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Systemic Responses of BALB/c Mice to Salmonella typhimurium Infection Xiaoyang Zhu,§,∥ Hehua Lei,§ Junfang Wu,§ Jia V. Li,‡ Huiru Tang,§,† and Yulan Wang*,§,⊥ §

Key Laboratory of Magnetic Resonance in Biological Systems, State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, Wuhan Centre for Magnetic Resonance, Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences, Wuhan 430071, P. R. China † State Key Laboratory of Genetic Engineering, Biospectroscopy and Metabolomics, School of Life Sciences, Fudan University, Shanghai 200433, P. R. China ‡ Computational and Systems Medicine, Department of Surgery and Cancer, Faculty of Medicine, Imperial College London, London SW7 2AZ, United Kingdom ⊥ Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Hangzhou 310058, P. R. China ∥ University of Chinese Academy of Sciences, Beijing 100049, P. R. China S Supporting Information *

ABSTRACT: Salmonella typhimurium is a bacterial pathogen that poses a great threat to humans and animals. In order to discover hosts’ responses to S. typhimurium infection, we collected and analyzed biofluids and organ tissues from mice which had ingested S. typhimurium. We employed 1H NMR spectroscopy coupled with multivariate data analysis and immunological techniques. The results indicate that infection leads to a severe impact on mice spleen and ileum, which are characterized by splenomegaly and edematous villi, respectively. We found that increased levels of itaconic acid were correlated with the presence of splenomegaly during infection and may play an important role in Salmonella-containing vacuole acidification. In addition, metabonomic analyses of urine displayed the development of salmonellosis in mice, which is characterized by dynamic changes in energy metabolism. Furthermore, we found that the presence of S. typhimurium activated an anti-oxidative response in infected mice. We also observed changes in the gut microbial co-metabolites (hippurate, TMAO, TMA, methylamine). This investigation sheds much needed light on the host− pathogen interactions of S. typhimurium, providing further information to deepen our understanding of the long co-evolution process between hosts and infective bacteria. KEYWORDS: infectious disease, itaconic acid, metabonomics, NMR, Salmonella typhimurium

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INTRODUCTION Salmonella typhimurium, a Gram-negative facultative bacterial pathogen capable of invading and replicating in host cells, is responsible for a variety of diseases, including human gastroenteritis as well as a systemic disease that resembles human typhoid fever in mouse models.1−3 The global prevalence of S. typhimurium-related diseases is high due to its excellent viability under various nutrition conditions, as well as an extensive host range, including both mammals and plants.4,5 S. typhimurium-induced acute gastroenteritis is lifethreatening to children and the elderly, as well as to immunocompromised individuals, such as people infected with HIV.6 There are an estimated 1.3 billion cases of salmonellosis per year and 3 million deaths globally.7 In the United States, 1.4 million people are thought to be infected with non-typhoidal Salmonella per year, resulting in nearly 15,000 hospitalizations and 400 deaths.8 Salmonella infection is also a significant cause of food-borne illnesses in the European Union, with 108,614 reported human cases in 2009.9 In addition, transmission of S. typhimurium among livestock in farms can also lead to enormous economic loss. A deeper insight into the influence © 2014 American Chemical Society

of S. typhimurium infection on host metabolism is therefore essential for controlling related diseases and reducing the socioeconomic cost. S. typhimurium targets the digestive tract and intestinal lumen of susceptible hosts through ingestion of contaminated food or water.10 In order to combat the resistance to S. typhimurium colonization from indigenous microbiotia, S. typhimurium compete with local microbes by exploiting host inflammation.11 S. typhimurium invades both phagocytic and non-phagocytic cells and becomes enveloped by the cell membrane to form a vacuolar compartment called the Salmonella-containing vacuole (SCV).12 In host cells, S. typhimurium directs SCVs’ maturation to protect itself from the host defense system via activation of NADPH oxidase, induction of nitric oxide, and fusion with the lysosome.13 To employ a host cell as a niche for multiplication, S. typhimurium utilizes bacterial effector proteins derived from a pair of type III secretion systems (TTSSs) called Salmonella pathogenicity islands (SPI-1 and SPI-2). SPI-1 encodes the Received: July 24, 2014 Published: September 11, 2014 4436

dx.doi.org/10.1021/pr500770x | J. Proteome Res. 2014, 13, 4436−4445

Journal of Proteome Research

Article

mice (n = 12) were inoculated intragastrically with 0.3 mL of Salmonella cell suspension (2.4 × 104 CFU per mouse), the other group (n = 12) employed as control animals were administrated intragastrically with 0.3 mL sterile saline per mouse.22 Urine and feces samples were collected the day before S. typhimurium inoculation, 1 day after infection, and then every other day until 9 days post infection. Sample collection was carried out between 8:30 a.m. and 12:00 p.m. to avoid potential metabolic variations owning to diurnal rhythm. Nine days after inoculation, all mice were sacrificed. Blood samples were collected in Eppendorf tubes and centrifuged at 1800g for 10 min. The supernatants (serum 250 μL) were transferred into 0.5 mL Eppendorf tubes. Spleens, liver, and intestinal tissues were also collected. All the samples were immersed in liquid nitrogen immediately after collection and stored at −80 °C until preparation for NMR acquisition.

TTSS and is indispensable for intruding into non-phagocytic cells, as well as establishing gastroenteritis in mouse models. Several hours after invasion, SPI-1 is down-regulated, and SPI-2 is up-regulated. SPI-2 is responsible for the maturation of SCVs and is necessary for replication in the intracellular compartments.3,14 Following colonization, bacteria adhere to and invade epithelial cells, survive in blood, proliferate in macrophages, and ultimately access systemic sites through the lymphatic and blood circulation systems.15 The bacterial infection processes, pathological effects, and impact on host hormone metabolism16 have all been extensively studied, but there has been less focus on the changing biochemical composition of host excreta and tissues during salmonellosis. Conventional biochemical techniques for monitoring changes in metabolism are time-consuming and fragmentary. However, recent techniques have been developed to detect and quantify small metabolites in complex biological samples. This has given rise to metabonomics,17 which offers an alternative method to discover biomarkers of diseases in biological samples.18 Metabonomics is capable of detecting a wide range of small metabolites simultaneously using 1H nuclear magnetic resonance (NMR) spectroscopy and chromatography coupled to mass spectrometry, providing a metabolic “fingerprint” for the collected samples. Multivariate statistical analysis can be utilized in spectral data analysis to aid visualization and characterization of changes which are associated with biological disruption caused by internal or external factors, including but not limited to pathogen infections19 and drug toxicity.20 Here, we have applied NMR-based metabonomics to investigate the impact of S. typhimurium infection on BALB/c mice, in order to further our understanding of the progress of salmonellosis as well as the metabolic changes in targeted tissues. Our research has provided important biochemical information, forming a solid basis for developing new methods for diseases diagnosis and treatment.

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Detection of IL-4 and IFN-γ in Serum and Histological Investigation

Serum samples were thawed at room temperature. The serum concentrations of IL-4 and IFN-γ were quantified by mouse IL4 high sensitive ELISA kits (BMS613HS, eBioscience) and mouse IFN-γ immunoassay kits (MIF00, R&D Systems Inc.), respectively. All measurements were strictly carried out following the procedures provided by manufacturers. For histological examination of tissue sections, tissue samples were fixed in 10% buffered neutral formalin, embedded in paraffin, and serially sectioned. Tissue sections were stained with hematoxylin and eosin. Images were captured using a Nikon E100 upright microscope. Bacterial Detection and Inhibition of Itaconic Acid to S. typhimurium

Bismuth sulfite (BS) Agar plates (BS, Qingdao Hope BiolTechnology Co. Ltd.) were used to detect S. typhimurium in feces and splenic tissues to ensure establishment of infection. The feces were kept in sterile saline solution containing 15% glycerol. Splenic tissues were homogenized in saline solution using a tissuelyser at 20 Hz for 90 s. Fecal or splenic tissue homogenates were placed on BS agar plates and cultured at 37 °C for 30 h to allow the formation of S. typhimurium colonies. Morphology of S. typhimurium colonies derived from agar plates with pure culture and the infected feces and spleen was monitored. In order to evaluate the effects of pH and various concentrations of itaconic acid on the growth of S. typhimurium, S. typhimurium was incubated in LB broth containing a range of concentrations (0, 0.73, 7.3, and 9.22 mM) of itaconic acid, with and without adjusting pH values to 7.2 or 4.5. Three biological repeats were performed for each sample, and OD600 values were taken every 2 h for 16 h.

MATERIALS AND METHODS

Bacterial Strain, in Vivo Experiment, and Sample Collection

S. typhimurium strain 14028 (ATCC) was obtained from Prof. Guo (Wuhan University) and cultured aerobically at 37 °C in Luria−Bertani (LB) broth (Sigma-Aldrich) overnight. Cultured bacteria were recovered by centrifuging at 10000g for 30 s, and then washed twice with sterile physiological saline21 and resuspended in saline (109 CFU/mL). The cell suspensions used for animal infection were diluted with sterile saline to desired concentration determined by plate counts on solid LB culture medium. The in vivo experiment was carried out at the specific pathogen free (SPF) animal experimental facility in Wuhan Institute of Physics and Mathematics in strict accordance with the National Guidelines for Experimental Animal Welfare (People’s Republic of China, 2006) and was approved by the Animal Welfare Committee of Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences (Permission No. S051-10-04-OU). A total of 24 SPF female BALB/c mice at the age of 6 weeks were purchased from Vital River Laboratories (Beijing, China). Mice were randomly and evenly divided into two groups and housed in ventilated plastic cages (12 per cage) under controlled conditions (temperature, 22 °C; humidity, 60%; light−dark cycle, 12 h−12 h) with free access to rodent food and water. After 3 weeks acclimatization, one group of

Sample Preparation and 1H NMR Spectroscopy

A total of 100 μL of urine was mixed with 400 μL of 10% D2O and 50 μL of phosphate buffer (1.5 M Na+/K+ buffer, pH = 7.4) containing 0.1% sodium 3-(trimethylsilyl) propionate2,2,3,3-d4 (TSP) as a chemical shift reference and 0.1% NaN3 as an antiseptic agent.23 After vortexing and centrifugation at 16000g, 4 °C for 10 min, 500 μL of supernatant was transferred into a 5 mm NMR tube. Serum samples (200 μL) was mixed with 400 μL of phosphate buffer saline solution (0.9% NaCl, 45 mM Na+/K+, 50% D2O). The mixture was centrifuged at 11000g, 4 °C for 10 4437



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Figure 1. Histological examination of intestinal, splenic, and liver tissues. Hematoxylin and eosin staining was performed on ileal, colonic, splenic, and liver sections from control (A,B,C,D) and S. typhimurium-infected (a,b,c,d) mice at day 9 post infection. Red arrows point the histological changes in ileum and spleen of infected mice. In panel a, enteritis is present in ileum characterized by edematous villi, which become shortened or broken; in panel c, the white pulp (Figure 1C,c, in red elliptical rings) of spleen enlarges in response to S. typhimurium-infection. In panels b and d, no significant changes were noted in the colon and liver of infected mice.

min, and then 550 μL of supernatant was transferred into a 5 mm NMR tube. Tissue samples were extracted by mixing approximately 50 mg of samples with 0.6 mL of a cold mixture of methanol and water (2:1, v:v) and homogenized by a tissuelyser for 90 s at 20 Hz, followed by three supersonic sessions of 60 s, with an interval of 60 s between sessions. Supernatants were collected following centrifugation of homogenate samples at 11000g and 4 °C for 10 min. This tissue extraction process was repeated three times, and the supernatants from the same sample were combined. Methanol was removed using a speed vacuum system, and samples were dried using a freeze-dryer overnight. The resulting animal tissue powder was reconstituted in 0.6 mL phosphate buffer (0.1 M Na+/K+ 50% D2O buffer, pH = 7.4, 0.05% TSP, and 0.1% NaN3), and 550 μL of supernatant was transferred into a 5 mm NMR tube following centrifugation at 16000g and 4 °C for 10 min. 1 H NMR spectra of urine and tissue extracts were acquired at 298 K using a Bruker AVIII 600 MHz NMR spectrometer (Bruker Biospin, Germany) with a cryogenic probe, operating at a proton frequency of 600.13 MHz. Standard onedimensional NMR experiments with water suppression (recycle delay−90°−t1−90°−tm−90°−acquisition) was employed. 1H NMR spectra of serum were recorded at 298 K on a Bruker AVII 500 MHz NMR spectrometer with a broad band inverse detection probe, operating at 500.13 MHz proton frequency. Spin−spin relaxation edited 1H NMR experiments using a Carr−Purcell−Meiboom−Gill (CPMG) pulse sequence with water saturation was performed for serum. A total spin−spin relaxation delay of 70 ms was used. The 90° pulse length was adjusted to about 10 μs. Sixty-four scans were accumulated into 32k data points, with a spectral width of 20 ppm for all the spectra. Two-dimensional NMR spectra (1H−1H COSY and TOCSY, 1H−13C HSQC and HMBC) were acquired on the selected samples to assist the spectral assignment.

extracts were referenced to TSP peak (δ, 0 ppm), whereas serum spectra were calibrated to the low field peak (δ, 5.233 ppm) of the doublets belonging to α-glucose. The spectra ranging from δ 0 to δ 10 ppm were integrated with an equal width of 0.002 ppm for urine and tissue extracts and 0.004 ppm for serum, using the AMIX package (V3.8, Bruker Biospin, Germany). Spectra containing water regions (4.5−6.2 ppm) were removed, and normalization to total intensity of the spectrum was carried out for urinary and serum spectra, while normalization to the wet weight was performed for the spectra of liver and ileal extracts. For the spectra of spleen extracts, normalization to dry weight was performed; the purpose of this procedure is to remove effects from different water content since some spleens show severe signs of splenomegaly. Principal component analysis (PCA) and orthogonal-projection to latent structures discriminant analysis (O-PLS-DA) of the spectral data were conducted using SIMCA-P+ software package (V12, Umetrics, Sweden).24,25 The quality of the models was represented by model parameters, such as Q2, indicating predictability of the model, and R2, denoting the interpretability of the model. The O-PLS-DA models were validated using a 7-fold cross-validation method, and ANOVA of the cross-validated residuals (CV-ANOVA) test.26 For visualization of the results, the loadings were back-transformed27 and plotted with color-coded coefficients for each variable using an in-house developed MATLAB script.

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RESULTS

Establishment of S. typhimurium Infection in Mice

Bismuth sulfite (BS) inhibits the growth of Gram-positive bacteria and the coliform bacterial groups, but it has no effect on Salmonella. S. typhimurium produces special colony units with a black metal luster appearance, due to the sediment of material derived from ferrous sulfate in the medium. Therefore, BS agar plates are used to detect existence of S. typhimurium in feces and spleen homogenates collected from both infected and control animals. Clear colonies of S. typhimurium were observed in BS agar plates, cultured with fecal extracts and spleen tissue samples from infected animals, confirming a successful establishment of S. typhimurium infection (Supporting In-

NMR Spectral Data Processing and Multivariate Pattern Recognition Analysis 1

H NMR spectra were corrected for phase and baseline deformation manually using the Topspin software package (V2.0, Bruker Biospin, Germany). Spectra of urine and tissue 4438



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Thirty metabolites were also identified in the serum, spleen, ileum, and liver samples (Figure S2, Table S1). The serum concentrations of D-3-hydroxybutyrate (3-HB), lactate, and glucose were lower in the S. typhimurium-infected mice compared with the controls. Decreased levels of creatine, leucine, and uridine could also be easily observed in the ileal extracts of the infected mice. Levels of 3-HB, lactate, and formate were elevated, whereas hypoxanthine levels decreased in the spleen of the infected mice with splenomegaly; this observation was coupled with the appearance of itaconic acid. In the liver, levels of some metabolites, such as glucose, glycogen, and taurine also altered due to the infection.

formation, Figure S1). No infection was observed in the uninfected control animals. Disease Progress and Histopathology

Mice began to show clinical symptoms at 3 days post S. typhimurium infection, such as reduced physical activity, reduced food intake, and overt ruffled fur. As the infection progressed, more serious symptoms, including a hunched and trembling appearance, were observed. By day 7 post infection, clinical symptoms disappeared in some mice, but persisted in others until the end of the experiment. Despite the controlled genetic background, environmental conditions, and infection dose, mice showed different degrees of physiological responses to the infection, indicating individual variations in the disease progress of salmonellosis. S. typhimurium infection induced histological changes in ileal and splenic tissues (Figure 1). Diffuse enteritis is present in ileum characterized by edematous villi, which either become shortened or broken (Figure 1a). The spleen is the largest peripheral lymphoid organ, which can purify blood, as well as function as an immune organ. The white pulp (mainly immune cells) of the spleen enlarges in response to antigens; this may be seen in Figure 1c. Hepatosplenomegaly is a symptom of severe S. typhimurium infection; however hepatomegaly did not appear in our in vivo experiment while splenomegaly did occur in about half of the infected mice. No significant changes were noted in the colon and liver of infected mice (Figure 1b,d). Nine days after the infection, serum IFN-γ and IL-4 levels of the infected mice were found to be significantly higher than that of the uninfected control animals (Figure 2A,B). The ratio of IFN-γ/IL-4 in serum was also higher in the infected mice compared with their non-infected counterparts (Figure 2C).

Sequential Metabolic Changes in Urine Associated with S. typhimurium Infection

In order to identify any important metabolite differentiating S. typhimurium-infected mice from the uninfected control animals, an O-PLS-DA method was employed in the analyses of the 1H NMR spectral data sets of urine samples obtained at specific time points (pre-infection and 1, 3, 5, 7, and 9 days post infection). Based on the values of R2X (goodness of fit), Q2 (robustness of model), and p values (CV-ANOVA), O-PLS-DA models obtained from the urinary profiles at days 1, 3, 5, and 9 post infection were valid (Supporting Information, Table S2). Color-coded coefficient plots of urine samples showed metabolic changes that were associated with a S. typhimurium infection (Figure 3). In these plots, the upward or downward

Figure 2. Serum concentrations of IFN-γ (A) and IL-4 (B), and the ratio of IFN-γ/IL-4 (C). con, uninfected control mice; stm, S. typhimurium-infected mice. The statistically significant differences were calculated using independent samples t test in SPSS 13.0. *p < 0.01. Figure 3. Scores plots (A,B,C,D) and corresponding loading plots (a,b,c,d) derived from O-PLS-DA of 1H NMR urinary spectra obtained from uninfected control (black) and S. typhimurium-infected mice (red) at different post infection time points (day 1: A,a; day 3: B,b; day 5: C,c; day 9: D,d). Signals pointing upward represent increased levels of metabolites in the infected groups compared with controls and vice versa; peaks with hot colors (e.g., red) exhibit higher correlations with the classification than that with cold colors (e.g., blue). For the key to metabolite numbering, refer to Table 1.

1 H NMR Spectra of Mouse Urine, Serum, Spleen, Liver, and Intestine Tissues

1

H NMR spectra of urine, serum, ileum, spleen, and liver tissues contain rich metabolic information (Supporting Information, Figure S2). A total of 30 metabolites (Supporting Information, Table S1) were detected in urinary samples, and the spectra were dominated by a number of organic acids, amines, and gutmicrobial co-metabolites. Visual inspection of the urinary spectra revealed obvious differences in metabolic compositions between the control and S. typhimurium-infected mice. For example, there were depleted levels of 2-keto-3-methy-valerate, 2-keto-isocaproate, 2-keto-isovalerate, TMAO, TMA, 2-ketoglutarate, trigonelline, and hippurate. Increased levels of urocanate and indoxyl sulfate were also observed in S. typhimurium-infected mice.

signal peaks represent respective elevated or depleted levels of metabolites in the infected animals compared with controls. Peaks colored in red exhibit a higher correlation with the classification than that in blue. Different sets of metabolites were found to be responsible for the differentiation at each time point (Table 1). Nine metabolites, including elevated levels of urocanate and decreased levels of keto-acids, 3-UP, TCA cycle 4439



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Table 1. Correlation Coefficientsa of Urinary Metabolites Indicate Significant Contribution to the Discrimination between Uninfected Control and S. typhimurium-Infected Mice at Four Post Infection Time Points

a b

metaboliteb

δ (ppm)

day 1

day 3

day 5

2-keto-3-methy-valerate (1) 2-keto-isocaproate (2) 2-keto-isovalerate (3) butyrate (4) 3-hydroxyisovalerate (5) TMAO (6) TMA (7) methylamine (8) citrate (10) 2-keto-glutarate (11) pyruvate (12) cis-aconate (13) creatine (14) urocanate (16) 3-ureidopropionate (17) hippurate (18) NMN (23) trigonelline (24) N-acetylglutamic acid (25) guanidinoacetate (27) acetamide (28)

0.90, 1.10 0.94, 2.62 1.13, 3.03 0.92, 1.62, 2.28 1.26, 2.35 3.27 2.88 2.61 2.55, 2.68 3.01, 2.45 2.38 3.12 3.04, 3.93 6.40, 7.30, 7.33 2.38, 3.31 3.97, 7.55, 7.64, 7.84 4.481, 8.90, 8.97, 9.29 4.44, 8.08, 8.81, 9.13 2.13, 2.79, 4.03 3.8 2.01

−0.56 −0.67 −0.57

−0.67 −0.70

−0.64

+0.92 −0.54 −0.79

day 9 +0.90 +0.78 +0.69 +0.84 +0.76 −0.84 −0.70 −0.65

−0.74 −0.53 −0.68 −0.55 −0.87 +0.87 −0.53

+0.72 −0.74 −0.74 +0.60 −0.58 −0.77 −0.81

−0.60

+0.76

Negative values indicate the decrease in the infected mice when comparing to the control mice, while positive values indicate the increase. Abbreviations used: TMAO, trimethylamine-N-oxide; TMA, trimethylamine; NMN, N-methylnicotinamide.

intermediates, and cis-aconate, were found in urine of the infected mice at day 1 post infection of S. typhimurium. At day 3 post infection, levels of keto-acids, urocanate, and gut microbiota-related metabolites such as TMAO, were altered. At day 5 and day 9 post infection, altered levels in TCA cycle intermediates disappeared and gut microbiota-related metabolites changed, especially at the last time point. Levels of trigonelline, TMAO, and methylamine (MA) were also consistently changed at 5 and 9 days post infection. S. typhimurium Infection-Induced Metabolic Changes in Serum, Spleen, Liver, and Intestine Tissues

OPLS-DA was used to extract the metabolic changes of serum and tissue (spleen, liver, jejunum, ileum, cecum, and colon) associated with the infection. Based on the cross validation of constructed models, metabolic profiles of the serum, liver, spleen, and ileum were markedly affected by the infection (Table S2). An O-PLS-DA comparison between spleen profiles of control and infected animals without splenomegaly suggested that there was no difference, hence only spleen profiles of six animals was employed for comparison. Color-coded coefficient plots of serum, liver, spleen, and ileum showed metabolic changes that were associated with S. typhimurium infection (Figure 4). The levels of citrate, malonate, and 3-HB decreased, accompanied by the elevated levels of lipids (Figure 4a, Table 2) in the serum of infected mice. The hepatic levels of taurine, glucose, and glycogen were increased in the infected mice (Figure 4b, Table 2). In spleen tissues, levels of two nucleotides, cytidine and hypoxanthine, were shown to be decreased, while levels of succinate, 3-HB, itaconic acid, uracil, AMP, lipids, and ADP were found to be increased (Figure 4c, Table 2). A reduction in the levels of alanine, phenylalanine, malonate, inosine, cytidine, and uridine was found in the ileum of infected mice, together with elevated levels of phosphocholine, glycerophosphocholine, and lipids (Figure 4d, Table 2).

Figure 4. Scores plots (A,B,C,D) and corresponding color-coded coefficient plots (a,b,c,d) derived from O-PLS-DA models of 1H NMR spectra obtained from serum (A,a), liver (B,b), spleen (C,c), and ileum (D,d) obtained from uninfected control mice (black) and S. typhimurium-infected mice (red). Signals pointing upward represent increased levels of metabolites in the infected groups compared with controls and vice versa. For the key to metabolite numbering, refer to Table 2.

Inhibition of Itaconic Acid to S. typhimurium Growth

Itaconic acid is an uncommon compound in mammalian metabolism and has not been previously reported for S. typhimurium infection. In order to evaluate the biological function of the elevations of itaconic acid in the spleen, we incubated S. typhimurium with various concentrations of itaconic acid. It appeared that high concentrations of itaconic acid suppressed the growth of S. typhimurium (Figure 5A). 4440



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Table 2. Correlation Coefficientsa of Metabolites in Serum, Liver, Spleen, Ileum That Changed Markedly When Comparing S. typhimurium-Infected Mice with Corresponding Control Mice metaboliteb citrate (10) ADP (20) itaconic acid (22) succinate (29) taurine (30) malonate (31) phosphocholine (33) GPC (34) 3-hydroxybutyrate (37) lipids (38) alanine (41) β-glucose (43) α-glucose (44) phenylalanine (46) uridine (48) glycogen (49) hypoxanthine (51) AMP (52) uracil (55) inosine (56) cytidine (60)

δ (ppm) 2.56, 8.61, 3.16, 2.41 3.27, 3.12 3.22, 3.23, 1.19, 0.85, 1.48, 4.65 5.23 7.18, 5.91, 5.43 8.20, 8.58, 5.81, 6.11, 5.92,

2,69 8.27, 6.15 5.38, 5.86

serum

liver

spleen +0.76 +0.92 +0.82

3.43

+0.56 −0.69

3.62, 4.24 4.32 4.15, 2.39 1.27, 5.30 3.78

ileum

−0.70

−0.58 +0.62 +0.59

−0.66 +0.77

+0.74 +0.81

+0.60 −0.58

+0.56 +0.56 −0.58 −0.60

7.32, 7.41 5.93, 7.89 +0.59 −0.78 +0.81 +0.76

8.22 8.23, 6.145 7.55 8.25, 8.35 7.86

−0.85

−0.67 −0.63

a

Negative values indicate the decrease in the infected mice when comparing to the control mice, while positive values indicate the increase. Abbreviations used: GPC, glycerophosphocholine; PE, phosphoethanolamine; GSH, glutathione; ADP, adenosine diphosphate; AMP, adenosine monophosphate.

b

added (Figure 5B). We further examined dependence of S. typhimurium growth on pH without the presence of itaconic acid (Figure 5C); clearly S. typhimurium exhibits slow growth in acidic conditions, particularly in the lag phase. In order to eliminate the effects of pH on growth, we adjusted pH of medium to 7.2 after addition of various concentrations of itaconic acid. The result showed that if pH-related impeded growth is eliminated, the presence of itaconic acid had little effects on the growth curves of S. typhimurium (Figure 5D).

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DISCUSSION In this study, we monitored the development of S. typhimurium infection in female BALB/c mice and investigated the infectionassociated metabolic changes in urine, serum, and tissues including spleen, liver, and intestine using a metabonomics approach. These results show the intruding and damaging effect of S. typhimurium infection on the host, as well as the metabolic responses of hosts to S. typhimurium infection. In this work, animals were infected with an S. typhimurium cell suspension through intragastric administration, which resulted in S. typhimurium replication in the intestinal lumen (Figure S1) and damage to the intestinal structure, which is characterized by edematous villi (Figure 1a). Concurrently, we observed increased levels of membrane metabolites, including phosphocholine and GPC, in the small intestine, which is in agreement with the damage to the intestinal structure. In the mean time, we also observed reduced levels of inosine and cytidine. Inosine is known to have anti-inflammatory effects.28 Inflammation can also stimulate expression of cytidine deaminase that converts cytidine to uridine.29 Therefore, the reduction in the levels of inosine and cytidine found in ileal tissue is highly likely to be associated with inflammation induced by S. typhimurium infection. However, it is noted that there is no significant damage to the colons of infected mice.

Figure 5. Effect of itaconic acid and pH values on the growth of S. typhimurium. (A) Growth curves of S. typhimurium in LB culture medium with different concentration of itaconic acid. (B) Initial pH values of LB culture medium with different concentration of itaconic acid. (C) Growth curves of S. typhimurium in LB culture medium with two different pH values without addition of itaconic acid (7.2 and 4.5). (D) growth curves of S. typhimurium in LB culture medium with different concentration of itaconic acid adjusted to the same pH value (7.2). The statistically significant differences were calculated using independent samples t test with SPSS 13.0. *p < 0.01.

Since itaconic acid is a strong organic acid, it is anticipated that the pH values of the growth medium will be affected. We found that pH value reduced to 3 when 9.22 mM of itaconic acid was 4441



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Figure 6. Schematic graph showing the function of itaconic acid. Macrophage produced itaconic acid, in order to inhibit S. typhimurium replication, however, the pH sensor of bacteria, such as SPI-2 T3SS, induced gene expression which is essential for intracellular replication. SCV, Salmonellacontaining vacuole; STM, S. typhimurium; TLR, toll-like receptor; LPS, lipopolysaccharide.

acid observed. In Mycobacterium tuberculosis-infected mice, itaconic acid can be detected in bacteria-targeted lungs with macrophage aggregation proving it to be a mammalian metabolite produced during macrophage activation38 and not the metabolite of bacteria or other normal mammalian cells.37 We additionally confirmed that the itaconic acid in the spleen is not originating from S. typhimurium cells (Supporting Information, Figure S3), where itaconic acid cannot be detected by LC-MS. Therefore, accumulation of itaconic acid in the swollen spleen is due to the lysed phagocytes; this view is supported by the histological examination of the spleen, where more immune cells, including macrophages, in the enlarged white pulp were found in the infected mice. Our in vitro experiments demonstrated that the ability of itaconic acid to inhibit the growth of S. typhimurium was mostly due to its nonspecific acidity. This is because isocitrate lyase, which can be inhibited by itaconic acid, is essential for surviving of S. typhimurium only when limited carbon source is available, such as when it relies on fatty acids or acetate as the sole carbon source entering the central carbon metabolism as acetylCoA.39−41 In the case of our in vitro experiment, the LB medium contains abundant glucose, hence itaconic acid showed limited inhibition on the growth of S. typhimurium. In the case of mouse infection with S. typhimurium, the function of itaconic acid is more complex. Although itaconic acid is a strong organic acid, S. typhimurium are able to survive in acidic conditions, which is highly dependent on the ability of a pH sensor,42 such as SPI-2 T3SS, which is activated in acidic conditions (Figure 6).43,44 The activation of SPI-2 T3SS is essential for S.

The indigenous gut microbiota that resides mainly in colon are able to defend against overt foreign pathogens including Salmonella. Existing literature on S. typhimurium infection demonstrates that mice pre-treated with antibiotics would have a more severe case of enterocolitis, compared to mild enteritis without any antibiotics treatment,30,31 highlighting the protective effect of the gut microbiota community against invasion of foreign pathogens. Therefore, different responses of intestinal segments to S. typhimurium infection in our in vivo experiment could be due to the variable host microbiota32,33 in fighting against foreign pathogens.10 After breaking through the gut barrier, bacterial cells migrate to other organs via lymph and blood circulation. In our in vivo experiment, the spleen which is the largest immune organ, was clearly intruded and damaged (Figure S1), which was manifested as splenomegaly. Most importantly, we found marked elevation in the levels of itaconic acid in spleen with splenomegaly. This close association of itaconic acid and splenomegaly is reported for the first time in S. typhimurium infection-induced inflammation. Immunoresponsive gene 1 is identified as the gene that codes for the enzyme that produces itaconic acid, which inhibits isocitrate lyase,34 an essential enzyme for the growth of bacteria such as Salmonella.35 Previous studies have reported that itaconic acid is present in fungi36 and can also be produced by mammalian macrophages, especially alongside of the stimulation of IFN-γ and bacterial antigens (e.g., lipopolysaccharides).37 In the current study, we found the levels of IFN-γ are markedly elevated in the infected mice, which is consistent with the elevated levels of itaconic 4442



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typhimurium replication in SCVs.45,46 In addition, S. typhimurium is able to decompose itaconic acid through itaconate coenzyme A (CoA) transferase, itaconyl-CoA hydratase, and (S)-citramalyl-CoA lyase.47,48 All these factors contribute to the incomplete inhibition of itaconic acid on the growth of S. typhimurium. Noticeable accumulations of glucose and glycogen are observed in the liver of infected mice due to presence of high levels of itaconic acid (Figure S3) since itaconic acid can suppress glycolysis by inhibiting the synthesis of fructose 2,6bisphosphate, an important enzyme in glycolysis.49 It is therefore inevitable for the host to shift energy metabolism, which is demonstrated in progressive changes in urinary metabolic profiles. Reduction in the levels of a range of urinary keto-acids was evident at the early stages of the infection, however, these keto-acids increased at 9 days post infection. 2keto-3-methy-valerate, 2-keto-isocaproate, and 2-keto-isovalerate are degradation products of isoleucine, leucine, and valine respectively; they can enter into TCA circle and be used as energy materials. In an attempt to fight the infection during the early stages, mice tend to consume more energy. In the case where there are not enough primary energy materials (such as glucose, glycogen, and lipids) to be utilized for supplying the TCA circle for energy consumption, amino acids were degraded to keto acids for the production of energy, in order to respond to the S. typhimurium infection. Consistent with this notion, we observed marked reduction in the levels of TCA cycle intemediates (day 1, citrate, 2-keto-glutarate, pyruvate, and cis-aconate). As the infection progressed, a few urinary metabolites were altered, which is consitent with visual inspection of apparent recovery of mice behavior. This suggested that the initial combat with the infection was effecitve. However, those bacteria that are able to sense low pH condition and subsequent induction of STM 1485 gene45 in host cells survived and began to replicate, resulting in second phase systemic infection. Concurrent with this, we observed metabolic changes in multiple organs (Table 2), for example, changes in the levels membrane metabolite (GPC and phosphocholine),50 which are consistent with shortened and broken ileal villi (Figure 1a). We also observed a simultaneous increase in the levels of urianry keto-acids (Table 2) at the later stage of the infection. Exhausting energy resources may contribute to this observation. Drastic up-regulation of IFN-γ was associated with S. typhimurium infected mice. Research shows that IFN-γ induced phagocytes can produce oxidizing substances.51,52 It is therefore anticipated that there is an association between prevalence of S. typhimurium infection with anti-oxidative activity of the mice. Indeed, we observed high levels of taurine in the liver of S. typhimurium infected mice. Taurine is a multifunctional metabolite and has been shown to have antioxidant53 and anti-inflammation54 effects, as well as balancing osmosis.55,56 The accumulation of taurine is related to its functions of antioxidant. Inflammation arose from S. typhimurium infection induced production of oxidizing substances, and taurine was used to eliminate these superfluous substances. In addition, trigonelline has been reported to exhibit potential antimicrobial effects against S. enterica.57 In the urine samples of mice, we observed that a S. typhimurium infection resulted in a shift of nicotinic acid (niacin) metabolism toward N-methylnicotinamide,58 reflected by increased levels of N-methylnicotinamide and decreased levels of trigonelline; the former is a methylation product of nicotinamide,59 whereas the latter is a methylation

metabolite of nicotinic acid.60 This metabolic shift caused by S. typhimurium infection may facilitate its viability inside the host. As discussed above, indigenous gut microbes act as protective agents against S. typhimurium inhabitation, preventing the colon from damage, which in turn will undoubtedly cause disturbances in gut microbiota. Since metabolic profiles of urine contain rich information on the co-metabolism between microbes and host,61,62 alterations in gut-microbial related metabolites are expected in the urine of infected mice. Here, we observed a reduction in the levels of urinary hippurate and a range of amines including methylamine, TMA, and TMAO. Hippurate is a glycine-conjugated benzoic acid that in turn is produced from gut microbiota degradation of phenols from ingested food,63,64 whereas amine products are metabolites of choline by gut microbiota.65−67 These observations suggested that the disturbance of gut microbiota is associated with S. typhimurium infection. In conclusion, the current study has characterized the impact of S. typhimurium infection on the host and disclosed the interactions that occur between this intracellular pathogen and its host (Figure 7). Our investigation showed the associations

Figure 7. Impact of S. typhimurium infection on hosts and their systemic responses.

between development of salmonellosis and organ-specific metabolic variations, with S. typhimurium infection in a mouse model. We observed changes in energy metabolism, which is in accordance with the development of the disease. Typhimurium infection caused splenomegaly that is associated with elevations of itaconic acid. In addition, we found that S. typhimurium infection caused oxidative stress to the host and disturbed the indigenous gut microbial community. These findings ultimately aid further understanding of the disease progress of salmonellosis and may potentially contribute to the control of S. typhimurium-associated diseases.

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ASSOCIATED CONTENT

S Supporting Information *

Experimental details; Figure S1, photographs of colonies of S. typhimurium; Figure S2, 1H NMR spectra of urine, serum, ileum, spleen, and liver from a control mouse and a S. typhimurium-infected mouse; Figure S3, representative chromatographic traces for the detection of itaconic acid; Table S1, assignment of the metabolites in urine, serum, liver, spleen, and ileum; and Table S2, model summary of O-PLS-DA of 1H NMR spectra at different time points from uninfected control 4443



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mice and S. typhimurium-infected mice. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +8644-27-87197143. Fax: +86 44-27-87199291. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to Prof. Guo (Wuhan University) for her generosity in supplying the bacterial strains. We acknowledge financial support from the National Natural Science Foundation of China (21375144, 21221064) and the Ministry of Science and Technology of China (2012CB934004).

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