Salmonella Metabolismo Durante El Desarrollo Bajo Condiciones Que Inducen Expresión de Virulencia

Molecular
BioSystems
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Salmonella modulates metabolism during growth

Cite this: DOI: 10.1039/c3mb25598k
under conditions that induce expression of virulence
genes†
Published on 22 March 2013 on http://pubs.rsc.org | doi:10.1039/C3MB25598K
Young-Mo Kim,za Brian J. Schmidt,zb Afshan S. Kidwai,c Marcus B. Jones,d

Downloaded by George Washington University on 18/04/2013 17:05:38.
Brooke L. Deatherage Kaiser,a Heather M. Brewer,e Hugh D. Mitchell,a

Bernhard O. Palsson,b Jason E. McDermott,a Fred Heffron,c Richard D. Smith,a
Scott N. Peterson,yd Charles Ansong,a Daniel R. Hyduke,b Thomas O. Metz*a and
Joshua N. Adkins*a
Salmonella enterica serovar Typhimurium (S. Typhimurium) is a facultative pathogen that uses complex
mechanisms to invade and proliferate within mammalian host cells. To investigate possible
contributions of metabolic processes to virulence in S. Typhimurium grown under conditions known to
induce expression of virulence genes, we used a metabolomics-driven systems biology approach
coupled with genome-scale modeling. First, we identified distinct metabolite profiles associated with
bacteria grown in either rich or virulence-inducing media and report the most comprehensive coverage
of the S. Typhimurium metabolome to date. Second, we applied an omics-informed genome-scale
modeling analysis of the functional consequences of adaptive alterations in S. Typhimurium metabolism
during growth under our conditions. Modeling efforts highlighted a decreased cellular capability to
both produce and utilize intracellular amino acids during stationary phase culture in virulence
Received 21st December 2012, conditions, despite significant abundance increases for these molecules as observed by our
Accepted 21st March 2013 metabolomics measurements. Furthermore, analyses of omics data in the context of the metabolic
DOI: 10.1039/c3mb25598k model indicated rewiring of the metabolic network to support pathways associated with virulence. For
example, cellular concentrations of polyamines were perturbed, as well as the predicted capacity for
www.rsc.org/molecularbiosystems secretion and uptake.
Introduction
Salmonella enterica serovar Typhimurium (S. Typhimurium) is a
highly infectious pathogenic bacterium that frequently causes
a
outbreaks of severe gastrointestinal infection (i.e. enteritis
Fundamental and Computational Sciences Directorate, Pacific Northwest
National Laboratory, Richland, WA 99352, USA. E-mail: [email protected],
Salmonella) that are geographically exacerbated via the rapid
[email protected]; Fax: +1 509 371-6555, +1 509 371-6546; modern food transport systems.1–3 Further, new difficult to
Tel: +1 509 371-6581, +1 509 371-6583 treat, drug-resistant isolates are emerging.4,5 Therefore, under-
b
Department of Bioengineering, University of California at San Diego, San Diego, standing the mechanisms of pathogen invasion and prolifera-
CA 92093, USA
c
tion within the hosts is critical to reduce health and economic
Department of Molecular Microbiology and Immunology, Oregon Health & Science
University, Portland, OR 97239, USA
impacts.6
d
J. Craig Venter Institute, Rockville, MD 20850, USA Current understanding of S. Typhimurium pathogenesis
e
Environmental Molecular Sciences Laboratory, Pacific Northwest National is being augmented and refined using systems biology
Laboratory, Richland, WA 99352, USA approaches7,8 namely whole genome mutagenesis,9,10 trans-
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
criptomics,11–17 and proteomics,18–24 which can provide global,
c3mb25598k
‡ These authors contributed equally to this work.
integrated insight into pathogenesis and virulence. However,
§ Current address: Sanford-Burnam Medical Research Institute, La Jolla, there are few reports to date of the application of metabolomics
CA 92037, USA. in the study of S. Typhimurium virulence.25–27
This journal is c The Royal Society of Chemistry 2013 Mol. BioSyst.

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Paper Molecular BioSystems
Collectively, metabolomics is a rapidly developing field in S. Typhimurium. A model-guided analysis of matching

devoted to identifying qualitative and quantitative differences transcriptomics data from analysis of cells grown in virulence-
in metabolites among biological samples.28 However, these inducing media suggests S. Typhimurium de-emphasizes
analyses have not yet been widely implemented in the study these pathways and prioritizes other subsystems important to
of pathogenic bacteria. With respect to S. Typhimurium, there virulence, including lipopolysaccharide (LPS) and fatty acid
are five reports of metabolomics analyses of intracellular or biosynthesis.
volatile metabolites in the context of biofilm formation25 or as a
potential diagnostic tool for determining food spoilage29–31 or Materials and methods
bacterial infection.32 Thus, analyses of changes in metabolite
profiles of S. Typhimurium during growth in virulence indu- Chemicals and materials
cing conditions is timely, particularly as complementary sys- All chemicals and reagents were purchased from Sigma-Aldrich
tems biology (e.g., transcriptomics and proteomics) data sets (St. Louis, MO) unless otherwise noted. 3,5-Dibromotyrosine
from the same growth conditions are available to integrate with was purchased from Tokyo Chemical Industry (Tokyo, Japan)
or assist in the analysis of metabolomics data. and used as an internal standard. Ammonium bicarbonate was
A unique benefit of systems biology approaches is the ability purchased from Merck (Darmstadt, Germany), while a mixture
to utilize relatively comprehensive datasets to globally inform of fatty acid methyl esters (FAMEs) and deuterated myristic acid
predictions made using genome-scale metabolic models.33 For were obtained from Agilent Technologies, Inc (Santa Clara, CA).
example, model-guided analysis of time-series microarray data Zirconium/silica beads (0.1 mm) were purchased from Biospec
from Pseudomonas aeruginosa isolated from a cystic fibrosis Products (Bartlesville, OK) and used for cell lysis. Deionized
patient yielded insight into tradeoffs between virulence factor and purified water was used to prepare buffer and standard
and biofilm production during infection.34 Global models of solutions (Nanopure Infinity ultrapure water system, Barnstead,
S. Typhimurium metabolism have been developed,35–37 yielding Newton, WA).
a better understanding of gene essentiality and resulting in
the identification of potential drug targets. However, an Sample preparation
in-depth analysis utilizing them as a framework to integrate S. Typhimurium wild type cells (ATCC 14028) were used
multi-omics data sets to gain a better understanding of meta- throughout this study and were taken from a single colony
bolic processes during in vitro virulence conditions has not on an agar plate, subsequently inoculated into 7 mL of Luria-
been performed. This is especially valuable where meta- Bertani (LB) media (comprised of 1% tryptone, 0.5% yeast
bolomics analysis of pathogens isolated from host cells can extract, and 1% sodium chloride at pH 7), and incubated
be challenging due to the broad dynamic range of metabolite overnight at 37 1C. The overnight culture was centrifuged and
concentrations and the inability to distinguish host from the supernatant discarded. The cell pellets were suspended in
pathogen metabolites. LB media and centrifuged again, and the supernatant was
We therefore performed intracellular metabolomics analyses discarded. For LB cultures, the cell pellets were subsequently
of S. Typhimurium grown in both a rich medium and a suspended in 2 mL of LB media and used to inoculate 700 mL
minimal medium that induces the expression of virulence of LB in a 2.8 L flask. After 160 min of growth, cells were
genes,38,39 including those on pathogenicity islands 1 and 2 harvested via centrifugation (4000 g) and washed twice with
(SPI-1 and SPI-2, respectively), which are responsible for encod- Dulbecco’s PBS (Mediatech) once cultures reached an OD600
ing two type III secretion systems that deliver effector proteins of +1.0.
essential for virulence. SPI-1 is induced by growth in rich To stimulate the Salmonella virulence program, cells were
medium and is required for intestinal infection, while SPI-2 is transferred to a low pH, low magnesium, and low iron (LPM)
induced in minimal acidic medium and is required for systemic medium comprised of 8 mM MgCL2, 0.5 mM ferric citrate, 5 mM
infection. The resulting data were interpreted in the context KCl, 7.5 mM NH4SO4, 0.5 mM K2SO4, 0.3% glycerol v/v,
of a constraints-based metabolic model of S. Typhimurium to 0.00001% thiamine, 337 mM H3PO4, and 80 mM 2-(N-morpho-
evaluate metabolic changes associated with the induction of the lino)ethanesulfonic acid at pH 5.8.38,39 Briefly, cell pellets from
virulence program.18,19,40,41 the 7 mL LB media were suspended in LPM media and
Model-guided data analysis suggested fewer nutritional centrifuged. The supernatant was discarded and cell pellets
dependencies for maintaining optimal growth in virulence were suspended in 2 mL of LPM media and used to inoculate
conditions, which may help to counter host nutrient sequ- 700 mL of LPM in a 2.8 L flask. Cells were harvested after 4 h or
estering strategies. Intriguingly, metabolites that were recently 20 h, then washed and isolated as above.
associated with immunoactivation and immunosuppression of Three biological replicates were performed for each of the
macrophages42 were identified in the current study as differ- conditions (Scheme 1) described above. Cell pellets were stored
entially abundant. Our results suggest that production and at 80 1C prior to thawing and were subsequently resuspended
consumption of these putative ‘immunomodulatory’ meta- in an appropriate volume of 100 mM ammonium bicarbonate
bolites may be regulated by the pathogen. Finally, following according to their wet weight to compensate for any differences
entry into stationary phase in virulence conditions, an increase in cell numbers. The cells were lysed by bead-beating, and the
in the intracellular abundances of amino acids is observed lysates were transferred into new tubes. Subsequently, 100 mL
Mol. BioSyst. This journal is c The Royal Society of Chemistry 2013

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GC-MS data-processing and analysis

GC-MS raw data files were processed using MetaboliteDetector.45
Retention indices (RI) of detected metabolites were calculated
based on analyses of a mixture of FAMEs (C8–C30), followed by
their chromatographic alignment across all analyses after
deconvolution. Metabolites were then identified by matching
GC-MS features (characterized by measured retention indices
and mass spectra) to the Agilent Fiehn Metabolomics Reten-
tion Time Locked (RTL) Library,44 which contains spectra
and validated retention indices for 700 metabolites. All meta-
bolite identifications were manually validated to reduce
deconvolution errors during automated data-processing and
to eliminate false identifications. In addition, an occurrence

filter was applied to remove those features that were detected
in fewer than 6 out of 18 GC-MS analyses. The curated data

matrix of identified metabolites, unidentified features, and
their abundances for each sample was then loaded into
DAnTE46 for statistical analysis. Data were first converted to
log 2 scale, followed by Pearson’s correlation and principal
component analyses (PCA) to assess the reproducibility of the
experiment and to identify natural clustering within the data,
Scheme 1 respectively.
During the TMS derivatization of metabolites, partial
derivatives having different numbers of TMS groups can be
aliquots of cell lysates were extracted with four volumes of
generated.47 In these instances, the most abundant GC-MS
chilled (20 1C) chloroform–methanol (2 : 1, v/v), and the
peak for each metabolite was selected, or the integrated areas
aqueous phases after centrifugation were transferred to glass
of two or three peaks were combined when those peaks had
vials and dried in vacuo (SpeedVac; Thermo Scientific, Waltham,
relatively similar abundances.
MA). All samples were kept at 80 1C prior to chemical deriva-
tization for GC-MS analysis.
Microarray analysis
Chemical derivatization of metabolites and GC-MS analysis Condition-matched S. Typhimurium cultures were utilized for
Dried metabolite extracts were briefly dried again to remove any mRNA extraction at the J Craig Venter Institute (JCVI), and gene
residual water prior to chemical derivatization as previously expression was quantified with JCVI S. Typhimurium 13k v8
described.43 To protect carbonyl groups and reduce the number two-channel spotted oligonucleotide microarrays. Data from
of tautomeric isomers, 20 mL of methoxyamine in pyridine 193 microarrays (75 replicates in LB, 70 replicates and 48
(30 mg mL1) were added to each sample, followed by incuba- replicates from 4 and 20 h in LPM media, respectively) were
tion at 37 1C with generous shaking for 90 min. To derivatize extracted using the limma package for R from Bioconductor,48
hydroxyl and amine groups to trimethylsilyated (TMS) forms, background-corrected with the normexp method,49 normalized
80 mL of N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) using the print-tip LOESS method, and adjusted by quantile
with 1% trimethylchlorosilane (TMCS) were then added to each normalization between the channels and arrays.
vial, followed by incubation at 37 1C with shaking for 30 min.
The samples were allowed to cool to room temperature and Data dissemination
were then analyzed by GC-MS in random order. For technical All raw and processed metabolomics data will be made
replicates, each of the derivatized samples was split into two available via the Metabolights metabolomics data repository
different vials and analyzed separately. (http://www.ebi.ac.uk/metabolights/) under accession number
An Agilent GC 7890A coupled with a single quadrupole MSD MTBLS35. Raw metabolomics data will also be made available
5975C (Agilent Technologies, Inc) was used for all analyses, and via the PNNL Biological MS Data and Software Distribution
separations were performed using a HP-5MS column (30 m Center (http://omics.pnl.gov/). Microarray data corresponding
0.25 mm 0.25 mm; Agilent Technologies, Inc). The sample to wild type Salmonella in LB and LPM media, as well as various
injection mode was splitless, and 1 mL of each sample was mutant strains, is available via the Gene Expression Omnibus
injected. The GC oven was held at 60 1C for 1 min after (http://www.ncbi.nlm.nih.gov/geo/) under Series GSE25441.
injection, and the temperature was then increased to 325 1C The previously published proteomics data referenced in the
by 10 1C min1, followed by a 5 min hold at 325 1C.44 The Discussion is available via the PNNL Biological MS Data
injection port temperature was held at 250 1C throughout the and Software Distribution Center under accession number
analysis. PNNL_1025.

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Model-guided analysis of omics datasets less active in the first condition. When 0 o |x| o 1, there is
A genome-scale reconstruction of S. Typhimurium’s metabolic overlap in the flux ranges for the reactions across the condi-
network35,50 was employed for integrated analysis of omics tions; i.e., the reactions may have the same activity.
datasets. Genome-scale reconstructions are biochemically, In order to develop insights into the biological subsystems
genetically, and genomically consistent knowledgebases that most impacted by the alterations in reaction flux, an enrich-
are amenable to conversion into mathematical models for ment analysis of the model reactions where |x| > 1, was
functional and structural analyses.51 Functional analysis refers performed. Subsystem data for model genes and metabolites
to simulating cellular phenotypes, such as growth rates, and is were downloaded from KEGG55,56 and enrichment analysis was
often undertaken with flux balance analysis (see Orth et al. for a performed with Fisher’s exact method and Benjamini and
review of FBA52). Because genome-scale reconstructions employ Hochberg’s false discovery rate correction in Gitools.57 The
a boolean formalism that relates genomic loci to enzymatic KEGG ‘‘metabolic pathways’’ subsystem was filtered from the
reactions they are increasingly used as a framework for analysis analysis.
of various omics data types.33 All simulations were performed In order to develop insight into metabolic alterations
with COBRA for Python35,50 (http://opencobra.sourceforge.net), between the two LPM time points, we performed structural
unless otherwise specified. analyses of transcriptomics and metabolomics data with the
We used transcriptomics and metabolomics data to con- model. Structural analysis refers to using the model as a ‘graph’
struct condition-specific models for S. Typhimurium grown in on which to organize the data to aid in identification of
LB and LPM at 4 h (LPM 4 h). Transcriptomics data were prominent features. Here, the model structure and the trans-
integrated with the model using the GIMME algorithm, as criptomics data were used to inform an analysis of reporter
previously described in Becker et al.53 In short, the GIMME metabolites.58,59 The Reporter Metabolites algorithm identifies
algorithm seeks to minimize discrepancies between trans- metabolites in a biochemical network that are associated with a
criptome data and the model pathways required to simulate a significant number of differentially expressed genes. The
specified objective. Here, we used growth rate as the simulation lmscFit function from the limma package48 for R60 was
objective for growth in LB and LPM 4 h. It was not possible to employed to calculate p-values for alterations in gene expres-
perform flux balance analysis for the LPM 20 h samples sion, and integrated network diagrams of significant altera-
because the culture had exited log-phase and there was no tions were drawn in Cytoscape.61
obvious simulation objective. Additionally, a qualitative analysis of alterations in sub-
We devised an algorithm (described in Schmidt et al. in system prioritization was conducted. We constructed a condition-
review) to incorporate the metabolomics data (ESI†). In short, specific model for the LPM 20 h condition subject to the omics
the algorithm requires that the condition-specific model is able measurements and the ability to produce biomass. Next, we
to carry flux through at least one enzymatic reaction for each optimized the LPM 4 and 20 h models for the turnover of each
detected metabolite subject to the transcriptomic measure- metabolite in our GC-MS data set. Then we compared theses
ments and a simulation objective. Due to numerical limitations turnover capacities for LPM 4 vs. LPM 20 h. Metabolites
of most simulation software, it is important to note that were classified as exhibiting ‘‘increased,’’ ‘‘maintained,’’ or
reaction fluxes must be greater than the solver tolerance to be ‘‘reduced’’ priority based on how the qualitative analysis pre-
considered active. Here, simulated reaction fluxes needed to dicted the maxmimal turnover in LPM 20 vs. 4 h. Subsystem
exceed the minimal tolerance (1 108) for the ILOG/CPLEX to enrichment analysis was also performed on the different
be considered non-zero. classes of metabolites using model KEGG identifiers to gain
To determine if the space of accessible metabolic fluxes insight into network alterations.
differed for the LB versus the LPM 4 h models, flux variability
analysis was employed.54 Flux variability analysis is a method Results
that determines the allowable range of reaction flux for a Overview of GC-MS-based S. Typhimurium metabolomics data
specific model given the growth medium composition and a
specified simulation objective. Alterations in flux ranges for To identify metabolic changes associated with the expression
reactions between conditions were characterized by the relative of virulence genes, we performed metabolomics analyses of
flux range change, defined as: S. Typhimurium grown in LB and LPM media (Scheme 1),
the latter of which has been shown to induce the expression
c2 c1
x¼ (1) of virulence genes.38,39 On average, 245 unique metabolite
ðr2 r1 Þ=2
features (annotated with measured mass spectra and retention
Here, ci indicates the center of the flux range and ri indicates indices) were detected across 18 GC-MS analyses, and 124 of
the width of the flux range for condition i. A critical cutoff to these were initially matched to entries in the Agilent Fiehn
employ when interpreting the change in flux is |x| > 1. If x > 1, Metabolomics RTL Library. After manual validation of database
the reaction in the first condition must carry more flux than in matches (i.e., inspection of mass spectral and retention index
the second condition; i.e., the reaction is more active in the first matches), 66 metabolites were found to be confidently identi-
condition. If x o 1, the reaction in the first condition must fied, representing the broadest coverage of the S. Typhimurium
carry less flux than in the second condition; i.e., the reaction is metabolome reported to date when compared to recent

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analyses of volatile30,32 or soluble25,29,31 metabolites from this abundant in LB prior to the shift to LPM media. Amino acids
organism. A data matrix of identified metabolites and their appear to be consumed during log-phase growth in LB medium
abundances is provided as Table S1 (ESI†). A representative but dramatically increase in abundance upon the shift to LPM
GC-MS chromatogram is shown in Fig. S1 (ESI†). Pearson’s medium, particularly at 20 h (Fig. 1). All metabolites with
correlation analysis of the curated data matrix (data not shown) statistically significant differences in abundance between
indicated that data from instrumental replicates were highly growth conditions as determined by t-test with Bonferroni
correlated (>0.99), whereas data from biological replicates correction65 are shown in Table 1.
within treatment groups were less correlated (averages of
0.95–0.98). Model-guided analysis
To explore the systems level phenomena contributing to varia-
Metabolome alterations induced by growth condition tions in the metabolome across growth media, we used our
The differences between growth conditions drove the segre- previously published genome-scale model of S. Typhimurium
gation of data in a principal components analysis scores plot metabolism as a platform for multi-omics data analysis.35 The
(Fig. 1). To better visualize the similarities and differences model exhibited good coverage of the experimental observa-
between growth conditions, we constructed a heatmap of tions – over 90% of the confidently identified metabolites
metabolite abundances for the 66 confidently identified meta- mapped to the model (Table 2), while 1265 of 1270 genes
bolites (Fig. 2). Each growth condition clearly shows differences represented in the model mapped to microarray data for
in metabolite abundance as identified by GC-MS. Metabolites S. Typhimurium grown in matched conditions.
involved in glycerol catabolism, such as glycerol, glycerol
Enrichment analysis of altered flux ranges
3-phosphate, dihydroxyacetone phosphate (p = 1.8 102),
and pyruvate (p = 2.3 102), accumulated in S. Typhimurium In order to characterize how S. Typhimurium adapts from
grown in the rich LB medium but were depleted in cells upon growth in LB to LPM media, we used metabolomics and
the switch to LPM medium. In contrast, glucose was detected in transcriptomics data to construct condition-specific models.
relatively lower abundance in cells grown in LB relative to LPM Then we identified reactions, and metabolite turnovers, with
media. These observations are consistent with previous reports flux ranges that were exclusive to each condition-specific model
that glucose is the preferred carbon source of S. Typhimurium (Table S2, ESI†). To identify underlying biological themes
when available62–64 and indicates flux through the glycerol associated with these metabolites, we characterized the reac-
catabolism pathway in LPM medium where glycerol is the sole tions and metabolites by subsystem enrichment analysis. In
carbon source. Metabolites comprising beta alanine meta- order to map the flux range results to impacted subsystems, we
bolism – beta-alanine (p = 1.7 103), diaminopropane used the identities of genes encoding for enzymes governing
(p = 9.9 103), spermidine (p = 9.4 107), and malonate reactions or KEGG compound identifiers for metabolites
(p = 1.4 103) – were also increased in LB relative to LPM (Tables S3 and S4, ESI†).
media. Malonate is a precursor in fatty acid biosynthesis, and KEGG pathway enrichment analysis of reactions with exclu-
various saturated (myristate) and mono-unsaturated fatty acids sive flux ranges (|x| > 1) identified differences in pyruvate
(oleate and palmitoleate, p = 4.4 103) were also more metabolism, glycolysis/gluconeogenesis, methane metabolism,
D-alanine metabolism, folate biosynthesis, D-glutamine and
D-glutamate metabolism, and nitrogen metabolism. Supporting
these findings, the abundances of pyruvate (p = 2.6 102),
glucose-6-phosphate (p = 8.5 103), and L-alanine (p = 5.1
103) were significantly altered upon the shift from LB to LPM
media (Table 2).
Similarly, model-guided analysis of metabolite turnover flux
changes (LPM versus LB) resulted in the identification of
changes in metabolites mapping to fatty acid biosynthesis,
peptidoglycan biosynthesis, folate biosynthesis, methane meta-
bolism, cysteine and methionine metabolism, glycolysis/gluco-
neogenesis, and nitrogen metabolism (Table S4, ESI†). These
results illustrate broad differences in metabolic processes as
S. Typhimurium adapts from growth in media rich in com-
plex nutrients to an acidic environment with glycerol and
ammonium as carbon and nitrogen sources, respectively.
Next, we performed a comparison of changes in the experi-
Fig. 1 Principal components analysis of the curated data matrix of identified
mentally quantified intracellular metabolite abundances with
metabolites, unidentified features, and their abundances. Similarities and differ-
ences in metabolite abundances due to growth condition drove the clustering
changes in the previously described modeled metabolite turn-
and segregation of data within and across treatment, respectively. The inset over flux. Forty-three of the metabolites that were quantified in
shows the variability captured by each principal component. both growth conditions were present in the metabolic model

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Fig. 2 Heatmap of confidently identified metabolites. The abundances of 66 confidently identified metabolites were z-score transformed to facilitate data
visualization and then plotted in a heat map. Each row corresponds to a unique metabolite, and each column corresponds to a technical replicate. Biological
triplicates were performed and were analyzed in duplicate. Red indicates high abundance, while blue shows low abundance. Black indicates that the metabolite was
not observed.
and were not dead-ends nor present in thermodynamically for immunostimulatory metabolites exhibited a preferential
infeasible loops66,67 (Fig. 3A). decrease in their allowable flux range (Fig. 3B, p = 6.0 102,
We have previously predicted that there are a number of rank-sum permutation test, x-axis). Our analysis supports the
metabolites whose production, or consumption, are implicated hypothesis that even though many cellular metabolites with a
in macrophage activation.42 Several predictions from our pre- putative propensity for macrophage activation increase with
vious study were well-supported in the literature. For example, respect to intracellular concentration in LPM medium, their
an increase in glucose oxidation in Bacillus Calmette–Guérin- rate of production and consumption decreases. Because our
activated macrophages has been reported to correspond with measurements and predictions pertain to intracellular concen-
increases in hydrogen peroxide production in response to a trations and fluxes, they do not necessarily reflect the external
number of additional stimuli, such as LPS.68 Seventeen of these environment. It is plausible that S. Typhimurium could be
were detected by metabolomics (Table S5, ESI†). Experimentally sequestering immunostimulatory metabolites by decreasing
quantified intracellular metabolites that were previously pre- export rates.
dicted to enhance macrophage activation42 were preferentially To investigate how S. Typhimurium’s internal state may
increased in LPM relative to LB media (Fig. 3B, p = 1.5 102, influence the external environment, we assessed the ability of
rank-sum permutation test, y-axis). Whereas, the turnover rates the condition-specific models to consume and produce

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Table 1 Metabolites identified as having statistically significant differences in abundance among growth conditions. Metabolites with statistically significant
differences in abundances between LB and LPM 4 h or LPM 4 and 20 h were determined by t-test. Bonferroni corrected p values o 0.05 are shown
Metabolite Bonferroni corrected p value Metabolite Bonferroni corrected p value

LB vs. LPM 4 h
Adenine 7.8 1003 Pantothenate 6.3 1006
L-Alanine 5.1 1003 Pyruvate 2.6 1002
L-Aspartate 3.1 1002 Putrescine 4.4 1005
Beta-alanine 1.7 1003 L-Serine 4.9 1002
1,3-Diaminopropane 9.9 1003 Spermidine 9.4 1007
Dihydroxyacetone phosphate 1.8 1002 L-Threonine 2.8 1005
D-Glucose-6-phosphate 8.5 1003 Tyramine 4.3 1003
Glycerate 1.3 1008 L-Tyrosine 3.8 1002
Glycine 2.5 1004 Uracil 4.6 1002
L-Lysine 1.4 1004 Urea 7.8 1005
Malonate 1.4 1003 Urocanate 2.7 1003
Nicotinate 2.1 1002 L-Valine 2.2 1004

Palmitoleate 4.4 1003
LPM 4 h vs. LPM 20 h

Adenine 1.5 1006 Glycine 2.8 1003
Adenosine 1.6 1003 L-Glutamate 7.2 1004
AMP 1.3 1003 L-Lysine 3.0 1006
L-Cysteine 5.9 1004 Palmitoleate 4.2 1001
Cytosine 1.4 1004 Phosphoenolpyruvate 1.4 1002
2 0 -DeoxyCMP 4.4 1005 L-Threonine 1.4 1002
2,6-Diaminopimelate 4.9 1002 Tyramine 2.2 1002
1,3-Diaminopropane 2.2 1007 L-Tyrosine 5.4 1003
Hypoxanthine 2.9 1002 Uracil 4.5 1002
Glycerol 3-phosphate 4.1 1005 Xanthine 2.6 1003
Table 2 Metabolomics coverage of the S. Typhimurium genome-scale model. Analysis of structural changes in log and stationary phase
Shown are the numbers of unique metabolites (1) identified, (2) mapped to the
model, (3) unblocked, and (4) unmapped to the model for each growth condition The marked differences in metabolomic profiles as S. Typhimurium
transitions from LB log to LPM log to LPM stationary phases
LB LPM 4 h (Fig. 2 and Table 1) may be indicative of metabolic alterations
Identified 65 64 that occur when S. Typhimurium exits the nutrient rich
Mapped to Model 59 59 intestine and attempts to colonize hostile host cells. Entry into
Unblocked 57 57
Unmapped to Model 6 5 stationary phase was previously shown to induce additional
Salmonella virulence genes and promote acid tolerance and
resistance to oxidative stresses.69,70
immunomodulatory amino acids, fatty acids, and polyamines To identify metabolic changes associated with the transition
(Fig. 4). In LPM medium, the consumption of glycerol and from a rich environment to a poor environment and the exit to
ammonium were essential (not shown) and agrees well with the stationary phase, we performed reporter metabolites analysis58
experimental observation of depletion of metabolites in the on the transcriptome data. The 10 metabolites associated with
glycerol catabolism pathway (Fig. 2). Glycerol and ammonium the most significant transcriptional perturbations are shown in
are the sole carbon and nitrogen sources, respectively, in LPM Fig. 5A and B for the transition from LB to LPM media at 4 h
medium. Whereas, in LB medium the consumption of thirteen and for LPM medium from 4 to 20 h, respectively. Network
different metabolites (L-threonine, L-tryptophan, L-phenylalanine, maps illustrating the integration of significantly altered meta-
L-methionine, L-tyrosine, L-leucine, L-serine, L-valine, L-lysine, bolites as quantified by GC-MS and the reporter method
L-histidine, L-arginine, L-glucose, and L-proline) was found to be between LB and LPM at 4 h as well as LPM at 4 and 20 h are
essential for near optimal (99%) growth (Fig. 2). shown in Fig. S2 and S3 (ESI†), respectively.
We have previously suggested that consumption of select Despite the experimentally observed large alterations in
‘‘immunoinhibitory’’ metabolites by macrophages can reduce cellular amino acid concentrations between 4 and 20 h
activation functions.42 In short, the consumption of select (Fig. 1), amino acids did not score among the metabolites with
metabolites by macrophages can inhibit or stimulate the the most significant alterations in related transcripts by the
macrophages capacity to produce NO, ATP, or NADH. In line reporter metabolite analysis (Fig. 5B). Instead, the most
with this suggestion, several inhibitory metabolites may be significant changes in the reporter analysis related to iron-
produced by S. Typhimurium in virulence conditions binding and glycerol metabolism. This result prompted us to
(Fig. 4B). Interestingly, secretion of putrescine was also found investigate whether the metabolites might increase or decrease
to be essential for near optimal growth in a manner consistent with regard to their maximum rate of production and con-
with the omics-constraints in LB medium. sumption (turnover) in LPM medium between 4 and 20 h.

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Fig. 4 Evaluation of the ability of S. Typhimurium to consume or secrete

immunomodulatory metabolites. Consumption (top frame) and secretion (bot-
tom frame) rates for S. Typhimurium were evaluated, subject to limits based on
metabolite solubility (maximum) and the numerical precision of the solver
(minimum). The results in LB (red bars) and LPM (blue bars) media are shown.
Note that the y-axis is on a logarithmic scale (mmol gDW1 h1). Metabolites
are grouped according to classification as immunoinhibitory (blue box,
bottom), immunostimulatory (red box, bottom), or alternative macrophage
activation (M2; green box, bottom). Exchanges determined to be essential for
optimal S. Typhimurium growth in agreement with the transcriptomics data
are denoted (*).
to lipopolysaccharide (LPS) biosynthesis were maintained at

20 h, but many pathways related to amino acid synthesis were
Fig. 3 A comparison of concentration and relative flux range changes on the reduced.
change from LB to LPM. The concentrations and relative flux range changes for all The broad increase in amino acid concentration between 4
experimentally paired metabolites were compared upon the change from LB to and 20 h but decreased priority further prompted us to com-
LPM media (A). S. Typhimurium was in log phase growth in both conditions.
pare amino acid components incorporated into S. Typhimurium
Specific metabolites with putative immunomostimulatory (red) and immunoin-
hibitory (blue) roles were also investigated (B). The concentrations of immunos- as biomass. Biomass is comprised of a number of macro-
timulatory metabolites were preferentially increased in LPM relative to LB molecular components, and the production of biomass effec-
media (p = 0.015, red versus blue group along y-axis). Immunostimulatory tively allows Salmonella to replicate. Modeled biomass
metabolites exhibited a preferential relative decrease in their allowable flux constituents include lipids, glycogen, LPS, peptidoglycan,
range (p = 0.060, x-axis).
amino acids, and nucleotides, and their quantitative contribu-
tion to S. Typhimurium’s mass has been reported previously.36
Several amino acid biomass components were implicated by
Metabolites were classified into one of three groups based on thermodynamically infeasible loops66 and therefore excluded
their change in turnover rate in LPM 4 vs. 20 h: ‘maintained’ from the analysis. The maximum production and consumption
(i.e. effectively no change), ‘increased’ turnover in LPM 20 h, (turnover) of amino acids were all not maintained from 4 to
and ‘decreased’ turnover in LPM 20 h. For each metabolite 20 h (Fig. 5D).
group, we performed enrichment analysis using KEGG sub- Interestingly, proline was the least impacted at 20 h and
system information to characterize impacted pathways nearly maintained. Glutamate and alanine were the only
(Fig. 5C). The only metabolites that exhibited an increase in amino acid components of biomass significantly altered by
prioritization by this method were periplasmic and cellular the reporter metabolite analysis (p = 5.0 1003 and
melibiose (not shown). 4.2 1002, respectively). The only amino acid that was a
KEGG subsystem enrichment analysis was performed to component of biomass and exhibited a statistically signi-
further characterize metabolites with maintained or reduced ficant decrease in concentration from 4 to 20 h was threonine
production and consumption (turnover) priority. Pathways related (p = 1.2 1002).

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Fig. 5 Exploratory, model-guided analysis of alterations in metabolism without growth as an objective. The top 10 reporter metabolites based on the structure of the
metabolic reconstruction and gene expression changes between LB medium and LPM medium at 4 h (A) as well as between LPM 4 and 20 h (B) are shown. Pathway
enrichment of metabolites with similar or reduced maximal turnover flux are presented and annotated as maintained or reduced prioritization, respectively (C).
Comparison of GC-MS results to reporter metabolite and maximal flux ratios was performed for the amino acids utilized in biomass synthesis (D). Maintained or
decreased prioritization in amino acids in LPM medium between 4 and 20 h after removing growth constraints is indicated by ‘M’ or ‘D’. Grey boxes indicate significant
p-values using a cutoff of 0.05. Metabolites with statistically significant differences in abundance between LPM 4 h and LPM 20 h as measured by GC-MS with p values
o 0.05 (see Table 2) are indicated by ‘#’. *The maximal turnover flux comparison for some biomass components was omitted since these components are implicated
by internal reaction loops.
Discussion genome-scale model of S. Typhimurium metabolism as a plat-

form for multi-omics data analysis. Although S. Typhimurium
In this study, we characterized the metabolic profile of grows more slowly during log-phase in LPM than LB media, our
S. Typhimurium grown under conditions designed to induce the condition-specific model suggests optimal growth in nutrient
virulence program38,39 and confidently identified 66 metabolites in rich LB requires the simultaneous uptake of 13 nutrients
a GC-MS-based metabolomics study, representing the most com- (L-threonine, L-tryptophan, L-phenylalanine, L-methionine,
prehensive coverage of this organism’s metabolome to date. L-tyrosine, L-leucine, L-serine, L-valine, L-lysine, L-histidine,
To gain mechanistic insight into the differences in meta- L-arginine, L-glucose, and L-proline). In contrast, S. Typhimurium
bolite abundances between growth conditions, we used a can consume these metabolites but is not singularly dependent

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on them to maintain optimal growth in LPM. If a similar Notably, S. Typhimurium is still in log phase growth at these
robustness is also present in the host environment, it may time points. These pathways mediate adaptations to cellular
facilitate S. Typhimurium virulence in two ways. First, the membrane components, but may have further influences on
ability to consume a variety of metabolites may help to counter adapting to a role as an intracellular pathogen. For example,
host strategies to sequester key growth substrates. Second, the peptidoglycan recycling has been reported to facilitate survival
ability to both consume and produce a diverse repertoire of in the phagosome environment and influence host activation.74
substrates may help to directly modulate host cell activation by The control of surface modification was continued in LPM 20 h,
providing the host cell with metabolites that inhibit activation when LPS and fatty acid synthesis pathways were among the
of immune responses42 or by metabolically draining the host few with maintained prioritization (Fig. 5C). LPS remodeling
cell. In support of the possibility that S. Typhimurium could has previously been demonstrated to both promote survival75
provide the host with nutrients, endosomes contain trans- and modulate macrophage activation.76
porters that may effectively transport metabolites to the macro- Acquisition of metabolic building blocks by a pathogen
phage’s cytoplasm.6,71 residing within a host is a complex interplay between the two,
The predicted secretion of putrescine in LB medium by the influenced by host sequestration of nutrients, such as iron,77
omics-constrained models is consistent with the experimentally and the pathogen’s ability to adapt to the available nutrient
observed increase in intracellular putrescine levels with sources within the host.78 LPM medium approximates some of
the change to LPM medium (p = 4.4 105). Intracellular the conditions known to exist in the hostile intracellular
putrescine may provide S. Typhimurium with protection environment, including limitation of iron, amino acid starva-
against osmotic stress in the intra-macrophage environment, tion, and low pH. S. Typhimurium responds to these signals in
as it has been shown to be a component of adaptation to specific ways, including upregulation of factors related to
osmotic stress modulated by magnesium in Escherichia coli.72 virulence and survival in the host, as detected in our previously
In addition to synthesis of putrescine, we predicted capacity published proteomics analyses of cells upon transition from LB
for its consumption in LPM medium (Fig. 4). Putrescine may be to LPM media at 4 h (Fig. 6A).79 Interestingly, we report here
available to intracellular pathogens residing in the host an increase in intracellular amino acids in LPM medium,
environment since macrophages can synthesize polyamines particularly at the 20 h time point (Fig. 1), consistent with a
by diverting arginine from the formation of nitric oxide, and previous report that de novo synthesis of amino acids occurs
macrophage intracellular putrescine concentrations increase in S. Typhimurium grown intracellularly.63 The intracellular
following stimulation with LPS.42 amino acids were not among the most strongly altered meta-
Our present findings indicate that there is the potential bolites based on transcriptional changes and assessed by a
for metabolic coupling between the host and S. Typhimurium, reporter metabolites analysis (Fig. 5B). However, many amino
not just for the pathogen to access carbon and energy sources, acids, such as those involved in biomass synthesis, were
but potentially for the pathogen to (1) exploit metabolites observed to exhibit a decrease in priority by a consideration
critically involved in adaptations to survival in the hostile of the potential impact of metabolic gene expression on their
phagocytic host environment and (2) inhibit or redirect host production and consumption (turnover, Fig. 5D), and this
cell activation. Indeed, in a murine model of L. donovani decrease in prioritization was apparent also at the subsystems
infection, putrescine administered in the drinking water level (Fig. 5C). Our results suggested that the accumulation of
of mice was able to penetrate to the phagolysosome com- amino acids was concomitant with decreases in cellular demand
partment and restore virulence to a mutant deficient in and supply.
polyamine synthesis, demonstrating host–pathogen metabolic Therefore, we analyzed our previously published79 proteo-
coupling.73 mics data obtained from S. Typhimurium grown under iden-
Additional observations of S. Typhimurium metabolism tical conditions to determine if proteins related to amino
during growth in the virulence-inducing medium, align well acid and protein synthesis were significantly (p o 0.05
with the intracellular metabolic capacity of macrophages deter- following unpaired t-test with Bonferroni correction) altered
mined in our previous study.42 For example, the measured in LPM medium between 4 and 20 h. At the 4 h time
increase in macrophage cysteine levels agreed well with the point, abundances of proteins integral to amino acid bio-
predicted increase in capability of S. Typhimurium to consume synthetic pathways were higher, suggesting that the initial
cysteine in LPM versus LB (Fig. 4) media. Further, statistically transition to the nutrient-poor LPM environment stimulates
significant increases in macrophage fructose, D-sorbitol, and these anabolic pathways (Fig. 6B). However, the transition to
42
D-mannitol suggested additional potential carbon sources stationary phase growth coincided with a decrease in protein
available for growth. An additional evaluation of S. Typhimurium synthesis in LPM at 20 h, as evidenced by a decreasing trend
nutrient consumption, as performed in the top panel of Fig. 4, for ribosomal proteins. In addition, the abundances of
predicted the capability to utilize all three in LPM medium tRNA synthetases and translation elongation factors were
(data not shown). decreased, suggesting translation was diminished (Fig. 6C).
In the context of virulence, the observed alterations in Taken together, these proteomics data suggest that increased
the turnover flux for metabolites involved in fatty acid amino acid production at 4 h, combined with a slowing of
and proteoglycan synthesis (Table S4, ESI†) are interesting. cell growth and protein synthesis at 20 h, contribute to the

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Fig. 6 Accumulation of intracellular amino acids in LPM conditions is supported by complementary proteomics data. Upon transition into LPM media at 4 h, the
abundances of virulence-related proteins increase (A). Proteins involved in the biosynthesis of amino acids were more abundant in LPM conditions at 4 h relative to
20 h (B). However, proteins related to translation, including ribosomal proteins, translation factors, and tRNA synthetases, were decreased during growth of
S. Typhimurium in LPM 20 h (C). Protein abundances were considered significantly different between LPM 4 and 20 h if p o 0.05 following an unpaired t-test and
Bonferroni correction. Each row corresponds to a unique protein, and each column corresponds to a technical replicate. Biological triplicates were performed and were
analyzed in duplicate.79 Red indicates high abundance, while blue shows low abundance. Statistically significant changes are marked with (*).
observed accumulation of amino acids within S. Typhimurium Acknowledgements

in LPM medium.
In summary, the analysis of metabolomics data from This work was funded by the National Institute of Allergy and
S. Typhimurium grown in rich and minimal media in the context Infectious Diseases under Interagency agreement Y1-AI-8401.
of a genome-scale model demonstrate a potential new mechanism DRH is supported in part by a Seed Award from the San Diego
to disrupt immunity in the host. Further, S. Typhimurium may Center for Systems Biology funded by NIH/NIGMS (GM085764).
sequester amino acids during later simulated intracellular Significant portions of the work were performed at the Environ-
growth, perhaps in order to support rapid outgrowth during mental Molecular Sciences Laboratory, a national scientific
the last phases of infection. user facility sponsored by the Department of Energy’s (DOE)

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Salmonella Metabolismo Durante El Desarrollo Bajo Condiciones Que Inducen Expresión de Virulencia

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Molecular

Salmonella modulates metabolism during growth

Young-Mo Kim,za Brian J. Schmidt,zb Afshan S. Kidwai,c Marcus B. Jones,d

Brooke L. Deatherage Kaiser,a Heather M. Brewer,e Hugh D. Mitchell,a

This journal is c The Royal Society of Chemistry 2013 Mol. BioSyst.

Paper Molecular BioSystems

Collectively, metabolomics is a rapidly developing field in S. Typhimurium. A model-guided analysis of matching

Mol. BioSyst. This journal is c The Royal Society of Chemistry 2013

Molecular BioSystems Paper

GC-MS data-processing and analysis

to eliminate false identifications. In addition, an occurrence

in fewer than 6 out of 18 GC-MS analyses. The curated data

This journal is c The Royal Society of Chemistry 2013 Mol. BioSyst.

Paper Molecular BioSystems

Mol. BioSyst. This journal is c The Royal Society of Chemistry 2013

Molecular BioSystems Paper

This journal is c The Royal Society of Chemistry 2013 Mol. BioSyst.

Paper Molecular BioSystems

Mol. BioSyst. This journal is c The Royal Society of Chemistry 2013

Molecular BioSystems Paper

Metabolite Bonferroni corrected p value Metabolite Bonferroni corrected p value

Nicotinate 2.1 1002 L-Valine 2.2 1004

LPM 4 h vs. LPM 20 h

This journal is c The Royal Society of Chemistry 2013 Mol. BioSyst.

Paper Molecular BioSystems

Fig. 4 Evaluation of the ability of S. Typhimurium to consume or secrete

to lipopolysaccharide (LPS) biosynthesis were maintained at

Mol. BioSyst. This journal is c The Royal Society of Chemistry 2013

Molecular BioSystems Paper

Discussion genome-scale model of S. Typhimurium metabolism as a plat-

This journal is c The Royal Society of Chemistry 2013 Mol. BioSyst.

Paper Molecular BioSystems

Mol. BioSyst. This journal is c The Royal Society of Chemistry 2013

Molecular BioSystems Paper

observed accumulation of amino acids within S. Typhimurium Acknowledgements

This journal is c The Royal Society of Chemistry 2013 Mol. BioSyst.

Paper Molecular BioSystems

Mol. BioSyst. This journal is c The Royal Society of Chemistry 2013

Molecular BioSystems Paper

44 T. Kind, G. Wohlgemuth, D. Y. Lee, Y. Lu, M. Palazoglu, 171–196.

This journal is c The Royal Society of Chemistry 2013 Mol. BioSyst.

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