Profiling of the perturbed metabolomic state of mouse spleen during acute and chronic toxoplasmosis

Abstract

Background: Toxoplasma gondii, a common opportunistic protozoan, is a leading cause of illness and mortality among immunosuppressed individuals and during congenital infections. Current therapeutic strategies for toxoplasmosis are not fully effective at curtailing disease progression in these cases. Given the parasite ability to influence host immunity and metabolism, understanding of the metabolic alterations in the host's immune organs during T. gondii infection may enhance the understanding of the molecular mechanisms that define the pathophysiology of T. gondii infection.

Methods: We investigated the global metabolic changes in the spleen of BALB/c mice at early and late stage of infection with T. gondii using LC-MS/MS-based metabolomics. Multivariate data analysis methods, principal components analysis (PCA) and partial least squares discriminant analysis (PLS-DA), were used to identify metabolites that are influenced by T. gondii infection.

Results: Multivariate analyses clearly separated the metabolites of spleen of infected and control mice. A total of 132 differential metabolites were identified, 23 metabolites from acutely infected versus control mice and 109 metabolites from chronically infected versus control mice. Lipids, hormones, lactones, acids, peptides, antibiotics, alkaloids and natural toxins were the most influenced chemical groups. There were 12 shared differential metabolites between acutely infected versus control mice and chronically infected versus control mice, of which 4,4-Dimethyl-5alpha-cholesta-8,14,24-trien-3beta-ol was significantly upregulated and ubiquinone-8 was significantly downregulated. Major perturbed metabolic pathways included primary bile acid biosynthesis, steroid hormone biosynthesis, biotin metabolism, and steroid biosynthesis, with arachidonic acid metabolism being the most significantly impacted pathway. These metabolic changes suggest a multifactorial nature of the immunometabolic responses of mouse spleen to T. gondii infection.

Conclusions: This study demonstrated that T. gondii infection can cause significant metabolomic alterations in the spleen of infected mice. These findings provide new insights into the molecular mechanisms that underpin the pathogenesis of T. gondii infection.

Keywords: Mass spectrometry; Metabolome; Non-targeted metabolomics; Pathway enrichment analysis; Spleen; Toxoplasma gondii.

Fig. 1

Gross and histopathological characteristics of spleen of mice infected with Toxoplasma gondii. a The spleen of mice during acute infection (AI) was enlarged compared to normal spleen size of mice in the control group (Con). b Mouse spleen during chronic infection (CI) showing slight splenomegaly compared to control (Con) mice. c Histopathology of spleen from acutely infected mouse showing a reduction in the white pulp with an expansion of the red pulp compartment. d Spleen of a chronically infected mouse showing regression of white pulp and red pulp. e Spleen of a healthy mouse showing no histopathological abnormalities. Scale-bars: c-e, 12.5 μm

Fig. 2

a The total ion current (TIC) chromatograms of spleen samples in the positive ion mode (ESI+). b PCA scores plot of mice spleens, including acutely infected (AI), chronically infected (CI) and uninfected control (Con) compared to quality control (QC) samples in the positive ion mode (ESI+). Clear separation was detected among the different mice groups and in relation to QC samples

Fig. 3

a, b Two dimensional PLS-DA score plots of the a acutely infected mice and b chronically infected mice vs control mice in the positive ion mode (ESI+). Each dot represents one spleen sample, projected onto first (horizontal axis) and second (vertical axis) PLS-DA variables. Mice groups are shown in different colors. The ellipse determines the 95% confidence interval. c, d Heatmaps of the differential metabolites of acutely infected mice c and chronically infected mice d vs control mice in the positive ion mode (ESI+). Red and green indicate values above and below the mean, respectively; black indicates values close to the mean

Fig. 4

A two-way Venn diagram showing the common and unique metabolites between acutely and chronically infected mice groups vs control mice. In total, we found 23 metabolites in acute infection vs control (blue), of which 12 metabolites could also be identified in chronically infected mice. Also, we detected 109 metabolites in the chronically infected mice vs control (yellow), 12 of which were shared between the groups

Fig. 5

Pathway analysis of the differential metabolites during acute and chronic infection. Metabolite features with putative identification were analyzed using MetaboAnalyst for potential impact on metabolic pathways in the acute phase a and chronic phase b. Small P-value and large pathway impact factor indicate that the pathway is greatly influenced, such as arachidonic acid metabolism pathway, which was highly impacted during acute and chronic infection. c A schematic illustration of the arachidonic acid metabolism pathway during chronic infection. Red and black circles represent upregulated and unaltered metabolites, respectively. As shown, arachidonic acid (AA), leukotriene A4 (LTA4), 14,15-HETE, and 15-deoxy-Δ^12,14-PGJ₂ were upregulated