Parallel Exploitation of Diverse Host Nutrients Enhances Salmonella Virulence

Pathogen access to host nutrients in infected tissues is fundamental for pathogen growth and virulence, disease progression, and infection control. However, our understanding of this crucial process is still rather limited because of experimental and conceptual challenges. Here, we used proteomics, microbial genetics, competitive infections, and computational approaches to obtain a comprehensive overview of Salmonella nutrition and growth in a mouse typhoid fever model. The data revealed that Salmonella accessed an unexpectedly diverse set of at least 31 different host nutrients in infected tissues but the individual nutrients were available in only scarce amounts. Salmonella adapted to this situation by expressing versatile catabolic pathways to simultaneously exploit multiple host nutrients. A genome-scale computational model of Salmonella in vivo metabolism based on these data was fully consistent with independent large-scale experimental data on Salmonella enzyme quantities, and correctly predicted 92% of 738 reported experimental mutant virulence phenotypes, suggesting that our analysis provided a comprehensive overview of host nutrient supply, Salmonella metabolism, and Salmonella growth during infection. Comparison of metabolic networks of other pathogens suggested that complex host/pathogen nutritional interfaces are a common feature underlying many infectious diseases

Transient silencing of APIP decreases the growth of the HeLa cells in MTA medium.

<p>(<b>A</b>) Schematic representation of the two mRNAs isoforms of APIP. Sequence positions of the shRNAs used in the study (sh1APIP and sh2APIP) are indicated by arrows. (<b>B</b>) Semi-quantitative RT-PCR analysis of APIP silencing 48 h and 144 h after transfection with plasmids expressing shRNAs. A plasmid expressing shRNA against β-galactosidase (shβGal) was used as a negative control. The GAPDH gene was used as an internal control. (<b>C</b>) Western blot analysis of APIP silencing 48, 72 and 144 hours after transfection with plasmids expressing shRNAs. All lanes were loaded with 80 µg of cell lysate proteins. Anti α-tubulin (α-tub) was used as a loading control. The bands present below APIP were nonspecifically stained with the anti-APIP antibody. (<b>D, E</b>) Cell growth analysis of HeLa cells transiently silenced for APIP. Alamarblue fluorescence is expressed relative to the fluorescence of HeLa cells transfected with the same plasmids and cultured in normal methionine media. (<b>D</b>) 96 hours after transfection with plasmids expressing shRNAs, the same number of cells was grown for 48 hours either in complete media, or in methionine free media complemented or not with MTA, and their end point growth was measured by Alamarblue fluorescence (<b>E</b>) 72 hours after transfection with plasmids expressing shRNAs, cells were transfected with plasmids expressing the N-terminally V5-tagged APIP protein (V5APIP) or the N-terminally V5-tagged chloramphenicol acetyltransferase protein (V5CAT). 24 hours later, the same number of cells was grown for 48 hours either in complete media or in methionine free media complemented or not with MTA, and their end point growth was measured by Alamarblue fluorescence.</p

Mutational analysis of APIP activity.

<p>(<b>A</b>) Sequence alignment of APIP.long with bacterial enzymes of the same family (class II aldolases). RHAD: Rhamnulose-1-phosphate aldolase (P32169, EC 4.1.2.19), ARAD: L-ribulose-5-phosphate 4-epimerase (P08203, EC 5.1.3.4), FUCA: L-fuculose-1-phosphate aldolase (P0AB87, EC 4.1.2.17). (<b>B, C, D</b>) Alamarblue cell growth analysis of APIP knockdown HeLa cells rescued with mutant forms of V5APIP in MTA medium. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052877#s3" target="_blank">Results</a> are expressed relative to the growth of the HeLa cells treated the same way in methionine medium (<b>B</b>) V5APIP mutated for the potential zinc binding site (V5APIP3HA) and V5APIP.short are not able to restore cell growth in MTA medium. Expression of V5APIP, V5APIP3HA and V5APIP.short was controlled by Western blot in APIP stable knockdown HeLa cells. (<b>C</b>) Co-expression of V5APIP.short did not affect the growth rescue in MTA conferred by V5APIP. V5CAT was used as control so that the amount of plasmid DNA used for transfection was constant in the three conditions. (<b>D</b>) No difference was observed in the rescue efficiency of V5APIP and V5APIP mutated at potential phosphorylation sites (V5APIP3SA and V5APIP3SD).</p

Stable knockdown of APIP specifically affects growth in MTA and depletes intracellular levels of methionine.

<p>(<b>A</b>) Western blot analysis of APIP stable knockdown HeLa cells. (<b>B</b>) Alamarblue cell growth analysis of control (shβGal) and APIP knockdown stable cell lines in MTA, MTOB, Hcy and SAM media. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052877#s3" target="_blank">Results</a> are expressed relative to the growth of the same cell lines in methionine medium. (<b>C, D</b>) Effects of APIP depletion on infection by wt <i>Shigella</i> and by a <i>Shigella</i> strain auxotrophic for methionine (<i>Shigella metA</i>). HeLa cells were switched to MTA for two hours and then infected in the same media with wt and mutant form of <i>Shigella flexneri</i> serotype 2a strain 2457T for 4 hours. Parameters of infection were monitored by flow cytometry as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052877#s2" target="_blank">material and methods</a>. (<b>C</b>) Measurement of infection rates (i.e percentage of infected HeLa cells) in each condition. No significant difference was observed. (<b>D</b>) Time course analysis of the <i>Shigella</i> load shows that the growth of <i>Shigella metA</i> is severely diminished in HeLa cells silenced for APIP in the absence of methionine in the cell culture medium.</p

APIP as candidate to perform the step 2 of the methionine salvage pathway in human.

<p>(<b>A</b>) Schematic overview of the methionine salvage pathway. Black arrows: human specific routes; Black dashed arrows: <i>Shigella</i> specific routes; Grey arrows: common routes. (<b>B</b>) Sequence alignment of human APIP long and short isoforms (APIP.long and APIP.short) with mtnB enzymes from the yeast <i>Saccharomyces cerevisiae</i> and the bacteria <i>Bacillus subtilis</i>. (<b>C</b>) Analysis of the expression pattern of APIP mRNA by semi-quantitative RT–PCR. Left panel: Expression in tissues. This experiment was performed using a commercially obtained pre-normalized human multiple tissue cDNA panel (Clontech, panel I). Right panel: Expression in cell lines. The experiment was performed using total RNA preparations derived from human cell lines. The GAPDH gene was used as an internal control. For APIP at least two bands were detected (arrows) and confirmed to be the short and long isoforms after sequencing (APIP.long: 729 bp; APIP.short: 628 bp). M: Size marker; S: APIP.short; L: APIP.long. (<b>D</b>) Immunofluorence analysis of APIP into HeLa cells show a major cytoplasmic staining.</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

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

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

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

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