mutant no changes in virulence were observed (Figure 4B). Altogether, these results indicate that deregulation of IesR-1 expression in S. Typhimurium impacts of the progression of the infection and, simultaneously, suggest that the regulation that IesR-1 exerts over the PSLT047 transcript may require interaction of both RNA molecules while they are co-expressed in a specific location of the virulence plasmid.

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