16 research outputs found
IesR-1 affects the capacity of <i>S.</i> Typhimurium to invade and control growth within the human fibroblast cell line BJ-5ta.
<p><i>S.</i> Typhimurium wild type (white bars) and its isogenic Î<i>iesR-1</i>/5âČ 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. (*) <i>P</i><0.05 in studentâs t test.</p
The sRNA IesR-1 is up-regulated in non-growing intracellular <i>S.</i> Typhimurium.
<p>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.<sub>600</sub> âŒ0.2). 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077939#pone.0077939-Headley1" target="_blank">[35]</a> and PCN <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077939#pone.0077939-Lober1" target="_blank">[34]</a> 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 <i>S.</i> 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 <i>phoP</i> 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 (âŒ200â300 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 âŒ600 nt in intracellular bacteria compatible with the expected size of IesR-1. A second transcript of âŒ275 nt was also detected specifically in intracellular bacteria, which as denoted by the RACE experiments (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077939#pone-0077939-g001" target="_blank">Figure 1</a> D) could correspond to 5âČ region of IesR-1. Note the lack of noticeable amount of these two molecules in wild-type bacteria grown extracellularly.</p
Identification of a novel sRNA in the virulence plasmid pSLT of <i>S.</i> Typhimurium.
<p>(A) Venn diagrams showing the number of oligonucleotide probes corresponding to intergenic regions (IGR) induced in three experimental conditions (intracellular wild-type, intracellular <i>phoP</i>, late stationary phase wild-type) as compared to wild-type bacteria growing in exponential phase of growth (see text and ref. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077939#pone.0077939-NunezHernandez1" target="_blank">[23]</a> for details). Significance threshold was established at log<sub>2</sub>-expression ratios â„2.0. The table refers to microarray data of the CNB1344-0995 probe expressed as log<sub>2</sub> 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 <i>PSLT048</i> (<i>tlpA</i>)<i>-pSLT047-PSLT046</i> (<i>mig5</i>) 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 (TAPâ) 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 âaâ and âbâ; (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.</p
A Novel Antisense RNA from the <i>Salmonella</i> Virulence Plasmid pSLT Expressed by Non-Growing Bacteria inside Eukaryotic Cells
<div><p>Bacterial small RNAs (sRNAs) are regulatory molecules playing relevant roles in response to environmental changes, stressful conditions and pathogenesis. The intracellular bacterial pathogen <i>Salmonella enterica</i> serovar Typhimurium (<i>S.</i> Typhimurium) is known to regulate expression of some sRNAs during colonization of fibroblasts. Here, we characterize a previously unknown sRNA encoded in the <i>S.</i> 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 <i>PSLT047- PSLT046</i> (<i>mig5</i>) intergenic region. The sRNA transcript was further identified as a 597-nucleotide molecule, which we named IesR-1, for â<u>I</u>ntracellular-<u>e</u>xpressed-<u>sR</u>NA-1âČ. IesR-1 expression is low in bacteria growing in axenic cultures across a variety of experimental conditions but displays a marked increase (âŒ200â300 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 <i>PSLT047-iesR-1</i>-<i>PSLT046</i> (<i>mig5</i>) region support a <i>cis</i>-acting regulatory mechanism of IesR-1 as antisense RNA over the <i>PSLT047</i> transcript involving interaction at their respective 3âČ 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 5âČ and 3âČ regions. Taken together, these data reveal that <i>S.</i> 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.</p></div
IS<i>6110</i> gene expression and determination of transposition dynamics in the MTBC.
<p>(<b>a</b>) Total IS<i>6110</i> expression in representative strains from the MTBC. Data are relative to BCG Pasteur. Columns and error bars are the average and standard deviation from three independent cultures. (<b>b</b>) IS<i>6110</i> RFLP from MTBC strains analysed in panel (a). (<b>c</b>) IS<i>6110</i> expression values normalised to the copy number content of this element. Columns represent normalised expression of IS<i>6110</i> according to the left Y-axis. Red squares show the IS<i>6110</i> copy number in each strain indicated in the right Y-axis. Normalised expression of BCG Pasteur is used as reference. (<b>d</b>) Expression per <i>IS6110</i> copy relative to the copy number content in MTBC strains. Data fit with an exponential curve (r<sup>2</sup> = 0.80) indicated by a grey shadowed line.</p
New insights into the transposition mechanisms of IS<i>6110</i> and its dynamic distribution between <i>Mycobacterium tuberculosis</i> Complex lineages
<div><p>The insertion Sequence IS<i>6110</i>, only present in the pathogens of the <i>Mycobacterium tuberculosis</i> Complex (MTBC), has been the gold-standard epidemiological marker for TB for more than 25 years, but biological implications of IS<i>6110</i> transposition during MTBC adaptation to humans remain elusive. By studying 2,236 clinical isolates typed by IS<i>6110</i>-RFLP and covering the MTBC, we remarked a lineage-specific content of IS<i>6110</i> being higher in modern globally distributed strains. Once observed the IS<i>6110</i> distribution in the MTBC, we selected representative isolates and found a correlation between the normalized expression of IS<i>6110</i> and its abundance in MTBC chromosomes. We also studied the molecular regulation of IS<i>6110</i> transposition and we found a synergistic action of two post-transcriptional mechanisms: a -1 ribosomal frameshift and a RNA pseudoknot which interferes translation. The construction of a transcriptionally active transposase resulted in 20-fold increase of the transposition frequency. Finally, we examined transposition in <i>M</i>. <i>bovis</i> and <i>M</i>. <i>tuberculosis</i> during laboratory starvation and in a mouse infection model of TB. Our results shown a higher transposition in <i>M</i>. <i>tuberculosis</i>, that preferably happens during TB infection in mice and after one year of laboratory culture, suggesting that IS<i>6110</i> transposition is dynamically adapted to the host and to adverse growth conditions.</p></div
The PhoP-Dependent ncRNA Mcr7 Modulates the TAT Secretion System in <i>Mycobacterium tuberculosis</i>
<div><p>The PhoPR two-component system is essential for virulence in <i>Mycobacterium tuberculosis</i> where it controls expression of approximately 2% of the genes, including those for the ESX-1 secretion apparatus, a major virulence determinant. Mutations in <i>phoP</i> lead to compromised production of pathogen-specific cell wall components and attenuation both <i>ex vivo</i> and <i>in vivo</i>. Using antibodies against the native protein in ChIP-seq experiments (chromatin immunoprecipitation followed by high-throughput sequencing) we demonstrated that PhoP binds to at least 35 loci on the <i>M. tuberculosis</i> genome. The PhoP regulon comprises several transcriptional regulators as well as genes for polyketide synthases and PE/PPE proteins. Integration of ChIP-seq results with high-resolution transcriptomic analysis (RNA-seq) revealed that PhoP controls 30 genes directly, whilst regulatory cascades are responsible for signal amplification and downstream effects through proteins like EspR, which controls Esx1 function, <i>via</i> regulation of the <i>espACD</i> operon. The most prominent site of PhoP regulation was located in the intergenic region between <i>rv2395</i> and <i>PE_PGRS41</i>, where the <i>mcr7</i> gene codes for a small non-coding RNA (ncRNA). Northern blot experiments confirmed the absence of Mcr7 in an <i>M. tuberculosis phoP</i> mutant as well as low-level expression of the ncRNA in <i>M. tuberculosis</i> complex members other than <i>M. tuberculosis</i>. By means of genetic and proteomic analyses we demonstrated that Mcr7 modulates translation of the <i>tatC</i> mRNA thereby impacting the activity of the Twin Arginine Translocation (Tat) protein secretion apparatus. As a result, secretion of the immunodominant Ag85 complex and the beta-lactamase BlaC is affected, among others. Mcr7, the first ncRNA of <i>M. tuberculosis</i> whose function has been established, therefore represents a missing link between the PhoPR two-component system and the downstream functions necessary for successful infection of the host.</p></div
IS<i>6110</i> in the MTBC.
<p>(<b>a</b>) Schematic phylogenetic relationships of MTBC members arisen from a most recent common ancestor (MRCA) after an evolutionary bottleneck. For <i>M</i>. <i>tuberculosis</i> different lineages and families are indicated. The position of IS<i>6110</i> sequences in fully assembled genomes in indicated by black dots. The arrow indicates the position of IS<i>6110</i> in the Direct Repeat region of the CRISPR-Cas locus, which is common to most MTBC strains. For the remaining 17 <i>M</i>. <i>tuberculosis</i> strains different from H37Rv, the number of IS<i>6110</i> sequences is indicated by a box plot (median = 17). (<b>b</b>) Box plots showing the IS<i>6110</i> copies in MTBC families. For each family, the lineage according to panel (a) is provided in parenthesis in the X-axis. For clarity, L4 have been subdivided into 5 different families according to spoligotyping.</p
Transposition in the laboratory and in a mouse infection model using reference <i>M</i>. <i>bovis</i> and <i>M</i>. <i>tuberculosis</i> strains.
<p>(<b>a</b>) Experimental model to measure transposition rates in <i>M</i>. <i>tuberculosis</i> H37Rv (15 IS<i>6110</i> copies) and <i>M</i>. <i>bovis</i> AF2122/97 (1 IS<i>6110</i> copy). Both strains are transformed with pIR-Km and used to inoculate liquid media or to intranasally infect C57BL/6 mice. After the indicated time points aliquots are plated in kanamycin and sucrose containing plates to ensure pIR-km loss and to recover colonies resulting from transposition. (<b>b</b>) Transposition frequencies in laboratory medium in <i>M</i>. <i>bovis</i> and <i>M</i>. <i>tuberculosis</i> are indicated by red and blue columns respectively. Error bars indicate the standard deviation of the mean value from three independent cultures. Transposition preferentially occurs in <i>M</i>. <i>tuberculosis</i> after the stationary phase reaching it maximum in the starvation period. (<b>c</b>) Expression of <i>M</i>. <i>tuberculosis</i> IS<i>6110</i> during exponential, stationary and starvation periods <i>in vitro</i>. Expression of ORF1 and ORF2 are indicated by dark and light blue columns respectively. Results are from three independent cultures. (<b>d</b>) Transposition frequencies during mouse infection with <i>M</i>. <i>bovis</i> or <i>M</i>. <i>tuberculosis</i> are indicated by red and blue columns respectively. Data from lung and spleen are shown and error bars indicate the standard deviation of the mean value from three independent mice. <i>M</i>. <i>bovis</i> does not exhibit increased transposition rates <i>in vivo</i> relative to liquid culture. Conversely <i>M</i>. <i>tuberculosis</i> show 10-fold higher transposition rates compared to exponential growth <i>in vitro</i>. In all cases, transposition frequencies were calculated relative to the total number of CFU in either cultures or mouse organs.</p
Summary of ChIP-seq results showing localization of PhoP binding sites in the <i>M. tuberculosis</i> genome.
<p>The table lists regions enriched in immunoprecipitated PhoP-DNA complexes from the H37Rv wild type strain as compared to the <i>phoP</i> mutant. Fold change expression values as determined by RNA-seq experiments are also reported for the flanking genes. Predicted or validated operons found to be deregulated in the <i>phoP</i> mutant are indicated.</p>a<p>p value <0.0001.</p>b<p>FDR 0.00%.</p>c<p>This column lists the predicted operons (according to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004183#ppat.1004183-Uplekar1" target="_blank">[17]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004183#ppat.1004183-Roback1" target="_blank">[18]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004183#ppat.1004183-Price1" target="_blank">[19]</a>), located downstream of PhoP binding sites, that were found to be deregulated in the <i>phoP</i> mutant compared to the wild type strain.</p>§<p>Region detected is between two genes transcribed in opposite direction.</p>ÂŁ<p>Region located in 3âČ-end of the gene.</p><p>ND: fold change expression levels of <i>phoP</i> were not quantified since the gene carries a deletion in the mutant strain.</p