Sulfadiazine Sodium Ameliorates the Metabolomic Perturbation in Mice Infected with Toxoplasma gondii

Abstract

In this study, we analyzed the global metabolomic changes associated with Toxoplasma gondii infection in mice in the presence or absence of sulfadiazine sodium (SDZ) treatment. BALB/c mice were infected with T. gondii GT1 strain and treated orally with SDZ (250 μg/ml in water) for 12 consecutive days. Mice showed typical manifestations of illness at 20 days postinfection (dpi); by 30 dpi, 20% had survived and developed latent infection. We used ultraperformance liquid chromatography-mass spectrometry to profile the serum metabolomes in control (untreated and uninfected) mice, acutely infected mice, and SDZ-treated and infected mice. Infection induced significant perturbations in the metabolism of α-linolenic acid, purine, pyrimidine, arginine, tryptophan, valine, glycerophospholipids, and fatty acyls. However, treatment with SDZ seemed to alleviate the serum metabolic alterations caused by infection. The restoration of the serum metabolite levels in the treated mice was associated with better clinical outcomes. These data indicate that untargeted metabolomics can reveal biochemical pathways associated with restoration of the metabolic status of T. gondii-infected mice following SDZ treatment and could be used to monitor responses to SDZ treatment. This study provides a new systems approach to elucidate the metabolic and therapeutic effects of SDZ in the context of murine toxoplasmosis.

Keywords: Toxoplasma gondii; biomarkers; metabolomics; mice; serum metabolites; sulfadiazine sodium.

FIG 1

Survival rates for control, untreated, and SDZ-treated mice plotted over time following T. gondii infection. (A) Survival of T. gondii-infected mice increased as the treatment duration increased. (B) A dose of 250 μg/ml was the optimal treatment dosage.

FIG 2

Schematic diagram of the experimental design. Serum samples from control untreated mice and treated mice (T6, T20, and T30 groups) were subjected to LC-MS/MS analysis.

FIG 3

Discrimination between treated and control mice based on ESI+-mode-derived metabolic phenotypes of serum. (A to C) PLS-DA score plots for the T6 group versus the control group (A), the T20 group versus the control group (B), and the T30 group versus the control group (C). (D to F) Heatmaps of the differential metabolites for the T6 group versus the control group (D), the T20 group versus the control group (E), and the T30 group versus the control group (F).

FIG 4

Discrimination between the control group, the T6 group, and the T20 group versus acutely infected (acute) mice based on ESI+-mode-derived serum metabolic profiles. (A to C) PLS-DA score plots for control versus acutely infected mice (A), the T6 group versus acutely infected mice (B), and the T20 group versus acutely infected mice (C). (D to F) Heatmaps of the top differential metabolites for control versus acutely infected mice (D), the T6 group versus acutely infected mice (E), and the T20 group versus acutely infected mice (F).

FIG 5

Global view of the temporal metabolic expression profiles during SDZ treatment. (A) Statistics for differentially expressed metabolites among samples. FC, fold change; Con, control. (B and C) Venn diagram analysis of differentially abundant metabolites between SDZ-treated (T20) and untreated acutely infected mice versus control (PBS-treated) mice. The numbers in the overlapping part of the circles represent common differentially abundant metabolites between the mouse groups in the ESI+ mode (B) and ESI– mode (C). (D) Top 10 enriched metabolite classes common for the acute group versus the control group and the T20 group versus the control group in ESI+ mode. (E) Top 10 enriched metabolite classes common for the acute group versus the control group and the T20 group versus the control group in ESI− mode. The x axis represents the number of metabolites for the chemical classes mentioned on the y axis.

FIG 6

Summary statistics for the annotated differential metabolites identified among different groups in ESI+ mode. (A) Ten largest categories for mice in the acute infection group versus the control group. (B) Ten largest categories for the T6 group versus the control group. (C) Eight largest categories for the T30 group versus the control group. The numbers in the Venn diagrams represent the numbers of the annotated differential metabolites.

FIG 7

Amino acid fluctuations during SDZ treatment. The x axis represents the mouse experimental groups and the y axis represents the relative abundance of the corresponding amino acids. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 8

Perturbed metabolic pathways in T. gondii infection and SDZ treatment. (A to C) Pathway impacts resulting from the differential metabolites determined using MetaboAnalyst 3.0. Small P values and high pathway impact factors indicate that the pathway is greatly influenced. (A) Acute group versus the control group. (B) T20 group versus the control group. (C) T30 group versus the control group. (D to F) Distribution of differential metabolites involved in amino acid metabolism. (D) Acute group versus the control group. (E) T20 group versus the control group. (F) T30 group versus the control group. The numbers in the Venn diagrams represent the numbers of the differential metabolites involved in amino acid metabolism.

FIG 9

Differential metabolites involved in purine metabolism (A) or pyrimidine metabolism (B). AICAR, aminoimidazolecarboxamide ribotide; AIR, 5-aminoimidazole ribonucleotide; CAIR, 4-carboxy aminoimidazole ribonucleotide; FAICR, formylaminoimidazole carboxamide ribotide; FGAM, formylglycinamidine ribonucleotide; FGAR, N-formylglycinamide ribonucleotide; GAR, glycinamide ribonucleotide; HIU, 5-hydroxyisourate; OMP, orotidine monophosphate; PRA, phosphoribosylamine; PRPP, phosphoribosyl pyrophosphate; SAICAR, succinylaminoimidazole carboxamide ribotide.