Mouse spleen colonization of <i>Salmonella</i> mutants with metabolic defects.

<p>The data represent competitive indices (CI) of mutants vs. wildtype <i>Salmonella</i> in spleen of individual mice at three (open symbols) or four days (filled symbols) post infection (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003301#ppat.1003301.s009" target="_blank">Table S3</a>). A log₂(CI) value of 0 (equivalent to a CI of 1) represents full virulence. Down triangles represent mutants with utilization defects, up triangles represent auxotrophic mutants. Grey symbols represent data from a previous study <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003301#ppat.1003301-Becker1" target="_blank">[34]</a> obtained in the same disease model. Red triangles represent data from an independently reconstructed <i>glpFK gldA glpT ugpB</i> mutant. The data provided evidence for access to a number of host nutrient which are shown in black (for detailed interpretation see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003301#ppat.1003301.s011" target="_blank">Table S5</a>). Nutrients with apparently low availability are shown in grey. Statistical analysis was carried out with the Benjamini-Hochberg false discovery rate (FDR) approach for multiple comparisons <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003301#ppat.1003301-Benjamini1" target="_blank">[45]</a> (***, FDR<0.001; **, FDR<0.01; *, FDR<0.05).</p

A common nutritional pattern for mammalian pathogens.

<p><b>A</b>) Presence of 254 nutrient utilization pathways in genomes of 153 mammalian pathogens (excluding all <i>Salmonella</i> serovars). Data were based on pathway annotations available in MetaCyc <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003301#ppat.1003301-Caspi1" target="_blank">[62]</a>. Degradation pathways for nutrients that support <i>Salmonella</i> in mouse spleen were highly overrepresented among pathogen genomes (<i>P</i><0.001; Mann-Whitney U test) suggesting similar nutritional preferences (filled circles; 1, purine nucleosides; 2, pyrimidine nucleosides; 3, fatty acids; 4, glycerol; 5, arginine; 6, N-Acetylglucosamine; 7, glucose; 8, gluconate). <b>B</b>) Depletion frequency of 118 biosynthesis pathways in mammalian pathogens. The values represent differences in pathway frequency in sets of 153 pathogens and 316 environmental bacteria (see text for explanation). Biosynthesis pathways for biomass components that <i>Salmonella</i> could obtain from the host were selectively depleted among pathogen genomes (<i>P</i><0.0001; Mann-Whitney U test) suggesting similar host supplementation patterns (filled circles; 1, tyrosine; 2, histidine; 3, arginine; 4, cysteine; 5, methionine; 6, tryptophan; 7, threonine; 8, valine; 9 leucine; 10, isoleucine; 11, proline; 12, pyridoxal; 13, purine nucleosides; 14, pyrimidine nucleosides; 15, glutamine; 16, thiamin; 17, pantothenate).</p

A quantitative genome-scale model of <i>Salmonella</i> nutrition, metabolism, and growth in infected mouse spleen.

<p>This schematic map shows available host nutrients, their respective uptake rates represented by color and font size, and their conversion to new <i>Salmonella</i> biomass through the <i>Salmonella</i> metabolic network (see text and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003301#ppat.1003301.s012" target="_blank">Tables S6</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003301#ppat.1003301.s013" target="_blank">S7</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003301#ppat.1003301.s014" target="_blank">S8</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003301#ppat.1003301.s015" target="_blank">S9</a> for detailed explanation and quantitative values). Symbols represent metabolites (squares, carbohydrates; pointing up triangles, amino acids; vertical ellipses, purines; horizontal ellipses, pyrimidines; pointing down triangles, cofactors; tees, tRNAs; circles, other metabolites; filled symbols, phosphorylated metabolites) and proteins (diamonds). The connecting lines present metabolic reactions. The brown lines represent the inner and outer membranes. An interactive map with detailed annotation of all reactions and the computational model in SBML format are available at <a href="http://www.biozentrum.unibas.ch/personal/bumann/steeb_et_al/index.html" target="_blank">http://www.biozentrum.unibas.ch/personal/bumann/steeb_et_al/index.html</a>. The model is also available in the supporting information (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003301#ppat.1003301.s019" target="_blank">Model S1</a>).</p

Large-scale experimental data are consistent with computational model predictions.

<p><b>A</b>) Validation of mutant phenotype predictions. The colors show the predicted gene relevance for spleen colonization (red, essential; orange, contributing; blue, non-detectable; see text for definitions). Comparison of model predictions with 738 experimental <i>Salmonella</i> mutant phenotypes revealed 92% prediction accuracy (inner dark colors) but also 61 discrepancies (pale outer colors). Numbers (correct/total number of experimentally validated predictions) are also given. <b>B</b>) Potential reasons for inaccurate phenotype predictions (redu, unrealistic redundancy; biom, incomplete biomass/maintenance issues; part, partially contributing functions; toxic, accumulation of toxic upstream metabolites; gap, missing enzyme; or exp, possibly inaccurate experimental data). For detailed descriptions see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003301#ppat.1003301.s016" target="_blank">Table S10</a>. <b>C</b>) Detection of enzymes with predicted differential relevance for optimal <i>Salmonella</i> in vivo growth. Enzyme relevance was classified by parsimonious enzyme usage flux-balance analysis (pFBA) (ess, essential enzymes; optima, enzymes predicted to be used for optimal in vivo growth; ELE, enzymatically less efficient enzymes that will increase flux if used; MLE, metabolically less efficient enzymes that will impair growth rate if used; zeroFlux, enzymes that cannot be not used in vivo). Filled bars represent enzymes that were detected by <i>Salmonella</i> ex vivo proteomics, open bars represent enzymes that were not detected. Statistical significance of the relationship between enzyme classes and the proportion of detected proteins was determined using the Chi square trend test. <b>D</b>) Feasibility of predicted reaction rates. For each reaction, the range of flux rates compatible with full <i>Salmonella</i> growth was determined using Flux-Variability Analysis. The circles represent the most economical state with minimal total flux (see text). Predicted reaction rates are compared to corresponding catalytic capacities calculated form experimental enzyme abundance and turnover numbers (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003301#ppat.1003301.s008" target="_blank">Table S2</a>). The reddish area represents infeasible fluxes. Reactions with substantial infeasible fluxes in the most economic simulated state are labeled (1, formyltetrahydrofolate dehydrogenase; 2, phosphoserine aminotransferase; 3, glycerol dehydrogenase). <b>E</b>) Predicted flux ranges and corresponding catalytic capacities after constraining all reactions to feasible fluxes (except for the three aminoacyl tRNA ligations mentioned in the text). <b>F</b>) Relative flux ranges of the initial unrestrained (straight line) and the enzyme capacity-restrained (dotted line) models. For each reaction, the flux range was divided by the respective flux value in the most economical state. Reactions that carried no flux in the most economical state were not considered. Statistical significance of the difference between both distributions was tested using the Mann-Whitney U test.</p

Nutrient limitation of intracellular <i>Salmonella</i> growth.

<p><b>A</b>) Schematic representation of external supplementation of intracellular <i>Salmonella</i> (red) in infected macrophages (grey). <b>B</b>) Increasing external nutrient availability accelerates intracellular <i>Salmonella</i> growth, and this depends on specific <i>Salmonella</i> nutrient utilization capabilities (open symbols, 0.5 g l⁻¹ glucose; filled black symbols, 1 g l⁻¹ glucose; filled grey symbols, 0.5 g l⁻¹ glucose 0.5 g l⁻¹ mannitol; circles, wildtype <i>Salmonella</i>; upward triangles, <i>Salmonella ptsG manX galP mglB</i>, deficient for high-affinity glucose transport; downward triangles, <i>Salmonella mtlAD</i>, deficient for high-affinity mannitol transport and degradation). Colony-forming units (CFU) at 10 h post infection for triplicate wells containing 300’000 RAW 264.7 cells are shown. <b>C</b>) Flux-balance analysis of nutrient excess scenarios. The computational model was set to incorporate various amounts of excess nutrients (beyond what was needed for cell maintenance and growth). Model parameters were adjusted to yield predictions that were consistent with experimental mutant and wildtype colonization data. Simulation of up to 18% nutrient excess was possible but required unrealistically high maintenance costs (shown in multiples of maintenance costs for axenic conditions). Simulated scenarios with nutrient excess beyond 18% were incompatible with experimental colonization data.</p

Nutrient utilization capabilities of <i>Salmonella</i> in infected mouse tissues.

<p>Colored names represent transporters and enzymes that were detected in <i>Salmonella</i> purified from mouse spleen (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003301#ppat.1003301.s007" target="_blank">Table S1</a>). The color shows enzyme abundance in copies per <i>Salmonella</i> cell. Grey proteins were not detected. Arrows represent metabolic reactions. Transport reactions are labeled with cylinders. Arrow colors show maximal catalytic capacities calculated from enzyme abundance and reported turnover numbers (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003301#ppat.1003301.s008" target="_blank">Table S2</a>). Grey arrows represent reactions, for which enzymes were not detected and/or turnover numbers were unavailable. Tsx is an outer membrane general nucleoside channel; NupC is a high affinity transporter for all nucleosides except guanosine and deoxyguanosine. An interactive map with detailed description of all detected metabolic capabilities is available at <a href="http://www.biozentrum.unibas.ch/personal/bumann/steeb_et_al/index.html" target="_blank">http://www.biozentrum.unibas.ch/personal/bumann/steeb_et_al/index.html</a>.</p

Schematic model for cellular immunity to <i>Salmonella</i>.

<p><i>Salmonella</i> (yellow) reside in intracellular vacuoles in infected host cells. <i>Salmonella</i> possesses internal (green) and surface-associated (red) antigens. <b>Left</b>) Live <i>Salmonella</i> shield internal antigens, but some of their surface-associated antigens are accessible for processing and presentation. As a consequence, T cells specific for <i>Salmonella</i> surface antigens can recognize these infected cells and initiate antibacterial immune effector mechanisms. In contrast, T cells specific for internal <i>Salmonella</i> antigens fail to detect host cells that contain exclusively intact <i>Salmonella</i>. <b>Right</b>) Dead <i>Salmonella</i> release internal antigens. As a consequence, both surface-exposed and internal antigens can be processed, presented, and recognized by cognate T cells. However, this recognition is unproductive for infection control since it targets <i>Salmonella</i> that are already dead.</p

Cellular and humoral immune responses of convalescent <i>Salmonella</i>-infected mice to recombinant <i>Salmonella</i> antigens.

<p><b>A</b>) Antigen-specific CD4 T cell frequencies as detected by CD154 upregulation (red) and IFNγ (green) or IL-17 (blue) secretion. The data represent means ± SE of three mice. Responses to <i>Salmonella</i> antigens in non-infected control mice were subtracted (see also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002966#ppat.1002966.s001" target="_blank">Fig. S1</a>). <b>B</b>) Serum antibody responses to <i>Salmonella</i> antigens. The data represent means ± SE of 11 convalescent mice (filled circles) and means ± SE for ten non-infected control mice (open circles).</p

CD4 T cell responses to <i>Salmonella</i> expressing an ovalbumin model antigen in various compartments.

<p><b>A</b>) Schematic overview of fusion proteins that target an immunodominant ovalbumin epitope (OVA) to various <i>Salmonella</i> cell compartments. <b>B</b>) Flow cytometric analysis of ovalbumin-specific CD4 T cell activation in a T cell receptor-transgenic adoptive transfer model. Mice were infected with control <i>Salmonella</i> expressing GFP (left) or <i>Salmonella</i> expressing LPP_OVA (right). Ovalbumin-specific transgenic CD4 T cells were detected with a clonotypic monoclonal antibody and analyzed for forward scatter and expression of the very early activation marker CD69. The dashed line was used to count CD4 T cell blasts. Similar observations were made for more than hundred mice in several independent experiments. <b>C</b>) Relationship between <i>Salmonella</i> Peyer's patches colonization and OVA-specific CD4 T cell induction in mice infected with <i>Salmonella</i> expressing high levels of LPP_OVA (filled circles) or low levels of GFP_OVA (open circles). Data represent means ± SEM's for groups of five to six animals from three independent experiments. CD4 T cell blasts correlated with <i>Salmonella</i> Peyer's patches colonization for both strains (Spearman test, <i>P</i><0.05 in both cases). The slopes of the two curves differed (ANCOVA; <i>P</i><0.05). <b>D</b>) OVA-specific CD4 T cell induction in mice infected with <i>Salmonella</i> expressing OVA at various levels (open circles, low abundance; filled circles, high abundance) in four different compartments. The dashed line represents CD4 T cell responses to saturating levels of cytosolic OVA. The star represents data for <i>Salmonella</i> expressing moderate levels of cytosolic OVA together with cholera toxin B and AIDA. Data represent means ± SEM's for groups of ten to twenty mice. Statistical significance of differences to <i>Salmonella</i> expressing saturating levels of cytosolic OVA were tested using Mann-Whitney U test (*, <i>P</i><0.05; **, <i>P</i><0.01).</p

Detection of intact and damaged <i>Salmonella</i> cells in infected mouse tissues.

<p><b>A</b>) Flow cytometry of a spleen homogenate infected with <i>Salmonella sifB::gfp</i> using 488 nm excitation. Gate 1 contains GFP-positive <i>Salmonella</i>. The inset shows the relationship between flow cytometry data and plate counts for individual mice, the dashed line represents a 1∶1 ratio. <b>B</b>) Confocal micrographs of liver cryosections infected with <i>Salmonella sifB::gfp</i> that were stained with antibodies to <i>Salmonella</i> lipopolysaccharide (red) and macrophage marker CD68 (blue). Individual color channels are shown with inverted grey scale for better visualization of weak staining. Micrographs represent typical observations for four independently infected mice. <b>C</b>) Confocal micrographs of lipopolysaccharide-positive particles that lack detectable GFP (even when contrast was increased compared to B). Such particles were absent in non-infected control sections.</p