Virulence of Salmonella enterica serovar typhimurium and innate antibacterial host responses

The bacterial species Salmonella enterica consists of a collection of closely related enteric bacteria giving rise to diverse diseases in a wide range of hosts. In the murine infection model Salmonella enterica serovar Typhimurium (S. Typhimurium) causes an invasive disease which in many aspects resembles human typhoid fever. The ability of this pathogen to survive and replicate within macrophages of the liver and spleen is a crucial virulence determinant which largely depends on the type III secretion system coded for by the Salmonella pathogenicity islands 2 (SPI-2). Innate immune recognition of bacterial pattern molecules such as the lipopolysaccharide (LPS) induces immediate defence responses such as production of reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI), synthesized by macrophages via the action of the NADPH phagocyte oxidase (phox) and inducible nitric oxide synthase (iNOS) respectively. The newly characterized phagocyte receptor TRAPC has been shown to trigger nitric oxide (NO) production in macrophages and dendritic cells upon receptor cross-linking, suggesting a possible role for TRAPC during bacterial infection. Surprisingly we could show that in combination with bacterial infection or LPS stimulation, cross-linking of TRAPC reduces macrophage NO production. The suppression of NO associated with a slight reduction of iNOS expression possibly mediated by a dampening the TLR-4 response. For S. Typhimurium and many other enteric pathogens, the ability to express complete LPS molecules has conventionally been regarded as a requirement for bacterial virulence, e.g. lack of the 0-antigen (the outermost part of the LPS molecule) has been shown to reduce the virulence of S. Typhimurium in murine infection models. However, S. Typhimurium has also been shown to down-regulate genes for LPS-synthesis and to reduce the chain length of the 0-antigen once it resides inside macrophage-like cells. This raises the question whether S. Typhimurium may benefit from expression of O-antigen-deficient LPS during intracellular stages of infection. To settle this issue the fitness of defined mutants devoid of O-antigen in macrophage-like cells was studied. O-antigen-deficient mutants inhibited iNOS activity in macrophage-like cells in an apparently SPI-2 dependent manner. Consequently the mutants displayed increased growth yields within these cells compared to wild type bacteria. Production of ROI as well as RNI is critical for control of disease proliferation in the murine salmonellosis model. In E.coli the thioredoxin and glutathione/glutaredoxin systems have been shown to mediate protection against oxidative stress. When studying the role of these systems in Salmonella we could show that whereas thioredoxin 1 (TrxA) is dispensable for resistance to oxidative or NO stress in vitro, it is essential for bacterial growth in both epithelial and macrophage-like cells as well as for virulence in vivo in the murine infection model. Whereas the level of replication within macrophage-like cells correlates directly to the redox potential of TrxA, in vivo virulence depends on both redox dependent and independent activities of TrxA. Moreover, TrxA was shown to be required for proper function of SPI-2 and for the ability of O-antigen deficient S. Typhimurium to inhibit iNOS activity

Thioredoxin 1 Promotes Intracellular Replication and Virulence of Salmonella enterica Serovar Typhimurium

The effect of the cytoplasmic reductase and protein chaperone thioredoxin 1 on the virulence of Salmonella enterica serovar Typhimurium was evaluated by deleting the trxA, trxB, or trxC gene of the cellular thioredoxin system, the grxA or gshA gene of the glutathione/glutaredoxin system, or the dsbC gene coding for a thioredoxin-dependent periplasmic disulfide bond isomerase. Mutants were tested for tolerance to oxidative and nitric oxide donor substances in vitro, for invasion and intracellular replication in cultured epithelial and macrophage-like cells, and for virulence in BALB/c mice. In these experiments only the gshA mutant, which was defective in glutathione synthesis, exhibited sensitization to oxidative stress in vitro and a small decrease in virulence. In contrast, the trxA mutant did not exhibit any growth defects or decreased tolerance to oxidative or nitric oxide stress in vitro, yet there were pronounced decreases in intracellular replication and mouse virulence. Complementation analyses using defined catalytic variants of thioredoxin 1 showed that there is a direct correlation between the redox potential of thioredoxin 1 and restoration of intracellular replication of the trxA mutant. Attenuation of mouse virulence that was caused by a deficiency in thioredoxin 1 was restored by expression of wild-type thioredoxin 1 in trans but not by expression of a catalytically inactive variant. These results clearly imply that in S. enterica serovar Typhimurium, the redox-active protein thioredoxin 1 promotes virulence, whereas in vitro tolerance to oxidative stress depends on production of glutathione

Thioredoxin 1 Participates in the Activity of the Salmonella enterica Serovar Typhimurium Pathogenicity Island 2 Type III Secretion System ▿

The facultative intracellular pathogen Salmonella enterica serovar Typhimurium relies on its Salmonella pathogenicity island 2 (SPI2) type III secretion system (T3SS) for intracellular replication and virulence. We report that the oxidoreductase thioredoxin 1 (TrxA) and SPI2 are coinduced for expression under in vitro conditions that mimic an intravacuolar environment, that TrxA is needed for proper SPI2 activity under these conditions, and that TrxA is indispensable for SPI2 activity in both phagocytic and epithelial cells. Infection experiments in mice demonstrated that SPI2 strongly contributed to virulence in a TrxA-proficient background whereas SPI2 did not affect virulence in a trxA mutant. Complementation analyses using wild-type trxA or a genetically engineered trxA coding for noncatalytic TrxA showed that the catalytic activity of TrxA is essential for SPI2 activity in phagocytic cells whereas a noncatalytic variant of TrxA partially sustained SPI2 activity in epithelial cells and virulence in mice. These results show that TrxA is needed for the intracellular induction of SPI2 and provide new insights into the functional integration between catalytic and noncatalytic activities of TrxA and a bacterial T3SS in different settings of intracellular infections

Salicylidene Acylhydrazides That Affect Type III Protein Secretion in Salmonella enterica Serovar Typhimurium▿

A collection of nine salicylidene acylhydrazide compounds were tested for their ability to inhibit the activity of virulence-associated type III secretion systems (T3SSs) in Salmonella enterica serovar Typhimurium. The compounds strongly affected Salmonella pathogenicity island 1 (SPI1) T3SS-mediated invasion of epithelial cells and in vitro secretion of SPI1 invasion-associated effector proteins. The use of a SPI1 effector β-lactamase fusion protein implicated intracellular entrapment of the protein construct upon application of a salicylidene acylhydrazide, whereas the use of chromosomal transcriptional gene fusions revealed a compound-mediated transcriptional silencing of SPI1. Salicylidene acylhydrazides also affected intracellular bacterial replication in murine macrophage-like cells and blocked the transport of an epitope-tagged SPI2 effector protein. Two of the compounds significantly inhibited bacterial motility and expression of extracellular flagellin. We conclude that salicylidene acylhydrazides affect bacterial T3SS activity in S. enterica and hence could be used as lead substances when designing specific inhibitors of bacterial T3SSs in order to pharmaceutically intervene with bacterial virulence

Distinct Translational Control in CD4⁺ T Cell Subsets

<div><p>Regulatory T cells expressing the transcription factor Foxp3 play indispensable roles for the induction and maintenance of immunological self-tolerance and immune homeostasis. Genome-wide mRNA expression studies have defined canonical signatures of T cell subsets. Changes in steady-state mRNA levels, however, often do not reflect those of corresponding proteins due to post-transcriptional mechanisms including mRNA translation. Here, we unveil a unique translational signature, contrasting CD4⁺Foxp3⁺ regulatory T (T_Foxp3+) and CD4⁺Foxp3⁻ non-regulatory T (T_Foxp3−) cells, which imprints subset-specific protein expression. We further show that translation of eukaryotic translation initiation factor 4E (eIF4E) is induced during T cell activation and, in turn, regulates translation of cell cycle related mRNAs and proliferation in both T_Foxp3− and T_Foxp3+ cells. Unexpectedly, eIF4E also affects Foxp3 expression and thereby lineage identity. Thus, mRNA–specific translational control directs both common and distinct cellular processes in CD4⁺ T cell subsets.</p></div

A translational signature that discriminates T_Foxp3+ and T_Foxp3− cells.

<p>(a–b) Polysome-associated mRNA levels differ from cytosolic mRNA levels in primary CD4⁺ T cell subsets <i>ex vivo</i> and post-activation. Shown are density scatter plots of polysome-associated vs. cytosolic mRNA data (a blue scale from light to dark represents increasing local density of data points; outliers are indicated as dots) for T_Foxp3− cells (a) and T_Foxp3+ cells (b) at the <i>ex vivo</i> and the activated condition. The solid and dotted lines indicate a >3-fold and >2-fold difference, respectively, in the density scatter plot. The number of mRNAs that show a >3-fold difference in each direction is indicated. (c) Substantial differences in levels of polysome-associated mRNA between T_Foxp3+ and T_Foxp3− cells. Density scatter plots (as in a–b) compare polysome-associated mRNA data between T_Foxp3+ and T_Foxp3− cells in both the <i>ex vivo</i> and <i>in vitro</i> activated conditions. A few genes known to be differentially expressed between T_Foxp3+ and T_Foxp3− cells are indicated (<i>Foxp3, Ctla4, Il2ra</i> [CD25] and <i>Tnfrsf18</i> [GITR]). As expected the differential expression of <i>Il2ra</i> is lost upon activation. (d) Differential translation in T_Foxp3+ vs. T_Foxp3− cells as identified with anota-RVM <i>ex vivo</i> and post <i>in vitro</i> activation. Significances (i.e. the −log10 p-value from the anota analysis used to identify differential translation) are compared to log2 translational fold changes (after correction for cytosolic mRNA levels).</p

eIF4E controls proliferation in T cell subsets.

<p>(a) Inhibition of eIF4E activity suppresses T_Foxp3− cell proliferation. eFluor 670-labeled T_Foxp3− cells were IL-2/TCR-activated for 72 h in the presence of increasing concentrations of the eIF4E inhibitor 4ei-1 (K_d = 0.80 µM). Proliferation was determined under each condition by eFluor 670 dilution assessed by flow cytometry (upper panel). The effect on proliferation was also assessed by comparing cell counts after 72 h under each condition (lower panel; the control was set to 100%). (b) Inhibition of eIF4E activity abrogates IL-2-mediated reversal of anergy in T_Foxp3+ cells. IL-2/TCR-activated eFluor 670 labelled T_Foxp3+ cells were cultured in the presence of 4ei-1, and proliferation was determined as described in (a). (c–d) IL-2/TCR-activated eFluor 670 labelled T_Foxp3− cells (c) or T_Foxp3+ cells (d) were cultured in the presence of 4ei-1 or 4ei-4. Proliferation was determined under each condition as described in (a). (a–d) Representative histograms from 4 independent experiments are shown (upper panels; the percentages of proliferating cells are indicated). Means and standard deviations of cell counts from 4 independent experiments are shown (lower panel). (e) Induction of T_Foxp3+ cell proliferation occurs independently of signalling through 4E-BPs. 4E-BPdko T_Foxp3+ and T_Foxp3− cells were plated and counted as described in (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003494#pgen-1003494-g005" target="_blank">Figure 5d</a>), and the fold increase in cell number was calculated and associated means and standard deviations (n = 2) are shown. Welch's two sample t-test was used to compare 4E-BPdko T_Foxp3+ cells cultured under different IL-2 concentrations. Also shown is a western blot of total protein extracts probed with antibodies for eIF4E in 4E-BPdko T_Foxp3+ and T_Foxp3− cells. Densitometry was used to quantify protein levels and obtained levels were normalized to β-actin (the normalized values were related to T_Foxp3− 72 h IL-2 100 U/ml which was set to 1 and are indicated above each lane). (f) Ki-67 and eIF4E co-expression in total CD4⁺ T cells isolated directly <i>ex vivo</i> from lymph nodes (left panel). Quantification of eIF4E expression is shown as Δ (eIF4E <i>vs.</i> isotype control) mean fluorescent intensity (MFI). Filled histograms represent staining with an isotype control. Quantification of eIF4E expression (ΔMFI) in Ki-67^+/− T_Foxp3− and T_Foxp3+ cells isolated directly <i>ex vivo</i> (right panel, mean and standard deviation is indicated, n = 3). (g–h) eFluor 670-labeled T_Foxp3− or T_Foxp3+ cells adoptively transferred into separate TCR β−/− mice were isolated from mesenteric (mes) and peripheral (per) lymph nodes (LN) followed by measurement of eFluor 670 and eIF4E expression four days post transfer. (g) Representative dot plots (n = 3) of T_Foxp3− and T_Foxp3+ cell proliferation relative to eIF4E expression in mesLN. Staining with an isotype control are shown as contour plots. (h) Quantification of eIF4E expression (ΔMFI) in cells that have (eFluor 670 low) or have not (eFluor 670 high) undergone cell division (means and standard deviations are indicated after per experiment normalization to T_Foxp3+ cells, n = 4–6). P-value (Welch two sample t-test) is indicated.</p

Genome-wide analysis of translationally regulated mRNAs in primary CD4⁺ T cell subsets.

<p>(a) Cytosolic mRNA was extracted and probed directly with DNA microarrays or processed using the polysome preparation technique where mRNAs are sedimented on a sucrose gradient and separated based on the number of ribosomes they associate with. Fractions containing mRNAs that engage ≥3 ribosomes were pooled and probed with microarrays to quantify mRNA levels. (b) Polysome UV-tracings from <i>ex vivo</i> and <i>in vitro</i> activated T_Foxp3+ and T_Foxp3− cells. Shown is the UV absorbance (254 nm) as a function of sedimentation. The large peak corresponds to the 80S ribosome peak and was used to align the polysome profiles so that fractions containing ≥3 ribosomes could be pooled from each sample. The part of the polysome profile that was pooled and used as the polysome-associated mRNA sample is indicated. (c) Assessment of data set quality. Shown is a dendrogram from a hierarchical clustering of all included samples (using Pearson correlations). Samples that are more similar cluster together. Cyto – cytosolic mRNA; poly – polysome-associated mRNA.</p

Inhibition of eIF4E activity results in spontaneous induction of Foxp3 expression in activated T_Foxp3− cells.

<p>T_Foxp3− cells were IL-2/TCR-activated for 72 h in the presence of increasing concentrations of 4ei-1 or the control pro-drug 4ei-4 in undifferentiating conditions, and Foxp3 expression (i.e. GFP) was assessed by flow cytometry. (a) Representative density plots from experiments using T_Foxp3− cells cultured in the presence of 4ei-1 from 4 independent experiments are shown. (b) Percentage Foxp3⁺ cells following treatment with 4ei-1 or 4ei-4 (shown are means and standard deviations, n = 4).</p

Translationally regulated mRNAs encode proteins are involved in ubiquitination, chromatin modification, or cell cycle pathways.

<p>Translational activity (from anota after correction for cytosolic mRNA levels) in T_Foxp3+ and T_Foxp3− cells <i>ex vivo</i> and post <i>in vitro</i> activation for individual mRNAs belonging to ubiquitination (a), chromatin modification (b) or cell cycle (c) pathways is shown. The colour scale represents translational activity in log2 scale.</p