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. 2013 Oct 31;8(10):e77939.
doi: 10.1371/journal.pone.0077939. eCollection 2013.

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

Jesús Gonzalo-Asensio  1 , Alvaro D Ortega, Gadea Rico-Pérez, M Graciela Pucciarelli, Francisco García-Del Portillo
Affiliations

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

Jesús Gonzalo-Asensio et al. PLoS One. .

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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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. 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. 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.
Figure 2
Figure 2. The sRNA IesR-1 is up-regulated in non-growing intracellular S. Typhimurium.
Relative levels of the sRNA IesR-1 were determined by reverse transcription and RT-qPCR. Data are relative to the transcript levels of IesR-1 in bacteria cultured with shaking in LB broth to early-exponential phase (O.D.600 ∼0.2). 16S ribosomal RNA was used as a reference gene. Bars indicate the mean ± standard deviation of three independent experiments. (A) IesR-1 expression in axenic cultures. Bacteria were cultured in LB broth and minimal media such as ISM and PCN with shaking to either exponential or stationary growth phases. A third condition consisting in growth with no shaking to stationary phase was also included in the analysis. (B) Expression of IesR-1 in non-growing intracellular S. Typhimurium collected at different post-infection times from rat and human fibroblasts (NRK-49F and BJ-5ta, respectively). For comparison, IesR-1 expression was also monitored in wild-type bacteria proliferating inside HeLa epithelial cells and in phoP mutant bacteria overgrowing within NRK-49F fibroblasts. The 0 h post-infection time point corresponds to bacteria grown overnight with no shaking in LB broth that were used to infect the eukaryotic cells. Note the pronounced up-regulation of IesR-1 (∼200–300 fold) in non-growing intracellular bacteria at late post-infection times, which contrasts with the moderate induction values registered in extracellular bacteria. Data are the mean and the standard deviation of three independent experiments. (C) Northern blot analysis showing the production of an sRNA of ∼600 nt in intracellular bacteria compatible with the expected size of IesR-1. A second transcript of ∼275 nt was also detected specifically in intracellular bacteria, which as denoted by the RACE experiments (see Figure 1 D) could correspond to 5′ region of IesR-1. Note the lack of noticeable amount of these two molecules in wild-type bacteria grown extracellularly.
Figure 3
Figure 3. 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.
Figure 4
Figure 4. Alteration in the integrity of the IesR-1 molecule causes a defect in virulence that is not restored by complementation in trans.
(A) Strains ΔiesR-1/5′::cat or ΔiesR-1/5′ were used in competition experiments in susceptible BALB/c mice. While both strains are devoid of the 5′ region of IesR-1, the strain ΔiesR-1/5′::cat overexpresses the 3′ region (lower part of the panel and Figure S3). Note that both strains are attenuated in virulence; (B) Competition experiments with strains ΔiesR-1/5′ overexpressing in trans from a construct positioned in the araE locus the entire IesR-1 molecule or exclusively the 5′ region (208 nt) of IesR-1 (lower part of the panel). Note that in any case the expression in trans of these molecules restored virulence. Groups of four to eight mice were inoculated with the indicated strain pairs by the intraperitoneal route. Liver and spleen were extracted 72 h post-infection and homogenated. Appropriate dilutions were plated onto LB plates with and without antibiotic and the competitive index (CI) calculated as the ratio between strains in the organs versus the ratio in the inoculum. p-values were obtained by one-sample student’s t test with log-transformed data and establishing 0 as hypothetical value.
Figure 5
Figure 5. 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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This work was supported by grants BIO2010-18885 and Consolider-CSD2008-00013-INTERMODS from the Spanish Ministry of Economy and Competitiveness; and, PIE-201320E020 from the ‘Agencia Estatal CSIC’ (to F.G.-P.). J.G.A. held a ‘Juan de la Cierva’ contract from the Spanish Ministry of Economy and Competitiveness. G.R.P. holds a FPU (Formación de Profesorado Universitario) fellowship from the Spanish Ministry of Education and Culture. A.D.O. is contracted under the CSD2008-00013-INTERMODS grant. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.