Model for the mechanism of stimulation of transcriptional responses by <i>S</i>. Typhimurium and <i>S</i>. Typhi.

<p>Model for the mechanism of stimulation of transcriptional responses by <i>S</i>. Typhimurium and <i>S</i>. Typhi.</p

<i>Salmonella enterica</i> serovar-specific transcriptional reprogramming of infected cells

<div><p>Despite their high degree of genomic similarity, different <i>Salmonella enterica</i> serovars are often associated with very different clinical presentations. In humans, for example, the typhoidal <i>S</i>. <i>enterica</i> serovar Typhi causes typhoid fever, a life-threatening systemic disease. In contrast, the non-typhoidal <i>S</i>. <i>enterica</i> serovar Typhimurium causes self-limiting gastroenteritis. The molecular bases for these different clinical presentations are incompletely understood. The ability to re-program gene expression in host cells is an essential virulence factor for typhoidal and non-typhoidal <i>S</i>. <i>enterica</i> serovars. Here, we have compared the transcriptional profile of cultured epithelial cells infected with <i>S</i>. Typhimurium or <i>S</i>. Typhi. We found that both serovars stimulated distinct transcriptional responses in infected cells that are associated with the stimulation of specific signal transduction pathways. These specific responses were associated with the presence of a distinct repertoire of type III secretion effector proteins. These observations provide major insight into the molecular bases for potential differences in the pathogenic mechanisms of typhoidal and non-typhoidal <i>S</i>. <i>enterica</i> serovars.</p></div

Type III secretion effector proteins impart specificity to the cellular responses to <i>S</i>. <i>enterica</i> serovars Typhi and Typhimurium.

<p>(<b>A</b> and <b>B</b>) Stimulation of MAPK, STAT3 and NF-κB activation after infection with <i>S</i>. Typhi (Ty) strains expressing the indicated <i>S</i>. Typhimurium effector proteins. Henle-407 cells were infected with the indicated <i>S</i>. Typhimurium (Ty) or <i>S</i>. Typhi (Ty) strains and the activation of JNK, Erk, or STAT3 was assessed by Western blot analysis using antibodies directed to the phosphorylated (activated) forms of these kinases (<b>A</b>). Alternatively, the activation of NF-κB in infected cells was measured in HEK-293T cells transfected with a NF-κB luciferase reporter construct (<b>B</b>). Values shown are relative to the activity of the reporter in uninfected control cells and represent the mean ± standard deviation of three independent measurements. *: indicate statistical significance (Students t-test; <i>p</i> ≤ 0.0001) <b>(C</b>) Stimulation of MAPK, STAT3 and NF-κB activation after infection with different mutant strains of <i>S</i>. Typhi (Ty). Henle-407 cells were infected with the indicated <i>S</i>. Typhi (Ty) deletion mutants and the activation of JNK, Erk, p38, or STAT3 kinases was assessed by Western blot analysis using antibodies directed to the phosphorylated (activated) forms of these kinases. Cell lysates were also probed for the levels of I-κB, a measure of NF-κB activation.</p

Pathway enrichment of upregulated genes after infection with <i>S</i>. Typhimurium or <i>S</i>. Typhi.

<p>Depicted is the interaction of genes whose expression increased at least 3 fold at 4 h (<b>A</b> and <b>B</b>) or 10 h (<b>C</b> and <b>D</b>) after infection with <i>S</i>. Typhimurium (<b>A</b> and <b>C</b>) or <i>S</i>. Typhi (<b>B</b> and <b>D</b>). The analysis was carried out with STRING 10.0 (<a href="http://string-db.org/" target="_blank">http://string-db.org/</a>) using high confidence (0.7) parameters. Nodes associated with STAT3, MAPK, or NF-κB signaling are denoted.</p

<i>Salmonella</i> typhimurium and <i>Salmonella</i> typhi stimulate specific gene expression patterns in cultured epithelial cells.

<p>(<b>A</b>) Venn diagram depicting the number of unique and common genes whose expression changed at least 3 fold at the indicated times after infection with <i>S</i>. Typhimurium (Tm) or <i>S</i>. Typhi (Ty). (<b>B</b>) Heat map of genes whose expression changed at least 8 fold at the indicated times after infection with wild type <i>S</i>. Typhimurium (Tm) or <i>S</i>. Typhi (Ty). The data are presented as row-normalized heat maps as indicated in the color scale. * p < 0.04, Student t test. (<b>C</b>) Expression of selected genes in infected Henle-407 cells at the indicated times after infection. Values (mean ± SD of 3 replicates) represent the GAPDH normalized transcript levels of selected genes in <i>S</i>. Typhimurium (Tm) or <i>S</i>. Typhi (Ty) infected cultured Henle-407 cells relative to levels of uninfected cells. (<b>D</b>) Western blot detection of selected proteins (SerpinB3 and TTP) in infected Henle 407 cells at the indicated time after infection with <i>S</i>. Typhimurium (Tm) or <i>S</i>. Typhi (Ty). Levels of tubulin in the different samples served as loading controls. (<b>E</b>) Immunofluorescence staining of endogenous SerpinB3 in Henle-407 cells after infection with <i>S</i>. Typhimurium (Tm) or <i>S</i>. Typhi (Ty). Cells were stained at the indicated times after infection with antibodies directed to SerpingB3 (green) or bacterial LPS (red), and DAPI (blue) to detect DNA.</p

<i>Salmonella</i> Modulation of Host Cell Gene Expression Promotes Its Intracellular Growth

<div><p><i>Salmonella</i> Typhimurium has evolved a complex functional interface with its host cell largely determined by two type III secretion systems (T3SS), which through the delivery of bacterial effector proteins modulate a variety of cellular processes. We show here that <i>Salmonella</i> Typhimurium infection of epithelial cells results in a profound transcriptional reprogramming that changes over time. This response is triggered by <i>Salmonella</i> T3SS effector proteins, which stimulate unique signal transduction pathways leading to STAT3 activation. We found that the <i>Salmonella</i>-stimulated changes in host cell gene expression are required for the formation of its specialized vesicular compartment that is permissive for its intracellular replication. This study uncovers a cell-autonomous process required for <i>Salmonella</i> pathogenesis potentially opening up new avenues for the development of anti-infective strategies that target relevant host pathways.</p></div

<i>Salmonella</i> stimulation of transcriptional responses in infected cells requires STAT3.

<p>(<b>A</b>) Interaction map of genes whose expression is stimulated by <i>S.</i> Typhimurium infection of Henle-407 cells. Shown is the interaction of genes whose expression increased at least 3 fold at 10 or 20 h after infection. The analysis was carried out with STRING 9.0 (<a href="http://string-db.org/" target="_blank">http://string-db.org/</a>) using the highest confidence (0.9) parameters. (<b>B–E</b>) <i>S.</i> Typhimurium induces STAT3 activation. Henle-407 cells were infected (MOI = 10) with wild-type <i>S.</i> Typhimurium for 1 h. Following chase in gentamicin containing medium, cells were lysed at the indicated times, separated by SDS-PAGE and probed by immuno blotting with antibodies to the phosphorylated (activated) form of STAT3 (P-Y705), unphosphorylated STAT3 (to detect total amount), and actin (loading control) (<b>B</b>). Fold activation of STAT3 (relative to uninfected cells) was quantified by scanning the blots with a LI-COR Odyssey imaging system standardizing the signals with the actin loading control. Values are the means (± SD) of three independent experiments (<b>C</b>). Alternatively, Henle-407 cells were infected (MOI = 5) for 1 h with wild-type <i>S.</i> Typhimurium, chased for 8 h in gentamicin supplemented medium, fixed, immuno stained for LPS (red), DNA (blue) and phosphorylated STAT3 (green), as indicated. Samples were then analyzed by epifluorescence microscopy (bar represents 10 µm) (<b>D</b>). The number of infected cells showing staining of phosphorylated STAT3 was determined and the values represent the mean (± SD) of three independent experiments in which at least 100 cells were quantified. *: indicates values that are statistically significantly different from uninfected controls (<i>p</i>≤0.01) (<b>E</b>). (<b>F</b>) STAT3 is required for the transcriptional responses to <i>S.</i> Typhimurium infection. Henle-407 cells treated with the STAT3 inhibitor S31-201 were infected (MOI = 10) with <i>S.</i> Typhimurium for 1 h, and chased for additional 3 h in gentamicin containing medium in the presence of DMSO or 100 µM S3I-201. mRNA levels of selected genes whose expression is induced by <i>S.</i> Typhimurium infection was analyzed by qRT-PCR. Values represent the mean (± SEM) of GAPDH normalized transcript levels of the indicated genes, relative to DMSO treated uninfected cells. *: indicates statistically significant differences (<i>p</i>≤0.0004). (n. i.: non infected).</p

<i>Salmonella</i> Typhimurium triggers a complex gene expression program in cultured Henle-407 cells.

<p>(<b>A</b>) Venn diagram depicting the number of unique and common genes whose expression changed at least 3 fold at the indicated times after <i>S.</i> Typhimurium infection. (<b>B</b>) Heat map of selected genes whose expression changed at least 12 fold at the indicated times after <i>S.</i> Typhimurium infection. (<b>C</b>) Western blot detection of selected proteins whose genes were significantly upregulated after <i>S.</i> Typhimurium infection. Henle-407 cells were infected (MOI = 10) with wild-type <i>S.</i> Typhimurium or the SPI-1 T3SS-defective <i>ΔinvA</i> mutant strain for 1 h. At the indicated times, cells were harvested, their lysates separated by SDS-PAGE and probed by immuno blotting with the specified antibodies to the indicated proteins of interest and to tubulin as a loading control. (n. i.: non infected).</p

Bacterially-induced reprogramming of host cell gene expression is required for efficient <i>Salmonella</i> replication.

<p><i>S.</i> Typhimurium infects intestinal epithelial cells by delivering effector proteins into the host cell, using its SPI-1 T3SS. The effector proteins SopB, SopE and SopE2 activate the small GTPases Cdc42, Rac1 and RhoG in a redundant manner, thus inducing membrane ruffling and bacterial uptake. Internalized bacteria reside in macropinosomes, which then maturate into <i>Salmonella</i> containing vacuoles (SCVs). Bacterial effector-stimulated small GTPases also activate members of the p21 activated kinase family (PAK). These serine/threonine kinases trigger downstream signaling of mitogen-activated protein kinases (MAPKs) and nuclear factor kappa B (NFκB) <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003668#ppat.1003668-Hobbie1" target="_blank">[11]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003668#ppat.1003668-Chen1" target="_blank">[40]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003668#ppat.1003668-Chen2" target="_blank">[43]</a>, resulting in activation of additional transcription factors. In addition, activated PAK proteins also phosphorylate members of the Abl kinase family, thereby triggering auto-phosphorylation of Abl proteins for full activation, subsequently leading to the activation the cytoplasmic transcription factor STAT3. Products of STAT3-controlled genes ultimately influence vesicular trafficking between cellular compartments and the SCV, contributing to the formation of <i>Salmonella</i> induced filaments (SIFs), which characterize a fully mature, replication-competent <i>Salmonella</i>-containing vacuole.</p

<i>Salmonella</i> Typhimurium activates STAT3 by a non-canonical pathway that requires Abl and PAK.

<p>(<b>A</b>) Culture supernatants from Henle-407 infected cells do not activate STAT3. Culture supernatants were obtained from Henle-407 cells 20 h after infection (MOI = 10) with either wild-type <i>S.</i> Typhimurium or the SPI-1 T3SS-defective <i>ΔinvA</i> mutant, filtered sterilized, and applied to uninfected Henle-407 cells. At different times after treatment cells were lysed, separated by SDS-PAGE and probed by immuno blotting with antibodies to the phosphorylated (activated) form of STAT3 (P-Y705), and tubulin (loading control). As a control, infected cells were analyzed for STAT3 activation in a similar fashion. (<b>B</b>) Activation of STAT3 by <i>S.</i> Typhimurium is JAK independent. Henle-407 cells were treated with the JAK inhibitor Tofacitinib at the indicated concentration and then infected (MOI = 10) with wild-type <i>S.</i> Typhimurium for 1 h, chased for additional 6 h in gentamicin containing medium in the presence of DMSO or Tofacitinib. Cells were then lysed and analyzed for STAT3 activation as indicated in (<b>A</b>). (<b>C</b>) Abl kinases are required for efficient <i>S.</i> Typhimurium-induced STAT3 activation. Cultured epithelial cells were pretreated with increasing concentrations of the STAT3 inhibitor Imatinib for 1 h, infected (MOI = 10) with wild-type <i>S.</i> Typhimurium, chased in the presence of the inhibitor, and at the indicated time cells were lysed and analyzed for STAT3 activation as described in (<b>A</b>). (<b>D</b>) <i>S.</i> Typhimurium-induced STAT3 activation requires PAK activity. Cultured epithelial cells were pretreated with increasing concentrations of the PAK inhibitor IPA-3 for 1 h, infected (MOI = 10) with wild-type <i>S.</i> Typhimurium, chased in the presence of the inhibitor, and at the indicated times cells were lysed and analyzed from STAT3 activation as indicated in (<b>A</b>). (<b>E</b>) Abl and PAK are required for the transcriptional responses to <i>S.</i> Typhimurium infection. Henle-407 cells were treated with inhibitors for Abl kinases (imatinib), PAK (IPA-3), JAK (Tofacitinib), and Src (PP1), infected (MOI = 10) with wild-type <i>S.</i> Typhimurium for 1 h, and chased for additional 3 h in gentamicin containing medium in the presence of DMSO or the inhibitors. mRNA levels of the selected indicated genes whose expression increase after <i>S.</i> Typhimurium infection were analyzed by qRT-PCR. Values represent the mean (± SEM) of GAPDH normalized transcript levels in infected cells relative to DMSO treated uninfected cells. *: indicates statistically significant differences (<i>p</i>≤0.02) of the indicated comparisons. (n. i.: non infected).</p