JOURNAL OF BACTERIOLOGY, Sept. 2011, p. 4719–4725 Vol. 193, No.

18
0021-9193/11/$12.00 doi:10.1128/JB.05132-11
Copyright © 2011, American Society for Microbiology. All Rights Reserved.

Metabolomics Reveals Phospholipids as Important Nutrient Sources

during Salmonella Growth in Bile In Vitro and In Vivo䌤
L. Caetano. M. Antunes,1 Sarah K. Andersen,2 Alfredo Menendez,1 Ellen T. Arena,1,3 Jun Han,4
Rosana B. R. Ferreira,1 Christoph H. Borchers,4 and B. Brett Finlay1,2,3*
Michael Smith Laboratories,1 Department of Biochemistry and Molecular Biology,2 and Department of Microbiology and
Immunology,3 The University of British Columbia, Vancouver, British Columbia, Canada, and University of
Victoria–Genome BC Proteomics Centre, University of Victoria, Victoria, British Columbia, Canada4
Received 21 April 2011/Accepted 5 July 2011

During the colonization of hosts, bacterial pathogens are presented with many challenges that must be
overcome for colonization to occur successfully. This requires the bacterial sensing of the surroundings and
adaptation to the conditions encountered. One of the major impediments to the pathogen colonization of the
mammalian gastrointestinal tract is the antibacterial action of bile. Salmonella enterica serovar Typhimurium
has specific mechanisms involved in resistance to bile. Additionally, Salmonella can successfully multiply in
bile, using it as a source of nutrients. This accomplishment is highly relevant to pathogenesis, as Salmonella
colonizes the gallbladder of hosts, where it can be carried asymptomatically and promote further host spread and
transmission. To gain insights into the mechanisms used by Salmonella to grow in bile, we studied the changes
elicited by Salmonella in the chemical composition of bile during growth in vitro and in vivo through a metabolomics
approach. Our data suggest that phospholipids are an important source of carbon and energy for Salmonella during
growth in the laboratory as well as during gallbladder infections of mice. Further studies in this area will generate
a better understanding of how Salmonella exploits this generally hostile environment for its own benefit.

To successfully colonize their hosts, bacterial pathogens
must be able to survive and adapt to the harsh conditions
encountered in some host environments. These include some
of the inherent physicochemical properties of host tissues as
well as specific host defenses. For intestinal pathogens, one of
the major barriers to overcome is the antibacterial action of
bile (5). Bile is produced in the liver, stored and concentrated
in the gallbladder, and excreted into the lumen of the small
intestine, where it aids in the digestion of dietary fat (4). The
antibacterial properties of bile are well documented, and some
organisms have adapted mechanisms to resist its action (8).
Salmonella enterica serovar Typhimurium is a bacterial patho-
gen with an intricate interaction with bile. Salmonella can
thrive in the gastrointestinal tract, liver, and gallbladder, all of
which are sites where bile is encountered in high concentra-
tions (4). The colonization of the gallbladder, the site with the
highest concentrations of bile, by Salmonella has important
implications not only for the pathogenesis of the disease but
also for carriage and transmission. Gallbladder colonization
during typhoid fever in asymptomatic individuals is a source of
bacterial shedding that aids in the transmission of this organ-
ism to new hosts (7).
Our group recently has shown that Salmonella can survive in
the lumen of the gallbladder, where it is exposed to a high
concentration of bile (15). Salmonella not only can survive the
antibacterial properties of bile but also can thrive in this harsh
environment, producing growth rates comparable to those
achieved in rich culture medium in the laboratory. Some of the
mechanisms used by Salmonella to resist the antibacterial ac-
tion of bile have been studied previously (3, 14, 16–21). How-
ever, most studies to date have focused on resistance to defined
mixtures of bile salts/acids and have not yet addressed the
interactions between Salmonella and “physiological” bile. Ad-
ditionally, the mechanisms used by Salmonella to successfully
grow using bile components as energy and carbon sources still
are unknown. To shed light on this issue, we performed a
metabolomic analysis of Salmonella growth in physiological
murine bile with the aim of identifying potential nutrient
sources used by this organism during the infection of the gall-
bladder. Our studies indicate that several host membrane lip-
ids are involved in Salmonella growth in this environment.
Specifically, the concentrations of multiple glycerophospholip-
ids, such as phosphatidylcholine (PC), phosphatidylethano-
lamine (PE), lysophosphatidylcholine (lyso-PC), and lysophos-
phatidylethanolamine (lyso-PE), were significantly decreased
upon Salmonella growth in bile, both in vitro and in vivo,
suggesting that these molecules can be used as growth sub-
strates by Salmonella. Our in vitro studies revealed that Salmo-
nella can readily use lyso-PC as the sole carbon source during
growth in minimal medium, supporting the notion that this
phospholipid can be used as a carbon source during growth in
the gallbladder. Further investigations of the nutritional re-
quirements for bacterial growth in bile will generate a better
understanding of how Salmonella exploits this normally anti-
bacterial host environment.

* Corresponding author. Mailing address: The University of Brit-

ish Columbia, 301-2185 East Mall, Vancouver, BC, V6T 1Z4, Can-
ada. Phone: (604) 822-2210. Fax: (604) 822-9830. E-mail: bfinlay
@interchange.ubc.ca.
䌤
Published ahead of print on 15 July 2011.
MATERIALS AND METHODS
Chemical reagents. Acetonitrile, water, formic acid, ammonium hydroxide,
streptomycin, dimethyl sulfoxide, L-histidine, and L-␣-lysophosphatidylcholine
(from egg yolk) were purchased from Sigma-Aldrich (St. Louis, MO). Casamino

4719
4720 ANTUNES ET AL.
Acids were obtained from Beckton, Dickinson, and Co. (Sparks, MD). An
electrospray (ES) tuning mix standard solution was purchased from Agilent
Technologies (Santa Clara, CA).
Salmonella growth in bile in vitro. Bile was extracted from eight C57BL/6 mice
(approximately 18 weeks old; The Jackson Laboratory, Bar Harbor, ME) and
used immediately. Salmonella enterica serovar Typhimurium SL1344 (23) was
grown overnight in Luria-Bertani (LB) broth containing 100 ␮g/ml of strepto-
mycin at 37°C with shaking. Cells were washed and resuspended in Dulbecco’s
phosphate-buffered saline (HyClone, Logan, UT), and 10 ␮l of bile was inocu-
lated with this solution at an approximate density of 5 ⫻ 106 cells/ml. The
experiment was performed in duplicate, and a total of two uninfected and two
infected bile samples were studied. Samples were incubated for 24 h at 37°C with
shaking. After incubation, aliquots were removed, diluted, and plated on LB agar
plates containing 100 ␮g/ml of streptomycin and incubated at 37°C overnight for
bacterial enumeration.
Salmonella growth in bile in vivo. Salmonella was grown overnight in LB broth
containing 100 ␮g/ml of streptomycin at 37°C with shaking. Cells were washed
and resuspended in Dulbecco’s phosphate-buffered saline (HyClone). Age- and
gender-matched C57BL/6 mice (approximately 20 weeks old; The Jackson Lab-
oratory) were infected with approximately 108 bacterial cells from this solution
by oral gavage. To investigate the impact of Salmonella infection on the chemical
composition of bile, four groups of three to four mice each either were infected
with Salmonella or kept uninfected (a total of 11 mice per treatment group). Five
days after infection, all mice were sacrificed and bile was collected. To ameliorate
the intrinsic issue of intersubject variability in our experiments, samples were
prepared by mixing equal volumes of bile from three to four mice, generating
three samples per treatment (uninfected and infected), which were used in the
subsequent steps. This also avoids the chance that any potential differences in
metabolite levels are masked by differences in bacterial loads in bile from dif-
ferent animals. Samples then were processed for bacterial enumeration by plat-
ing serial dilutions on LB agar plates containing 100 ␮g/ml of streptomycin and
incubated at 37°C overnight.
Sample preparation. After appropriate incubation, bile samples were centri-
fuged for 5 to 10 min at 13,000 ⫻ g and 7 ␮l of the supernatant was removed,
dried at room temperature in a centrifuge equipped with a vacuum pump, and
frozen at ⫺80°C until used.
Direct infusion (DI)-FT-ICR-MS. For metabolic fingerprinting, the dried ex-
tracts were suspended in a mixture of acetonitrile and water (200 ␮l of 50%
acetonitrile for in vitro samples and 400 ␮l of 75% acetonitrile for in vivo
samples), vortexed, and cleared by centrifugation. To reduce ionization suppres-
sion or enhancement effects, extracts were further diluted 1:20 (in vitro) or 1:6 (in
vivo) with 50% acetonitrile containing either 0.2% formic acid (for positive-ion
mode) or 0.5% ammonium hydroxide (for negative-ion mode) and spiked with
predefined amounts of the ES tuning mix solution as the internal standard for
mass calibration. The solutions then were infused through a syringe pump (KDS
Scientific, Holliston, MA) at a flow rate of 2.5 ␮l/min into a 12-T Apex-Qe hybrid
quadrupole Fourier transform ion cyclotron mass spectrometer (FT-ICR-MS)
(Bruker Daltonics, Billerica, MA) equipped with an Apollo II electrospray ion-
ization (ESI) source, a quadrupole mass filter, and a hexapole collision cell. Data
were recorded in both positive- and negative-ion modes using broadband detec-
tion with a data acquisition size of 1,024 kb/s and within m/z 150 to 1,000. Typical
ESI-MS parameters were a capillary electrospray voltage of 3,600 to 3,750 V,
spray shield voltage of 3,300 to 3,450 V, source ion accumulation time of 0.1 s,
and collision cell ion accumulation time of 0.2 s. Survey scan mass spectra in each
ion mode were averaged from the accumulation of 200 scans per spectrum, and
duplicate acquisitions per sample were performed.
Data processing. Raw mass spectrometry data were processed using a custom-
developed software package as described elsewhere (10). First, raw mass spectra
acquired from each sample group were batch processed using a home-written
VBA script within the instrument vendor’s data analysis software, DataAnalysis,
to do automatic internal mass calibration with the reference masses of the spiked
calibration standards and a known contaminant, N-butylbenzensulfonamide.
Monoisotopic peaks corresponding to the isotopic pattern distributions then
were automatically determined, and those with a signal-to-noise ratio of at least
3 were picked. Their m/z values were converted to neutral masses by subtracting
or adding 1.007276 for positive- or negative-ion mode, respectively. The resulting
mass lists from all of the mass spectra within each set of uninfected/infected
groups detected in positive- or negative-ion modes were further processed with
another customized software program developed with LabVIEW (National In-
struments, Austin, TX). The first step of this software is to remove the masses
corresponding to adduct ions (M⫹Na)⫹ and (M⫹K)⫹ for the positive-ion mode
and (M⫹Cl)⫺ for the negative-ion mode from the mass lists based on the
expected mass differences for these ions within 2 ppm to yield a list of unique
J. BACTERIOL.
biochemical component masses together with their peak intensities. The peak
intensities of all the monoisotopic neutral masses subsequently were normalized
to the total ion intensity calculated from a mass spectrum. Masses observed in
both samples of at least one of the sample groups (uninfected or infected) were
aligned and combined into unique metabolite features from the masses that
matched within 2 ppm across all data. Finally, a two-dimensional data matrix
(mass versus relative intensity) was generated for each sample group and saved
in a format amenable to further data analysis. To identify differences in metab-
olite composition between uninfected and infected samples, we first selected
metabolites that were present on one set of samples (uninfected or infected) but
not the other. Additionally, we averaged the intensities of the remaining masses
in each group, calculated the ratios between averaged intensities of metabolites
from uninfected and infected mice, and selected those showing changes of at
least 2-fold. To assign possible metabolite identities to the masses selected as
described above, the monoisotopic neutral masses of interest were queried
against the human metabolome database (HMDB; http://www.hmdb.ca) (22)
with a tolerance of 0.001 Da.
Salmonella growth on lysophosphatidylcholine. For growth studies, we used
M9 medium (0.1 mM CaCl2, 2 mM MgSO4, 22 mM KH2PO4, 42 mM Na2HPO4,
8.6 mM NaCl, and 18.7 mM NH4Cl) containing either 0.1% (wt/vol) Casamino
Acids or 200 mM histidine. L-␣-lysophosphatidylcholine was suspended in di-
methyl sulfoxide at 100 mg/ml and added to culture media at a final concentra-
tion of 1 mg/ml. Salmonella was grown overnight in LB broth containing 100
␮g/ml of streptomycin at 37°C with shaking. Cells were washed with Dulbecco’s
phosphate-buffered saline (HyClone) and diluted 1:100 in the appropriate cul-
ture medium. Samples were incubated at 37°C with shaking, and growth was
assessed through measurements of culture optical density at 600 nm.
Statistical analysis. Data were analyzed by unpaired t tests with 95% confi-
dence intervals using GraphPad Prism, version 4.0 (GraphPad Software Inc., San
Diego, CA).
Mass spectrometry data accession number. Data have been deposited in the
GEO database (http://www.ncbi.nlm.nih.gov/geo) under accession number
GSE-30404.
Ethics statement. Animal experiments were approved by the Animal Care
Committee of the University of British Columbia and performed in accordance
with institutional guidelines.
RESULTS
To study the effect of Salmonella in vitro growth on the
chemical composition of bile, we extracted fresh bile from
mice, inoculated it with Salmonella, and compared the chem-
ical composition of uninfected and infected samples through
DI-FT-ICR-MS. During the 24 h of incubation, Salmonella
replicated extensively in bile, reaching a density of 8.4 ⫻ 109
cells/ml (standard error of the mean of 1.87 ⫻ 109) from an
initial inoculum of 5 ⫻ 106 cells/ml, in accordance with our
previous report (15). DI-FT-ICR-MS analysis of uninfected
and infected samples enabled us to detect 1,244 metabolites
with distinct masses (Table 1). To identify metabolites showing
differences in intensities between uninfected and infected sam-
ples, we screened our data set for masses that were present in
only one of the sample groups (uninfected or infected). Addi-
tionally, we calculated average intensities and fold changes
between the two data sets and identified masses displaying
differences of 2-fold or more. Of the 1,244 total masses de-
tected, 447 masses (35.9%) were affected, according to the
criteria described above (Table 1). These masses then were
used to query the HMDB to predict putative metabolite iden-
tities. Of the 447 masses that were changed upon Salmonella
growth, 49 (11%) had hits in the HMDB; 36 were decreased
upon Salmonella growth, whereas 13 were increased (Tables 1
and 2). Because our aim was to study potential carbon and
energy sources used by Salmonella, we focused our analysis on
the masses whose levels were decreased upon bacterial growth.
Table 2 shows the signal intensities for all metabolites whose
VOL. 193, 2011
TABLE 1. Overview of the effects of Salmonella growth on bile composition
No. of distinct masses detected
Growth
Negative Positive
type Overlappinga Total
ionization ionization
In vitro 410 869 35 1,244
In vivo 244 312 12 544
a
Masses were considered to overlap when they differed by less than 0.001 Da.
levels were decreased after Salmonella growth in bile. As can
be seen from these data, glycerophospholipids were the most
overrepresented metabolite category (15 out of 25 significantly
affected masses [P ⬍ 0.05]; 60%), with many masses being as-
signed to phosphatidylcholine (PC), phosphatidylethanolamine
(PE), lysophosphatidylcholine (lyso-PC), and lysophosphatidyl-
ethanolamine (lyso-PE). PC and lyso-PC were the most
overrepresented metabolites among the metabolites affected;
of the 25 metabolites significantly affected, 11 were assigned as
one of these compounds.
The data presented above suggested that Salmonella uses
phospholipids, mainly PC and lyso-PC, as sources of carbon
and energy during growth in bile in vitro. However, whether or
not this was relevant for Salmonella growth during gallbladder
infections remained to be determined. To investigate this pos-
sibility, we analyzed the composition of bile from uninfected
mice and compared it to bile from mice that had been infected
with Salmonella. After 5 days of infection, Salmonella was
present at various levels in the three samples analyzed (from
1.6 ⫻ 103 to 9.2 ⫻ 108 cells per ml). DI-FT-ICR-MS analysis
allowed the detection of 544 metabolites (Table 1). We then
analyzed this data set as described above to identify metabo-
lites showing differences in intensities between uninfected and
infected samples. This revealed that 318 metabolites (58.5%)
had their levels changed by infection (Table 1). Again, we
queried the HMDB to determine putative metabolite identi-
ties. Sixty-two out of the 318 masses affected by Salmonella
were putatively identified (19.5%), with 23 being decreased
and 39 being increased upon infection (Tables 1 and 3). Table
3 shows signal intensities of the metabolites that were de-
creased after Salmonella infection. Once again, we found that
glycerophospholipids were the most overrepresented category
of metabolites decreased by Salmonella growth (11 out of 21
significantly affected masses [P ⬍ 0.05]; 52.4%). PC and
lyso-PC were abundant within our data set, with 10 out of the
21 metabolites significantly affected being putatively identified
as one of these compounds. These results suggest that Salmo-
nella degrades PC and lyso-PC during growth in gallbladder
infections, and that our in vitro results do not represent a
phenomenon restricted to growth in the laboratory.
The metabolomics data described above suggest that glyc-
erophospholipids are used as substrates by Salmonella during
growth in bile. To obtain further evidence that Salmonella can
grow on these compounds, we performed in vitro growth stud-
ies in minimal medium using lyso-PC as the sole carbon source
or in addition to other carbon sources. To do so, we monitored
the optical density (at 600 nm) of Salmonella cultures growing
in M9 minimal medium in the absence or presence of lyso-PC.
Because the Salmonella strain used in our studies is a histidine
auxotroph, we added either 200 mM L-histidine or 0.1% Casa-
METABOLOMICS OF SALMONELLA GROWTH IN BILE 4721
No. showing change in intensity
No. putatively % Putatively
% Changed
Decreased Increased Total identified identified
254 193 447 49 35.9 11
174 144 318 62 58.5 19.5
mino Acids as histidine sources. Figure 1A shows that lyso-PC
enhanced Salmonella growth when medium containing
Casamino Acids was used. In these experiments, Casamino Acids
function not only as a source of histidine but also as the source
of other amino acids that can be used by Salmonella as carbon
and energy sources. Although Salmonella was able to grow in
this medium without the addition of lyso-PC, this phospholipid
clearly enhanced bacterial growth (Fig. 1A). In medium con-
taining histidine but with no other amino acids that could be
used as carbon and energy sources, no growth was observed in
the absence of lyso-PC. However, when lyso-PC was added to
these cultures, Salmonella exhibited robust growth, confirming
that lyso-PC and potentially other glycerophospholipids pres-
ent in bile can be used as the sole carbon and energy sources.
To obtain a relative measure of how well Salmonella grows on
lyso-PC, we performed growth curves using either lyso-PC or
glucose as the sole carbon and energy source. Figure 1B shows
that Salmonella grows significantly better on lyso-PC than on
glucose, even though the glucose concentration used (10 mg/
ml) was 10 times higher than that of lyso-PC (1 mg/ml). Ex-
ponential growth in lyso-PC began after 6 h of incubation,
whereas the same occurred only after approximately 12 h of
incubation in glucose. Taken together, our data show that
phospholipids are rich carbon and energy sources that can be
used for Salmonella growth, and that these likely are important
growth substrates for Salmonella in bile.
DISCUSSION
Bile is an important body fluid whose most widely recog-
nized function is aiding in the digestion of dietary fats.
Although this is an important function of bile, this complex
mixture of organic molecules is involved in many other
physiological processes. Bile is important for the elimination of
cholesterol, regulation of gene expression, induction of mucin
secretion, control of electrolyte absorption, and other func-
tions (11). Of relevance to this study, bile shows strong anti-
bacterial activity and is involved in the control of commensal
microbial growth in the gastrointestinal tract as well as defense
against invading pathogens. Some microbes, however, have
evolved mechanisms to cope with the deleterious effects of bile
(5, 8). This is true for both intestinal commensals and patho-
gens.
Salmonella enterica serovar Typhimurium is an intestinal
pathogen with specific mechanisms that allow it to resist bile
(3, 14, 16–21). The induction of drug efflux pumps by bile salts
has been shown to play a role in Salmonella resistance to both
bile and antibiotics (18). DNA repair also is critical for bile salt
resistance in Salmonella (17). Lastly, the major virulence reg-
ulator PhoPQ in Salmonella also is critical for resistance to bile
4722 ANTUNES ET AL. J. BACTERIOL.

TABLE 2. Putative identities of metabolites decreased by Salmonella growth in bile in vitro

Chemical Signal intensityb

Common name Mass (Da) HMDBc no.
formula Uninfected Infected

Ascorbic acid C6H8O6 176.0322 0.56 0.25 00044

D-Glucurono-6,3-lactone 06355
3-Oxododecanoic acid C12H22O3 214.1569 0.24 0.07 10727
L-Isoleucyl-L-proline C11H20N2O3 228.1474a 1.3 0 11174
L-Leucyl-L-proline 11175
Myristic acid C14H28O2 228.2089 0.61 0.3 00806
2,6,10-Trimethylundecanoic acid 02221
Glycerophosphocholine C8H20NO6P 257.1028a 6.73 0.5 00086
Norophthalmic acid C10H17N3O6 275.1117a 3.99 0.19 05766
␥-Glutamylglutamine 11738
L-Cysteinylglycine disulfide C8H15N3O5S2 297.0453a 2.67 0 00709
3-Oxooctadecanoic acid C18H34O3 298.2508 1.4 0.5 10736
Capsaicin C18H27NO3 305.199 0.16 0.07 02227
8,11,14-Eicosatrienoic acidd C20H34O2 306.256 0.58 0.17 02925
Adrenic acid C22H36O2 332.2715 0.65 0.31 02226
14,15-DiHETrEd C20H34O4 338.2455a 0.35 0 02265
12-Keto-tetrahydro-leukotriene B4 02995
Tetracosanoic acid C24H48O2 368.3652 0.61 0.28 02003
Pentacosanoic acid C25H50O2 382.3811 0.49 0.14 02361
7-Dehydrocholesterol C27H44O 384.3392 3.93 1.81 00032
Vitamin D3 00876
Cholestenone 00921
8-Dehydrocholesterol 02027
Desmosterol 02719
Zymosterol intermediate 2 06271
Previtamin D3 06500
Lumisterol 3 06505
Tachysterol 3 06560
5,6-Trans-vitamin D3 06719
5a-Cholesta-7,24-dien-3b-ol 06842
5a-Cholesta-8-en-3-one 12178
Cysteineglutathione disulfide C13H22N4O8S2 426.0877a 6.3 0 00656
Ubiquinone Q4 C29H42O4 454.3092a 0.67 0.19 06710
LysoPC(14:0)d C22H46NO7P 467.3013a 2.48 0 10379
LysoPE关0:0/18:2(9Z,12Z)兴d C23H44NO7P 477.2856a 7.42 2.04 11477
LysoPC关16:1(9Z)兴d C24H48NO7P 493.317a 26.25 12.34 10383
LysoPE关0:0/20:2(11Z,14Z)兴d C25H48NO7P 505.3168a 7.22 2.88 11483
LysoPE关0:0/22:5(4Z,7Z,10Z,13Z,16Z)兴d C27H46NO7P 527.3015a 1.34 0 11494
LysoPE关0:0/22:4(7Z,10Z,13Z,16Z)兴d C27H48NO7P 529.3171a 3.24 0.68 11493
LysoPC关20:5(5Z,8Z,11Z,14Z,17Z)兴d C28H48NO7P 541.317a 15.48 0 10397
LysoPC关20:4(5Z,8Z,11Z,14Z)兴d C28H50NO7P 543.3321a 290.86 95.65 10395
(3a,5b,7a)-23-Carboxy-7-hydroxy-24-norcholan- C30H48O10 568.325a 1.35 0.58 02430
3-yl-b-D-glucopyranosiduronic acid
Deoxycholic acid 3-glucuronide 02596
Cholic acid glucuronide C30H48O11 584.3199a 4.7 0.79 02577
25-Hydroxyvitamin D2-25-glucuronide C34H52O8 588.3656 2.28 1.05 10342
25-Hydroxyvitamin D2 25-(beta-glucuronide) 10360
Oxidized glutathione C20H32N6O12S2 612.1519a 8.75 0 03337
PC关15:0/18:2(9Z,12Z)兴d C41H78NO8P 743.5464a 1.04 0.12 07940
PE关14:0/22:2(13Z,16Z)兴d 08843
PC关15:0/18:1(11Z)兴d C41H80NO8P 745.5628a 0.34 0 07938
PE关14:0/22:1(13Z)兴d 08842
N-dimethyl-PE关16:0/18:1(9Z)兴 10568
PC关14:0/20:4(5Z,8Z,11Z,14Z)兴d C42H76NO8P 753.5301a 1.06 0 07883
PE关15:0/22:4(7Z,10Z,13Z,16Z)兴d 08910
PC关15:0/20:4(5Z,8Z,11Z,14Z)兴d C43H78NO8P 767.5459a 0.47 0 07949
PE关16:0/22:4(7Z,10Z,13Z,16Z)兴d 08943
PC关14:1(9Z)/22:2(13Z,16Z)兴d C44H82NO8P 783.5782a 23.64 0 07921
PC关16:0/22:4(7Z,10Z,13Z,16Z)兴d C46H84NO8P 809.5942a 3.82 0 07988
PC关18:2(9Z,12Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)兴d C48H80NO8P 829.5624a 0.3 0 08156
a
Significantly changed (P ⬍ 0.05).
b
Average intensities of four measurements (two samples with duplicate acquisitions) are shown.
c
Human metabolome database (www.hmdb.ca).
d
Several derivatives could be assigned to these masses; only one representative of each class of molecules is presented. PC, phosphatidylcholine; PE, phosphatidyl-
ethanolamine; DiHETrE, dihydroxy-eicosatrienoic acid.
VOL. 193, 2011 METABOLOMICS OF SALMONELLA GROWTH IN BILE 4723

TABLE 3. Putative identities of metabolites decreased by Salmonella growth in bile in vivo

Chemical Signal intensityb

Common Name Mass (Da)a HMDBc no.
formula Uninfected Infected

Glycerophosphocholine C8H20NO6P 257.1028e 2.6 0.54 00086

Tetracosahexaenoic acid C24H36O2 356.2715e 2.73 0.32 02007
7-Ketodeoxycholic acid C24H38O5 406.2719e 4.88 0 00391
3,7-Dihydroxy-12-oxocholanoic acid 00400
3-Oxocholic acid 00502
3a,7a,12b-Trihydroxy-5b-cholanoic acidd C24H40O5 408.2876e 275.59 0.6 00312
Allocholic acid 00505
Alpha-muricholic acid 00506
6a,12a-Dihydroxylithocholic acid 00527
Cholic acid 00619
Hyocholic acid 00760
Muricholic acid 00865
Ursocholic acid 00917
1b-Hydroxycholic acid C24H40O6 424.2824e 1.07 0 00307
3b,4b,7a,12a-Tetrahydroxy-5b-cholanoic acidd 00311
1b,3a,7b-Trihydroxy-5b-cholanoic acidd 13158
Deoxycholic acid glycine conjugate C26H43NO5 449.314e 1.51 0 00631
Chenodeoxycholic acid glycine conjugate 00637
Glycoursodeoxycholic acid 00708
Chenodeoxyglycocholic acid 06898
Coprocholic acid C27H46O5 450.3345e 1.77 0.51 00601
3a,7a,12a-Trihydroxy-5b-cholestanoic acid 03873
LysoPE关0:0/20:2(11Z,14Z)兴d C25H48NO7P 505.3159 0.88 0.2 11483
LysoPC关18:1(9Z)兴d C26H52NO7P 521.348e 152.4 48.61 02815
LysoPE(关0:0/22:6(4Z,7Z,10Z,13Z,16Z,19Z)兴d C27H44NO7P 525.2853e 5.08 2.33 11496
LysoPC关20:4(5Z,8Z,11Z,14Z)兴d C28H50NO7P 543.3318e 121.98 36.27 10395
Triterpenoid C30H48O7S 552.312e 1.59 0 04309
LysoPC关22:6(4Z,7Z,10Z,13Z,16Z,19Z)兴d C30H50NO7P 567.332e 157.31 58.88 10404
Protoporphyrinogen IX C34H40N4O4 568.3043 4.88 2.14 01097
Cholic acid glucuronide C30H48O11 584.3197e 1.59 0 02577
Oxidized glutathione C20H32N6O12S2 612.1517e 0.53 0.15 03337
PC(14:0/16:0)d C38H76NO8P 705.5308e 1.85 0 07869
PE(15:0/18:0)d 08892
N-methyl-PE(16:0/16:0)d 10567
PC关14:0/18:2(9Z,12Z)兴d C40H76NO8P 729.5308e 12.16 5.85 07874
PE关20:2(11Z,14Z)/15:0兴d 09285
PC关14:0/18:1(11Z)兴d C40H78NO8P 731.5468e 54.63 10.28 07872
PE关20:1(11Z)/15:0兴d 09252
N-methyl-PE关16:0/18:1(9Z)兴d 10569
PC(16:0/16:0)d C40H80NO8P 733.5624e 14.39 6.52 00564
PE(20:0/15:0)d 09219
PC关18:2(9Z,12Z)/15:0兴d C41H78NO8P 743.5471 e
7.27 1.81 08132
PE关14:0/22:2(13Z,16Z)兴d 08843
PC关18:1(9Z)/15:0兴d C41H80NO8P 745.5629e 6.06 0 08099
PE关14:0/22:1(13Z)兴d 08842
N-dimethyl-PE关16:0/18:1(9Z)兴d 10568
PC关14:0/20:2(11Z,14Z)兴d C42H80NO8P 757.5626e 306.2 0 07880
PE关22:2(13Z,16Z)/15:0兴d 09549
N-methyl-PE关18:1(9Z)/18:1(9Z)兴d 10565
a
Masses also affected by Salmonella growth in bile in vitro are in boldface and italics.
b
Average intensities from six measurements (three samples with duplicate acquisitions) are shown.
c
Human metabolome database (www.hmdb.ca).
d
Several derivatives could be assigned to these masses; only one representative of each class of molecules is presented.
e
Significantly changed (P ⬍ 0.05). PC, phosphatidylcholine; PE, phosphatidylethanolamine.

salts (21). It is important to note that all of these studies have
been performed using defined mixtures of bile salt and acids.
Therefore, although these findings are important to our un-
derstanding of the mechanisms allowing Salmonella to resist
the antibacterial action of bile, these studies provide limited
knowledge of the interactions that occur between Salmonella
and physiological bile during infection.
Besides resisting the deleterious effects of bile, Salmonella
can use bile components as growth substrates; Salmonella
growth rates in physiological bile are the same as, if not higher
than, those in rich culture medium (15). Because Salmonella
can colonize the gallbladder of hosts, both resistance to bile
and its use as a growth substrate have important implications
for Salmonella pathogenicity. However, the mechanisms used
by Salmonella to grow in bile remain unidentified. To identify
potential growth substrates in bile, we set out to detect organic
molecules whose levels in bile were decreased after Salmonella
growth. Bile is a complex mixture of molecules, and identifying
4724 ANTUNES ET AL.
A pholipids, are significantly decreased following Salmonella
2.0
Optical density (600 nm)
1.6
1.2
0.8
0.4
0
0 5 10 15 20 25
Time (hours)
B 0.8
Optical density (600 nm)
0.6
0.4
0.2
0 lipids, in particular PC and lyso-PC, are used by Salmonella
0 5 10 15 20 25
Time (hours)
FIG. 1. Salmonella growth curves in minimal medium with various
carbon and energy sources. (A) Salmonella was cultured in M9 mini-
mal medium containing 200 mM histidine (His; circles) or 0.1% Casa-
mino Acids (CAA; squares). Cultures without lyso-PC are represented
by gray symbols, whereas cultures containing lyso-PC are indicated by
black symbols. Results shown represent the averages from three cul-
tures, and bars indicate the standard deviations. (B) Salmonella was
grown in M9 minimal medium with 200 mM histidine (His). Cultures
with no addition are represented by diamonds. Circles indicate cul-
tures with glucose (Glu; 10 mg/ml), whereas squares indicate cultures
with lyso-PC (1 mg/ml). Results shown represent averages from three
cultures, and bars indicate the standard deviations.
and quantifying levels of its components requires high-throughput
methods of molecular characterization. We recently used high-
throughput metabolomics through DI-FT-ICR-MS to study
changes in the host intestinal and hepatic metabolome due to
antibiotic treatment and Salmonella infection (1, 2). DI-FT-
ICR-MS is a powerful technology that allows the detection and
quantification of thousands of chemical species in complex
samples (9, 10). Therefore, we applied DI-FT-ICR-MS to
study changes in the chemical composition of physiological bile
elicited by Salmonella growth in vitro and in vivo. Our metabo-
lomics results showed that many of the components decreased
by Salmonella growth could be putatively identified as glycero-
phospholipids. The main components of bile are bile acids,
phospholipids, cholesterol, proteins, and biliary pigments
(6). Phospholipids constitute the second most abundant compo-
nent of bile, accounting for 22% of the total composition.
Therefore, phospholipids would represent an attractive target
for Salmonella utilization. Indeed, Salmonella previously has
been shown to use phosphatidylserine that is present in intes-
tinal mucus as the sole source of carbon and nitrogen (12). In
this study, we show that PC and lyso-PC also can be used as
carbon and energy sources during growth in bile; the concen-
trations of these compounds, as well as those of other phos-
J. BACTERIOL.
growth. We also show that, in vitro, lyso-PC can be a rich
carbon and energy source for Salmonella growth.
His Although altogether our results strongly suggest that Salmo-
nella uses PC and lyso-PC as growth substrates, it is important
His, Lyso-PC
to mention that other processes could result in decreased levels
CAA of phospholipids in bile during gallbladder infections. For in-
CAA, Lyso-PC stance, it is possible that Salmonella infection alters phospho-
lipid synthesis, transport, or degradation, and this also would
result in changes in the phospholipid content of bile. In this
context, it is worth noting that lyso-PC and other phospholipids
have critical host signaling functions. Lyso-PC can activate
signaling cascades that culminate in the activation of inflam-
matory processes, among others (13). Therefore, it is tempting
to speculate that, in vivo, Salmonella benefits from the degra-
dation of lyso-PC beyond the use of this molecule as a carbon
His
and energy source. However, this hypothesis remains to be
His, Glu investigated.
His, Lyso-PC Our work shows that metabolomics can be used as a tool to
identify important metabolic functions in microbes and gener-
ate hypotheses as to how they could be important during dis-
ease. The data presented here suggests that glycerophospho-
during growth in bile. Because Salmonella actively colonizes
the gallbladders of hosts during infection, growth in bile is an
important component of this microorganism’s virulence strat-
egies. By quickly multiplying in host bile, Salmonella is able to
create a niche from which it can successfully spread to other
hosts through the reseeding of the intestinal tract during bile
secretion and fecal shedding. This opens the possibility that
interfering with microbial lipid metabolism is of therapeutic
value during systemic Salmonella infections, particularly during
the colonization of the gallbladder.
ACKNOWLEDGMENTS
This work was funded by the Canadian Institutes of Health Re-
search, Crohn’s and Colitis Foundation of Canada, Genome Canada,
and Genome British Columbia. L.C.M.A. and R.B.R.F. are supported
by postdoctoral fellowships from the Canadian Institutes of Health
Research. B.B.F. is an HHMI International Research Scholar and the
University of British Columbia Peter Wall Distinguished Professor.
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