https://orcid.org/0000-0003-0768-6773
This is an uncorrected proof.
Figures
Abstract
Toxoplasma gondii (T. gondii) is an opportunistic protozoan parasite capable of infecting nearly all warm-blooded animals, including humans. Infection with T. gondii often triggers potent inflammatory responses that can lead to severe and potentially life-threatening tissue damage. Based on the mechanistic relationship between the gut microbiota and the host immune system, this study explores the metabolic regulatory network orchestrated by the gut microbiota during T. gondii infection. Using intraperitoneal infection models with both a wild-type ME49 strain and an attenuated ME49Δα-amy strain, we report for the first time a pivotal role for N-acetyl-D-glucosamine (GlcNAc) in modulating parasite-induced inflammation. Integrated analysis of 16S rRNA sequencing and metabolomic profiling revealed that GlcNAc, a gut microbiota-associated metabolite, was significantly enriched in mice infected with the ME49Δα-amy strain. Exogenous administration of GlcNAc to T. gondii-infected mice resulted in the marked downregulation of key pro-inflammatory cytokines, including TNF-α, IL-1β, IL-6, and IL-12, and a significant upregulation of the anti-inflammatory cytokines IL-10 and TGF-β. Moreover, GlcNAc treatment substantially reduced parasite burden and alleviated infection-associated weight loss. These findings not only elucidate the immunomodulatory function of microbiota-related metabolites in the context of zoonotic parasitic infections but also provide a novel theoretical foundation for the development of microbiota-targeted therapeutic strategies against toxoplasmosis. Collectively, our work offers important insights that may inform public health interventions aimed at controlling and preventing zoonotic parasitic diseases.
Author summary
Toxoplasma gondii is a widespread parasite that infects humans and animals, often through contaminated food or water. Infection can cause severe inflammation and tissue damage. In this study, we investigated how gut bacteria influence the body’s response to this parasite. We discovered that a molecule produced by gut microbiota, named N-acetyl-D-glucosamine (GlcNAc), plays a key role in reducing harmful inflammation during infection. When we gave GlcNAc to infected mice, it lowered inflammation, reduced the number of parasites, and prevented weight loss. These findings help explain how our body’s natural microbes can fight infectious diseases. More broadly, this work suggests that targeting gut metabolites like GlcNAc could lead to new treatments for infections caused by parasites and other inflammatory diseases, benefiting both human and animal health.
Citation: Yang Y, Zhou C, Yang C, Yang Z, Xu S, Ma X, et al. (2026) Gut microbiota-associated metabolite N-acetyl-D-glucosamine alleviates systemic inflammatory responses induced by acute Toxoplasma gondii infection. PLoS Negl Trop Dis 20(3): e0014108. https://doi.org/10.1371/journal.pntd.0014108
Editor: Mehmet Aykur, Tokat Gaziosmanpaşa University Faculty of Medicine: Tokat Gaziosmanpasa Universitesi Saglik Bilimleri Fakultesi, TÜRKIYE
Received: September 19, 2025; Accepted: March 2, 2026; Published: March 13, 2026
Copyright: © 2026 Yang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: 16S rRNA gene data has been presented in the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) database under accession number PRJNA1330808. Metabolomics raw data have been deposited in Mendeley Data (https://data.mendeley.com/datasets/drcj4xbmkk/1).
Funding: This work was supported by the National Natural Science Foundation of China (grant NO. 32503069 to JY), and the Yunnan Fundamental Research Projects (grant NO. 202401AT070212, 202301AU070109 and 202301BD070001-069 to JY). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
T. gondii is an obligate intracellular parasitic protozoan with a broad host range, capable of invading the nucleated cells of nearly all warm-blooded animals, thereby causing toxoplasmosis in both humans and animals [1]. Tachyzoites represent the rapidly proliferating and virulent stage of T. gondii. During acute infection, tachyzoites disseminate extensively via the bloodstream and lymphatic system to multiple host tissues and organs, leading to systemic infection [2]. In immunocompetent individuals, T. gondii infection is usually asymptomatic. After invasion, tachyzoites differentiate into bradyzoites that reside within tissue cysts, establishing a persistent chronic infection [3]. However, in immunocompromised or immunosuppressed hosts, reactivation of latent infection can result in severe and potentially fatal acute disease. The inflammatory immune response triggered by tachyzoites may lead to tissue-damaging pathologies, including enteritis, encephalitis, retinochoroiditis, and even mortality [4–6].
It has been reported that parasite invasion and proliferation induce potassium ion efflux, which activates the NLRP3 inflammasome and other inflammatory signaling pathways, thereby triggering a substantial release of IL-1β during early acute T. gondii infection [7]. Meanwhile, specific protein components secreted by the parasite can trigger classical inflammatory pathways in immune cells, such as intestinal epithelial cells and lamina propria macrophages, thereby promoting the production of various pro-inflammatory cytokines including IL-6, IL-12, and TNF-α [8]. Concurrently, the invasion of T. gondii can activate the NF-κB signaling pathway in intestinal epithelial cells and lamina propria macrophages, promoting the production of pro-inflammatory cytokines such as IL-6, IL-12, and TNF-α. This inflammatory milieu facilitates parasite penetration [9,10]. Elevated levels of these cytokines culminate in a cytokine storm within the host intestine, which induces apoptosis of intestinal epithelial cells and downregulates the expression of tight junction proteins, including ZO-1 and occludin. These alterations compromise the integrity of the intestinal mucosal barrier, increase intestinal permeability, and ultimately lead to lethal enteritis [11]. Despite the severity of these pathological consequences, there remains a lack of widely adopted vaccines for the prevention of T. gondii infection in humans globally, and the range of effective therapeutic agents remains limited [12]. Developing a therapeutic strategy that effectively targets acute infection stage of the T. gondii while ensuring safety and cross-species applicability remains a critical unmet need. Currently, the standard clinical treatment for toxoplasmosis involves the combination of pyrimethamine and sulfadiazine. However, this regimen is associated with a high failure rate and a range of adverse effects, including hepatotoxicity, nephrotoxicity, and suppression of hematopoietic stem cell proliferation and differentiation, thereby limiting its suitability as an ideal therapeutic option [13].
Through the production of metabolites and signaling molecules, as well as direct physical interactions, the gut microbiota profoundly influences the development, function, and homeostasis of the host immune system, thereby regulating both immune and metabolic processes. Alterations in the composition and metabolic profiles of gut microbiota are closely associated with the host’s physiological status and significantly influence the progression and outcomes of parasitic infections [14,15]. Accumulating evidence demonstrates that gut microbiota can modulate the colonization of parasites and their pathogenic potential within the host [16]. For example, secondary bile acids produced by gut microbes activate the Takeda G protein-coupled receptor 5 (TGR5), suppress the NF-κB signaling pathway in hepatic macrophages, and reduce the secretion of pro-inflammatory cytokines such as TNF-α and IL-6, thereby mitigating inflammation induced by parasitic infections [17]. Earlier studies have also shown that the metabolite α-linolenic acid attenuates intestinal inflammation caused by T. gondii infection by inhibiting the expression of pro-inflammatory cytokines IFN-γ, TNF-α, IL-1β, and IL-6 via the MyD88/NF-κB signaling pathway [18]. Accumulating evidence indicates that T. gondii infection can disrupt the composition and functional balance of gut microbiota by directly depleting beneficial bacterial populations or inducing cytokine-mediated suppression of antimicrobial peptides (AMPs), ultimately leading to microbial dysbiosis and exacerbating infection [19,20]. Notably, in the intraperitoneal tachyzoite infection model, significant alterations in gut microbiota structure and intestinal inflammation have also been documented, suggesting that systemic inflammatory signals can profoundly influence the intestinal microecology via the gut-immune axis [21,22].
At present, research on the specific mechanisms by which gut microbiota-associated metabolites regulate the inflammatory response during acute T. gondii infection still remains unclear. This study aims to investigate the differences in the host’s microbiota metabolic network and the immunomodulatory roles of microbial metabolites in combating acute T. gondii infection following challenge with strains of differing virulence. In our previous study, the α-amylase (α-amy) gene was deleted in the T. gondii ME49 strain using CRISPR/Cas9 technology to generate the attenuated mutant, designated as ME49Δα-amy [23]. Therefore, we established acute infection models using the wild-type virulent ME49 strain and the low-virulence ME49Δα-amy strain. Integrated analysis of 16S rRNA gene sequencing data revealed that the composition of the gut microbiota, particularly the relative abundance of beneficial bacterial groups such as Turicibacter and Ruminococcus, showed a significant negative correlation with pro-inflammatory factors, while demonstrating a significant positive correlation with anti-inflammatory factors and tight junction proteins. Furthermore, we identified GlcNAc, a metabolite associated with the intestinal microbiota via the metabolomics. To further evaluate its functional role, we established a murine model of oral GlcNAc administration during T. gondii infection and demonstrated that GlcNAc treatment significantly suppressed the expression of pro-inflammatory cytokines and upregulated the levels of anti-inflammatory cytokines. Moreover, GlcNAc administration markedly reduced parasite burden and effectively alleviated infection-induced weight loss. This study represents the first report on the protective role of GlcNAc in antagonizing T. gondii infection, offering novel insights into the complex interplay between the intestinal microbiota and parasitic infections.
Methods
Ethics statement
All experimental procedures involving animals were approved by the Life Scientific Ethic Committee of Yunnan Agricultural University (Approval number: 202303090).
Animals and parasites
Seven-week-old female ICR mice were obtained from the Laboratory Animal Center of Kunming Medical University (Kunming, China). All animals were maintained under pathogen-free conditions at a constant temperature of 22°C and 50–60% humidity, with a 12:12 h light–dark cycle, and provided with ad libitum access to standard rodent chow and fresh water. Prior to the initiation of experimental procedures, the mice were acclimatized to the vivarium environment for one week. Tachyzoites of the wild-type II virulent strain ME49 and the ME49Δα-amy mutant were propagated in confluent monolayers of human foreskin fibroblasts obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), and the host cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 2% fetal bovine serum.
Establishment of mouse model and sample collection
Female ICR mice that completed the acclimation period were randomly and evenly assigned to two groups and intraperitoneally infected with 10² tachyzoites of either the ME49 or ME49Δα-amy strain. On day 10 post-infection, serum samples were collected for Toxoplasma antigen-based ELISA to confirm successful infection. Mice that tested positive were selected for subsequent experimental procedures. On day 10 post-infection, the selected mice were humanely euthanized, and ascites, heart, liver, spleen, lungs, and colon tissues were carefully isolated and collected under aseptic conditions to minimize internal tissue contamination. Fecal samples were also collected on the same day, immediately flash-frozen in liquid nitrogen, and stored at -80°C. Subsequent analyses included 16S rRNA gene sequencing and metabolomics profiling.
To investigate the effect of GlcNAc on inflammation induced by T. gondii in host tissues, female ICR mice that completed the acclimation period were pretreated with an antibiotic solution (ATB) following a previously described protocol [24]. The treatment involved providing sterile drinking water supplemented with ampicillin (1 mg/mL), streptomycin (5 mg/mL), vancomycin (0.25 mg/mL) and colistin (1 mg/mL) for three consecutive days. Following ATB pretreatment, the mice were randomly assigned to three experimental groups and designated as Control, ME49 and ME49 + GlcNAc (Solarbio, Beijing, China). Mice from ME49 + GlcNAc group received oral administration of 300 mg/kg GlcNAc [25]. The control and ME49 groups were administered an equal volume of phosphate-buffered saline (PBS) for three days. Subsequently, mice in groups ME49 and ME49 + GlcNAc were intraperitoneally inoculated with 10² ME49 tachyzoites to establish an inflammatory model, whereas group Control received intraperitoneal injection of PBS. On day 10 post-infection, serum samples were collected from mice in groups ME49 and ME49 + GlcNAc for Toxoplasma antigen-based ELISA to confirm successful infection, and only infected mice were selected for subsequent experiments. Serum, ascites, heart, lung, liver, spleen, and colon tissues were collected from the infected mice for further downstream analyses.
Histopathological analysis
On the 10th day post-infection, colon, liver, and lung tissues were collected under aseptic conditions, fixed in 4% paraformaldehyde, and embedded in paraffin. The embedded tissues were sectioned into 4 μm slices using a microtome and stained with hematoxylin and eosin (H&E). The histopathological changes were evaluated with a light microscopy (Olympus, Japan). Heart tissue was scored on a scale from 0 to 4: 0 indicated no pathology; 1 for <25% myocardial involvement; 2 for 25–50%; 3 for 50–75%; and 4 for 75–100% involvement [26]. Pathological changes in the lung, liver, and spleen were assessed using a previously established semi-quantitative scoring system to evaluate tissue damage, and the standard nonlinear scoring system assigns values from 0 to 5 based on the severity of histopathological changes [27]. Furthermore, the amount and depth of inflammation in the colon tissue was evaluated using a 0–3 score range and the extent of crypt damage was assessed using a 0–4 score range. The final score for each parameter per section was calculated as the product of its severity grade and the percentage of tissue involvement [28].
Extraction of total RNA and real-time quantitative PCR (RT-qPCR)
Heart, liver, spleen, lung and colon tissues were collected and homogenized in TRIzol reagent, then transferred to fresh 1.5 ml centrifuge tubes. After adding 200 μl chloroform, the mixture was vortexed and centrifuged at 14,000 × g for 15 minutes. The supernatant was carefully transferred to an RNase-free centrifuge tube and mixed with an equal volume of isopropanol, followed by centrifugation at 14,000 × g for 10 minutes to precipitate RNA. The supernatant was discarded, and the RNA pellet was washed with 75% cold ethanol and centrifuged at 14,000 × g for 5 minutes. The RNA was then resuspended in RNase-free water. RNA was reverse transcribed into cDNA using the EasyScript One-Step gDNA Removal and cDNA Synthesis SuperMix (TRANSGEN Biotech, AE311) according to the manufacturer’s instructions. Gene expression levels were analyzed by real-time reverse transcription polymerase chain reaction (RT-qPCR) using the qTOWER3 G IVD fluorescence quantitative gene amplification system (Analytik Jena, Germany). Relative RNA expression levels were normalized and quantified using the 2−ΔΔCT method, with TNF-α, IL-1β, IL-6, IL-10, IL-12 and TGF-β mRNA normalized to β-actin as internal controls. Primer sequences are provided in Table 1.
Immunofluorescence
Colon tissue samples from each group of mice were sectioned into 4 μm-thick slices using a microtome. The sections were deparaffinized and rehydrated through sequential immersion in: three changes of an eco-friendly dewaxing solution (10 min each), followed by three changes of absolute ethanol (5 min each), and a final rinse in distilled water. Antigen retrieval was performed, after which the sections were washed three times with PBS (pH 7.4), 5 minutes per wash. Endogenous peroxidase activity was blocked by incubation with 3% hydrogen peroxide solution at room temperature in the dark for 25 minutes, followed by three additional PBS washes. After air-drying, the sections were incubated with blocking solution (3% BSA) for 30 minutes. The blocking solution was then removed, and the sections were incubated with the primary antibody (ZO-1 and occludin) overnight at 4°C in a humidified chamber. After three PBS washes, the sections were treated with the corresponding HRP-conjugated secondary antibody (HRP-labeled goat anti-rabbit IgG) at room temperature for 50 minutes. Following another three PBS washes, the sections were incubated with TSA fluorescent dye in the dark at room temperature for 10 minutes, and then washed three times with TBST (5 minutes each). Nuclear staining was performed using DAPI at room temperature in the dark for 10 minutes, followed by three PBS washes. The sections were then incubated with autofluorescence quenching agent B solution for 5 minutes, and rinsed under running water for 10 minutes. Finally, the sections were mounted using anti-fade mounting medium. The average immunofluorescence intensity of each target protein was quantified using ImageJ software (version 1.8).
16S rRNA gene sequencing
Bacterial genomic DNA was extracted from collected fecal samples using the QIAamp DNA isolation kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. DNA concentration was quantified using a Nanodrop spectrophotometer, and the integrity of the extracted DNA was assessed via 1.2% agarose gel electrophoresis. The V3–V4 hypervariable regions of the 16S rRNA gene were amplified by PCR using specific barcode primers (338F: 5’- ACTCCTACGGGAGGCAGCAG-3’, 806R: 5’-GGACTACHVGGGTWTCTAAT-3’), and the resulting amplicons were purified and quantified using fluorescence-based methods. Sequencing libraries were prepared using the TruSeq Nano DNA LT Library Prep Kit (Illumina) and subjected to high-throughput sequencing. Raw sequencing data were filtered to remove contaminants and low-quality reads. The processed data were analyzed using the QIIME2 dada2 pipeline. Alpha diversity, including species richness and diversity, was assessed using the Chao1 and Shannon indices, respectively, based on the distribution of amplicon sequence variants (ASVs). To enable comparisons across samples with differing sequencing depths, the absolute sequence counts for each taxon were normalized to their relative abundance within each sample. Specifically, the abundance of a given bacterial genus in a sample is expressed as the percentage of sequences assigned to that genus relative to the total number of high-quality sequences obtained for that sample. Principal coordinates analysis (PCoA) was performed to visualize β-diversity among microbial communities by reducing the dimensionality of the microbial dataset and displaying sample distribution along principal axes. Additionally, LEfSe (Linear Discriminant Analysis Effect Size) analysis was conducted by integrating non-parametric Kruskal-Wallis and Wilcoxon rank-sum tests with LDA to identify differentially abundant taxa across groups. Functional profiling was performed by statistically analyzing the abundance of level-2 functional pathways in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database.
Metabolomics analysis
Fecal samples were thawed on ice and mixed with methanol-based internal standards, followed by grinding and vortexing to extract metabolites. Following homogenization, samples were centrifuged at 18,000 × g for 20 minutes. The supernatant was collected and treated with derivatization reagents. After the derivatization reaction was completed, the reagents were removed, and the derivatized products were resuspended in 53% methanol. The mixture was centrifuged again, and the resulting supernatant was collected for subsequent LC-MS analysis.
Metabolite profiling was performed using an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) system (ACQUITY UPLC-Xevo TQ-S, Waters Corporation, Milford, MA, USA). Metabolite separation was achieved using an ACQUITY UPLC BEH C18 system, equipped with a 1.7 μM VanGuard pre-column (2.1 × 5 mm) and an analytical column (2.1 × 100 mm). The mobile phase consisted of water (with 0.1% formic acid) and acetonitrile/IPA (70:30), delivered at a flow rate of 0.40 mL/min. For mass spectrometric detection, the source and desolvation temperatures were set to 150 °C and 550 °C, respectively, with a desolvation gas flow of 1000 L/hr to optimize metabolite profiling. The raw data were processed using TMBQ software (v1.0, Metabo-Profile, Shanghai, China), which included principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) to identify differentially abundant metabolites. Metabolites with a variable importance in projection (VIP) score > 1, P value < 0.05, and a fold change (FC) ≥ 2 or ≤ 0.5 were considered statistically significant. A volcano plot was generated using the R package “ggplot2”, and hierarchical clustering heatmap was constructed using the “pheatmap” package in R. Finally, the relative abundance of genomic data and raw metabolite values were log-transformed, and Spearman correlation coefficients were calculated using R software.
Determination of N-acetyl-D-glucosamine
The serum levels of GlcNAc were measured based on Morgan Elson method [29]. Following adjustment to pH 10 with 4 M NaOH and dilution to volume with distilled water, 250 µL aliquots of the serum samples and standard were reacted with 250 µL of acetylacetone reagent (1 mL acetylacetone in 300 mL of 0.5 M sodium carbonate) at 100°C for 20 min. After the addition of 1 mL absolute ethanol to ensure the complete recovery of the reaction mixture, 250 µL of N,N’-dimethylaminobenzaldehyde solution was introduced, with a subsequent incubation at 65°C for 10 min. The absorbance of the final solution was measured at 530 nm after cooling and vigorous shaking.
Cell cultures and treatments
RAW264.7 cells, obtained from the ATCC (Maryland, USA), was maintained in DMEM supplemented with 10% fetal bovine serum (FBS) at 37°C with 5% CO₂. For infection experiments, cells were challenged with tachyzoites of T. gondii type II strain ME49 (MOI = 5). The parasite was propagated in human foreskin fibroblast cells cultured in DMEM with 2% FBS under the same conditions. The experiment included the following groups: (1) Control (PBS-treated); (2) ME49 infection only; and (3) ME49 + GlcNAc, where cells were pre-treated with 5mmol/L GlcNAc for 12 h prior to ME49 infection for 24 h [30].
Determination of parasite burden in peritoneal fluids
The peritoneal cavity of mice infected with T. gondii was flushed with PBS to collect peritoneal fluids. The peritoneal fluids were centrifuged at 3000 × g for 10 minutes at 4°C, after which the supernatant was removed and the pellet containing T. gondii tachyzoites and shed host cells was retained. Subsequently, the pellet was washed three times with PBS and resuspended in PBS. Genomic DNA was extracted from the collected ascites using the TIANamp Genomic DNA Kit (Tiangen Biotech, Beijing, China) according to the manufacturer’s instructions. To generate a standard for absolute quantification, tachyzoites of the ME49 strain were counted, serially diluted, and processed in parallel for DNA extraction. The sequences of the internal reference primers used for quantitative PCR were as follows: forward primer: 5’-TCGGTGACGAAGCCCAAA-3’; reverse primer: 5’-AGTTCGTTGTAGAAGGTGTGA-3’. The parasite load in each sample was determined by interpolation from the standard curve, which was established based on the linear relationship between Ct values and the logarithm of parasite numbers.
Statistical analysis
Statistical analysis was carried out using GraphPad Prism 9 software (GraphPad Software Inc., La Jolla, CA, USA). Changes in body weight were assessed by two-way ANOVA. Quantitative data are expressed as mean ± SEM. Comparisons between groups were performed using one-way ANOVA or Student’s t-test, as appropriate. A p-value < 0.05 was considered statistically significant.
Results
The extent of tissue inflammation following infection with tachyzoites varied significantly with the virulence level of the strain
To evaluate tissue damage induced by infection with tachyzoites of differing virulence in mice, histopathological analysis was performed on the liver, lung, colon, heart, and spleen. The results showed that mice infected with ME49 tachyzoites exhibited more severe Kupffer cell hyperplasia, hepatic congestion, and portal inflammation in the liver. In the lungs, prominent edema, alveolar congestion, hemorrhage, and thickening of the alveolar septum were observed. Cardiac tissue displayed focal inflammatory infiltrates and mild myocardial disorganization in ME49-infected mice, whereas the spleen showed marked white pulp hyperplasia and increased red pulp congestion. In contrast, the ME49Δα-amy infection group exhibited relatively mild inflammatory lesions across all tissues, with reduced pathological changes in the heart and spleen (Fig 1A). The pathological scores in the ME49Δα-amy group were significantly lower than those in the ME49 group across all examined tissues (Fig 1B-1F). Furthermore, expression levels of the pro-inflammatory cytokines TNF-α, IL-1β, IL-6, and IL-12 were significantly lower, and anti-inflammatory cytokines IL-10 and TGF-β were markedly upregulated in the ME49Δα-amy group compared to the ME49 group (Fig 1G–1L). Immunofluorescence analysis also revealed a significant reduction in the fluorescence signals of the tight junction proteins ZO-1 and occludin in the colon tissues of ME49-infected mice compared to those infected with ME49Δα-amy (Fig 2). Together, these findings indicate that the low-virulence ME49Δα-amy strain causes less severe inflammatory damage across multiple organs, and that infection with ME49 and ME49Δα-amy tachyzoites elicits distinct inflammatory responses in mouse tissues.
A. H&E staining of the mice heart, lung, liver, spleen and colon tissue samples (n = 3 per group). B-F. Histological scores of heart, lung, liver, spleen and colon tissues. G-L. The mRNA expression levels of TNF-α, IL-1β, IL-6, IL-12, IL-10, and TGF-β were measured by qRT-PCR (n = 3 per group). The Data are presented as the mean ± SEM of three independent experiments. Statistical significance was done with the Student’s t-test. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05.
The expression levels of occludin and ZO-1 were measured by immunofluorescence. Occludin protein was labeled with a green fluorophore, ZO-1 protein was labeled with a red fluorophore, and the cell nucleus was labeled with a blue fluorophore (n = 3 per group). The Data are presented as the mean ± SEM of three independent experiments. Statistical significance was done with the Student’s t-test. ****p < 0.0001.
The gut microbiota structure of mice infected with tachyzoites of different virulence levels exhibited significant differences
Analysis of alpha diversity, as measured by the Shannon and Chao1 indices, revealed no significant differences between the ME49 and ME49Δα-amy tachyzoite-infected groups (Fig 3A and 3B). This indicated that the overall species richness and evenness of the gut microbiota were comparable under these two infection conditions. Principal coordinate analysis (PCoA) of weighted UniFrac distances revealed distinct microbial community structures between the two infection groups (Fig 3C). At the phylum level, the ME49Δα-amy group showed higher relative abundances of Bacteroidetes and Firmicutes, while the relative abundance of Proteobacteria was reduced compared with the ME49-infected group (Fig 3D). At the family level, the abundance of Ruminococcaceae was increased in the ME49Δα-amy group, whereas Enterobacteriaceae exhibited an opposite trend (Fig 3E). At the genus level, Bacteroides and Shigella were the dominant bacterial genera in the ME49Δα-amy group (Fig 3F). Further analysis indicated enrichment of various Gram-positive anaerobic bacteria primarily belonging to the phylum Firmicutes in the ME49Δα-amy group, such as Turicibacter and Ruminococcus (Fig 3G). Linear discriminant analysis of effect size (LEfSe) analysis identified differentially abundant bacterial taxa, and the results manifested that the relative abundance of Ruminococcaceae and Turicibacteraceae significantly increased after ME49Δα-amy tachyzoites infection (Fig 3H). KEGG pathway enrichment analysis demonstrated significant enrichment of pathways related to infectious diseases, carbohydrate metabolism, amino acid metabolism, lipid metabolism, and metabolism of cofactors and vitamins among the differentially abundant microbial species (Fig 3I). To investigate the possible relationship between gut microbiota and inflammatory phenotypes following T. gondii tachyzoite infection, Spearman correlation analysis was performed to evaluate associations among pro inflammatory cytokines, tight junction proteins, and gut bacterial taxa. The results demonstrated that several bacterial genera, including Clostridium, Ruminococcus, Turicibacter, Helicobacter, Desulfovibrio, and Alistipes, showed significant negative correlations with pro-inflammatory factors. Conversely, these genera exhibited significant positive correlations with anti-inflammatory factors as well as with the tight junction proteins Occludin and ZO-1 (Fig 3J). These results suggest that infection with ME49 and ME49Δα-amy strains leads to distinct gut microbiota profiles, with the ME49 infection group exhibiting more pronounced depletion of beneficial intestinal bacteria.
A. Microbial community diversity (measured by Shannon index). B. Microbial community abundance (measured by Chao1 index). C. Principal coordinate analysis (PCoA) based on the weighted UniFrac distance matrix. D. Relative abundances of fecal bacterial at phylum level. E. Relative abundances of fecal bacterial at family level. F. Relative abundances of fecal bacterial at genus level. G. Heat map of the top 50 microbiota at genus level in fecal samples from mice infected with ME49 or ME49Δα-amy tachyzoites. H. Differentially abundant taxa of fecal microbiota was analyzed by LEfSe. LDA score ≥ 2. I. KEGG pathway analysis of differentially expressed genes. J. Heatmap of Spearman’s correlation between gut microbiota abundance and inflammatory factors. *p < 0.05, **p < 0.01.
The metabolite N-acetyl-D-glucosamine is closely linked to regulating T. gondii-induced inflammation responses
Accumulating evidence suggests that metabolites associated with the intestinal microbiota modulate inflammatory responses through diverse mechanisms and play a critical role in regulating the progression of various inflammation-related diseases [31,32]. To examine whether the disparity in inflammatory severity following infection with two T. gondii strains of distinct virulence is linked to changes in gut microbial metabolites, a non-targeted metabolomics strategy via UPLC-MS/MS was utilized. Metabolite classification revealed that 32.92% of the compounds were short-chain fatty acids (SCFAs), 31.47% were amino acids, 13.75% were fatty acids, and 7.91% were benzenoids (Fig 4A). PLS-DA was performed to evaluate intergroup differences between the ME49 and ME49Δα-amy groups. The clustering of data points indicated a clear and statistically significant separation in metabolic profiles between the two groups (Fig 4B and 4C). To identify potential biomarkers, we further analyzed metabolites that exhibited significant differences in relative abundance. Compared to the ME49 group, seven metabolites (Methylglutaric acid, N−acetyl−D−glucosamine, 2 − Hydroxybutyric acid, 4 − Aminohippuric acid, Fructose, Hydroxypropionic acid, and Glucose) were significantly upregulated and five (UDCA, Indole−3 − methyl acetate, bMCA, TUDCA, 2 − Hydroxybutyric acid, and GCA) were downregulated in the ME49Δα-amy group (Fig 4D). Carbohydrate metabolites, fructose, glucose and GlcNAc, were markedly enriched in ME49Δα-amy-infected mice, showing a significantly higher relative abundance than in the ME49 group (Fig 4E–4H). The results of the enrichment analysis of Pathway-associated metabolite sets (SMPDB) database visualized by bubble chart revealed that these differential metabolites were abundant in amino sugar metabolism, propanoate metabolism, and trehalose degradation (Fig 4I). To investigate the pathway through which gut microbiota-derived GlcNAc alleviates systemic inflammation caused by acute T. gondii infection, we quantified serum GlcNAc levels. As shown in Fig 4J, serum GlcNAc levels were significantly higher in mice infected with the knockout strain compared to those infected with the wild-type strain. Spearman correlation analysis was carried out to assess associations between differentially abundant bacterial taxa and altered metabolites during infection. The results revealed a positive correlation between GlcNAc levels and the abundance of Ruminococcus and Turicibacter, both members of the phylum Firmicutes (Fig 5). Together, these findings suggest that the attenuated strain may reshape the commensal gut microbiota, enhance GlcNAc synthesis, and subsequently elevate circulating GlcNAc levels, which may contribute to mitigating systemic inflammation induced by acute T. gondii infection.
A. Total metabolome classifications of compounds with differential metabolites. B-C. Fecal metabolome profiles were clustered using PLS-DA. D. Volcanic map analysis of differential metabolites. E. Metabolite relative abundances were clustered by UPGMA and visualized in a heatmap using Z-scores. F-H. The concentrations of fecal Fructose, Glucose, and N-Acetyl-D-glucosamine were displayed as box and dot plots. I. SMPDB pathway enrichment analysis according to the markedly altered metabolites. J. The concentration of GlcNAc was measured in serum. Four independent biological replicates were analyzed for each group. Statistical significance was done with the Student’s t-test. **p < 0.01.
The heatmap displays correlation coefficients between differentially enriched bacterial taxa and metabolites in response to ME49Δα-amy tachyzoites infection. Red represents positive correlation, and blue represents negative correlation. ***p < 0.001, **p < 0.01, *p < 0.05.
Inhibitory effect of GlcNAc on T. gondii-induced inflammation in RAW264.7 cells
The impact of GlcNAc on the inflammatory response triggered by T. gondii was assessed through in vitro experiments. Comparative analysis showed that cells treated with GlcNAc exhibited a pronounced reduction in the mRNA expression of multiple proinflammatory cytokines, specifically TNF-α, IL-1β, IL-6, and IL-12, following infection. In contrast, the expression of the anti-inflammatory cytokines IL-10 and TGF-β was significantly enhanced in the GlcNAc-treated group compared to the ME49-infected group (Fig 6).
The mRNA expression levels of TNF-α (A), IL-1β (B), IL-6 (C), IL-12 (D), IL-10 (E), and TGF-β (F) were measured by qRT-PCR. The Data are presented as the mean ± SEM of three independent experiments. Statistical significance was done with the one-way ANOVA. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05.
Oral administration of GlcNAc can alleviate systemic inflammation caused by T. gondii infection
To investigate the role of GlcNAc in modulating the inflammatory response induced by T. gondii infection, SPF mice pretreated with antibiotics for three days were orally administered GlcNAc. Subsequently, the ME49-infected group and the ME49 + GlcNAc intervention group were established via intraperitoneal injection of ME49 tachyzoites, while the control group received PBS. On day 4 post-infection, mice in the ME49 group exhibited significant weight loss, which worsened as the infection progressed. In contrast, the ME49 + GlcNAc group showed minimal change in body weight (Fig 7A). qPCR analysis of ascitic fluid samples revealed a significant reduction in parasite load in the GlcNAc-treated group compared to the ME49 group (Fig 7B). Histopathological analysis indicated severe structural damage in the colon of the ME49 group, including extensive crypt loss and inflammatory cell infiltration in the lamina propria. However, the ME49 + GlcNAc group exhibited milder lesions, with inflammation largely restricted to the submucosa. In extraintestinal tissues such as the heart, lungs, liver, and spleen, the ME49 group displayed substantial pathological damage, including hemorrhage and extensive inflammatory cell infiltration, which were markedly attenuated in the ME49 + GlcNAc group (Fig 7C–7H). Further assessment by qPCR showed that GlcNAc treatment significantly suppressed the expression of pro-inflammatory cytokines TNF-α, IL-1β, IL-6, and IL-12, while upregulated IL-10 and TGF-β in the heart, liver, spleen, lungs, and colon tissues (Fig 7I–7N). Moreover, immunofluorescence staining demonstrated that the expression levels of the tight junction proteins ZO-1 and occludin were significantly higher in the ME49 + GlcNAc group than in the ME49 group (Fig 8). In summary, these findings indicate that oral administration of GlcNAc effectively alleviates T. gondii infection-induced weight loss, systemic inflammation, and parasite burden, while preserving intestinal barrier integrity and reducing tissue damage and epithelial injury.
A. Body weight changes of mice (n = 10 per group). B. The parasite loads in peritoneal fluids were determined by quantitative PCR (n = 3 per group). C. H&E staining of the mice heart, lung, liver, spleen and colon tissue samples. D-H. Histological scores of heart, lung, liver, spleen and colon tissues (n = 3 per group). I-N. The mRNA expression levels of TNF-α, IL-1β, IL-6, IL-12, IL-10, and TGF-β were measured by qRT-PCR (n = 3 per group). The Data are presented as the mean ± SEM of three independent experiments. Statistical significance was done with the one-way ANOVA. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05.
The expression levels of occludin and ZO-1 were measured by immunofluorescence. Occludin protein was labeled with a green fluorophore, ZO-1 protein was labeled with a red fluorophore, and the cell nucleus was labeled with a blue fluorophore (n = 3 per group). The Data are presented as the mean ± SEM of three independent experiments. Statistical significance was done with the one-way ANOVA. ****p < 0.0001.
Discussion
Currently, due to the lack of safe and effective interventions, the health of both humans and animals remains threatened by toxoplasmosis. The current standard treatment for acute toxoplasmosis relies on sulfonamide antibiotics, which, while effective, are associated with drawbacks such as drug resistance and adverse effects [33]. In contrast, our study explores GlcNAc, a gut microbiota-associated metabolite, as a potential host-directed immunomodulator. This approach aims to mitigate infection-induced pathology by bolstering host defense mechanisms, offering a complementary strategy that differs from direct antiparasitic action. The intestinal microbiota, as the largest microbial ecosystem coexisting with the host, plays a crucial role in maintaining host health and modulating immune responses [34]. Accumulating evidence has demonstrated that the intestinal microbiota and its derived metabolites are key players in resisting T. gondii infection. These microbial components can inhibit the invasion of T. gondii and alleviate the associated pathological damage through multiple mechanisms; their regulatory capacity is pivotal in determining host resistance to parasitic infection [20,35]. However, little is known about differences in the metabolic networks of the gut microbiota following infection with parasite strains of varying virulence. Therefore, we established infection models in mice by intraperitoneal injection of low-virulence ME49Δα-amy tachyzoites and wild-type ME49 tachyzoites. Our findings revealed that the two strains induced distinct patterns of tissue inflammation by altering the composition of the gut microbiota. Using 16S rRNA sequencing combined with metabolomic profiling, we identified GlcNAc as a key differential metabolite, whose abundance was significantly and positively correlated with the abundance of beneficial intestinal bacteria. Furthermore, we demonstrated that GlcNAc supplementation effectively reduced parasite burden and alleviated systemic inflammation triggered by T. gondii infection.
The rapid replication of tachyzoites throughout the host organism initiates the onset of acute infection, the severity of which is closely associated with the virulence of the infecting T. gondii strain [36]. Infections with ME49Δα-amy and ME49, two strains with differing pathogenic potentials, exhibit distinct clinical courses and intestinal pathological changes. These variations are closely linked to alterations in the composition and structure of the intestinal microbiota. Through its strong proliferative capacity and ability to disrupt the intestinal barrier, the wild-type ME49 strain induces gut microbiota dysbiosis, ultimately leading to severe barrier dysfunction and inflammatory responses, which was consistent with our observations [37].In contrast, ME49Δα-amy has been shown to confer long-term immune protection against both tachyzoites and bradyzoites of T. gondii infection [38]. The mechanism underlying this protective effect may be mediated through the modulation of the intestinal microbiota. The microbial and metabolomic analyses were conducted at 10 days post-infection, a time point within the acute phase where significant and rapid microbiota remodeling in response to enteric challenge is well-documented, providing a relevant context for the observed differences driven by parasite virulence [18,39]. The gut-organ axis, a bidirectional communication system between the intestine and distant organs, has garnered significant scientific interest, with the intestinal microbiota serving as a central regulatory component. Previous studies have reported that T. gondii infection promotes the expansion of Shigella-like pathogens while reducing the abundance of beneficial microbial taxa, a pattern that aligns with our findings [19].In the present study, mice infected with ME49Δα-amy tachyzoites exhibited a marked increase in the relative abundance of Bacteroidetes and Firmicutes, along with a significant reduction in Proteobacteria. Notably, this modulation is evident not only following intraperitoneal infection with tachyzoites but also after oral infection with cysts, where the ME49Δα-amy strain promotes a distinct and potentially more stable microbial community. Specifically, oral administration of ME49Δα-amy cysts resulted in a microbiota profile significantly different from that induced by the ME49 strain, marked by the enrichment of beneficial genera such as Lactobacillus, Bacteroides, and [Prevotella] [18]. In contrast, intraperitoneal infection with tachyzoites led to a unique enrichment pattern, with overall compositional structures and key signature taxa clearly distinguishable from those observed in the cyst-induced phase. Although some beneficial genera are detected under both infection routes, differences in their relative abundance and community context suggest that the ME49Δα-amy strain may dynamically reshape the gut microenvironment through route-specific mechanisms, thereby exerting protective effects across different modes of infection. The mechanisms by which these beneficial bacteria exert anti-parasitic effects include the interaction of probiotics with Toll-like receptors (TLRs), which subsequently inhibits the activation of NF-κB and MAPK signaling pathways, thereby modulating mucosal immune responses [40–43]. Moreover, Bacteroides species have been shown to activate CD4+ T cells, regulate the Th1/Th2 immune balance, and suppress the progression of colitis [44]. The proliferation of probiotic bacteria can also inhibit the growth of pro-inflammatory pathogens, thereby exerting a positive regulatory influence on the overall composition of the intestinal microbiota [45]. In our study, a strong positive correlation was observed between the genera Ruminococcus and Turicibacter and GlcNAc, a metabolite with anti-inflammatory properties during infection. As symbiotic organisms, Firmicutes are known to contribute to anti-inflammatory responses and the maintenance of immune homeostasis through various mechanisms, such as modulating intestinal tight junction protein expression and generating anti-inflammatory metabolites [46]. This finding highlights the potential functional roles of these bacterial taxa in mitigating T. gondii infection. Such insights may offer novel avenues for developing microbiota-targeted therapeutic strategies against T. gondii.
Previous studies have demonstrated the existence of a complex interaction between the immune system and the gut microbiota during parasitic infections. The gut microbiota can directly inhibit the colonization or proliferation of parasites through the production of metabolites that either physically disrupt or metabolically interfere with parasitic activity [47]. Gut-derived metabolites serve as crucial mediators of the immune-regulatory functions exerted by the gut microbiota. GlcNAc, a key amino sugar related to the gut microbiota, participates in protein glycosylation modification, regulates immune cell metabolism and signaling pathways, and thereby exerts multi-faceted immunomodulatory effects. However, no studies have yet reported the regulatory role of GlcNAc in inflammation induced by T. gondii infection. Earlier reports have indicated that GlcNAc is essential for the growth of Akkermansia muciniphila, a commensal bacterium critical for maintaining intestinal barrier integrity [48]. Furthermore, Alves et al. demonstrated that GlcNAc suppresses the function of innate immune effector cells and effector T cells through metabolic reprogramming, resulting in reduced secretion of pro-inflammatory cytokines such as IL-17a, IFN-γ, and TNF-α, along with increased IL-10 production, thereby alleviating immune-mediated tissue damage. This observation aligns with our findings of attenuated tissue inflammation in GlcNAc-treated mice [49]. The role of GlcNAc in mediating host resistance to infection via modulation of immune cell proliferation and activity has been well established [50]. Our findings further demonstrate that GlcNAc treatment significantly reduces inflammatory damage in both the intestine and other organs, reinforcing its therapeutic potential for systemic inflammation caused by T. gondii infection. Moreover, we observed a significant upregulation in the expression levels of tight junction proteins ZO-1 and occludin following GlcNAc administration, suggesting that GlcNAc can reverse intestinal barrier dysfunction induced by T. gondii and indicating its protective and therapeutic effects against toxoplasmosis. GlcNAc not only directly inhibits immune cells and cytokine secretion to limit tissue damage, but also remodels the gut microbiota toward a beneficial composition, which reinforces intestinal barrier integrity and suppressing inflammation [51].
It is important to acknowledge the limitations of this study. While we established a strong association between GlcNAc, the gut microbiota, and the amelioration of systemic inflammation, the precise molecular mechanisms by which GlcNAc exerts its anti-inflammatory effects specifically against T. gondii infection remain to be fully elucidated. Furthermore, the correlative nature of our microbiome-metabolome data means that while we identify GlcNAc as a key differential metabolite linked to a beneficial microbiota, definitive proof of a direct causal relationship from bacterial shifts to metabolite change to host phenotype requires future experimental validation. Our study primarily describes the phenotypic outcomes and correlative relationships. Future investigations should focus on delineating the exact signaling pathways through which GlcNAc modulates immune cell responses during toxoplasmosis. Additionally, research into whether GlcNAc directly affects parasite viability or invasion, or exerts its effects solely through host immunomodulation, would provide deeper mechanistic insights. Addressing these questions will be crucial for translating these findings into targeted therapeutic strategies.
In conclusion, our findings indicate that alterations in the intestinal microbiota structure may be associated with the differential inflammatory responses elicited by infection with T. gondii strains of varying virulence. Infection with T. gondii strains of distinct virulence is associated with changes in gut microbial composition and corresponding shifts in the abundance of certain metabolites, which may be driven by a virulence-dependent reduction in intestinal damage and a modulated immune response that favors beneficial bacterial proliferation and activity. Our data suggest that GlcNAc, potentially in relation to the gut microbiota, may contribute to the balance between pro- and anti-inflammatory cytokines, thereby alleviating systemic inflammation caused by T. gondii infection via the gut-organ axis through systemic circulation. Collectively, these observations provide insights into the potential immunomodulatory role of gut microbiota-associated metabolites during zoonotic parasitic infections and could inform future exploration of microbiota-targeted strategies against toxoplasmosis.
Acknowledgments
We thank Metabo-Profile Biotechnology (Shanghai) Co., Ltd for the technical assistance and bioinformatic analysis of the data.
References
- 1. Kochanowsky JA, Koshy AA. Toxoplasma gondii. Curr Biol. 2018;28(14):R770–1. pmid:30040931
- 2. Lambert H, Hitziger N, Dellacasa I, Svensson M, Barragan A. Induction of dendritic cell migration upon Toxoplasma gondii infection potentiates parasite dissemination. Cell Microbiol. 2006;8(10):1611–23. pmid:16984416
- 3. Chen Y, Zhao Y-F, Yang J, Jing H-Y, Liang W, Chen M-Y, et al. Selenium alleviates lipopolysaccharide-induced endometritis via regulating the recruitment of TLR4 into lipid rafts in mice. Food Funct. 2020;11(1):200–10. pmid:31845693
- 4. Dian S, Ganiem AR, Ekawardhani S. Cerebral toxoplasmosis in HIV-infected patients: a review. Pathog Glob Health. 2023;117(1):14–23. pmid:35694771
- 5. Sauer A, Schaeffer M, Serrar Y, Kodjikian L. Ocular toxoplasmosis. J Fr Ophtalmol. 2024;47(10):104334. pmid:39490112
- 6. Saadatnia G, Golkar M. A review on human toxoplasmosis. Scand J Infect Dis. 2012;44(11):805–14. pmid:22831461
- 7. Gov L, Schneider CA, Lima TS, Pandori W, Lodoen MB. NLRP3 and Potassium Efflux Drive Rapid IL-1β Release from Primary Human Monocytes during Toxoplasma gondii Infection. J Immunol. 2017;199(8):2855–64. pmid:28904126
- 8. Qiu J, Xie Y, Shao C, Shao T, Qin M, Zhang R, et al. Toxoplasma gondii microneme protein MIC3 induces macrophage TNF-α production and Ly6C expression via TLR11/MyD88 pathway. PLoS Negl Trop Dis. 2023;17(2):e0011105. pmid:36730424
- 9. Melo MB, Jensen KDC, Saeij JPJ. Toxoplasma gondii effectors are master regulators of the inflammatory response. Trends Parasitol. 2011;27(11):487–95. pmid:21893432
- 10. Guiton PS, Sagawa JM, Fritz HM, Boothroyd JC. An in vitro model of intestinal infection reveals a developmentally regulated transcriptome of Toxoplasma sporozoites and a NF-κB-like signature in infected host cells. PLoS One. 2017;12(3):e0173018. pmid:28362800
- 11. Resta-Lenert S, Barrett KE. Probiotics and commensals reverse TNF-alpha- and IFN-gamma-induced dysfunction in human intestinal epithelial cells. Gastroenterology. 2006;130(3):731–46. pmid:16530515
- 12. Dunay IR, Sibley LD. Monocytes mediate mucosal immunity to Toxoplasma gondii. Curr Opin Immunol. 2010;22(4):461–6. pmid:20537517
- 13. Montoya JG, Liesenfeld O. Toxoplasmosis. Lancet. 2004;363(9425):1965–76. pmid:15194258
- 14. Berrilli F, Di Cave D, Cavallero S, D’Amelio S. Interactions between parasites and microbial communities in the human gut. Front Cell Infect Microbiol. 2012;2:141. pmid:23162802
- 15. Agirman G, Yu KB, Hsiao EY. Signaling inflammation across the gut-brain axis. Science. 2021;374(6571):1087–92. pmid:34822299
- 16. Sardinha-Silva A, Alves-Ferreira EVC, Grigg ME. Intestinal immune responses to commensal and pathogenic protozoa. Front Immunol. 2022;13:963723. pmid:36211380
- 17. Wang Y-D, Chen W-D, Yu D, Forman BM, Huang W. The G-protein-coupled bile acid receptor, Gpbar1 (TGR5), negatively regulates hepatic inflammatory response through antagonizing nuclear factor κ light-chain enhancer of activated B cells (NF-κB) in mice. Hepatology. 2011;54(4):1421–32. pmid:21735468
- 18. Yang J, Liu S, Zhao Q, Li X, Jiang K. Gut microbiota-related metabolite alpha-linolenic acid mitigates intestinal inflammation induced by oral infection with Toxoplasma gondii. Microbiome. 2023;11(1):273. pmid:38087373
- 19. Raetz M, Hwang S-H, Wilhelm CL, Kirkland D, Benson A, Sturge CR, et al. Parasite-induced TH1 cells and intestinal dysbiosis cooperate in IFN-γ-dependent elimination of Paneth cells. Nat Immunol. 2013;14(2):136–42. pmid:23263554
- 20. Luo X, Yang X, Tan S, Zhang Y, Liu Y, Tian X, et al. Gut microbiota mediates anxiety-like behaviors induced by chronic infection of Toxoplasma gondii in mice. Gut Microbes. 2024;16(1):2391535. pmid:39182245
- 21. Lv QB, Ma H, Wei J, Qin YF, Qiu HY, Ni HB. Changes of gut microbiota structure in rats infected with Toxoplasma gondii. Front Cell Infect Microbiol. 2022;12.
- 22.
Prandovszky E, Li Y, Sabunciyan S, Steinfeldt CB, Avalos LN, Gressitt KL. Toxoplasma gondii-induced long‐term changes in the upper intestinal microflora during the chronic stage of infection. 2018;2018(1):2308619.
- 23. Yang J, Yang C, Qian J, Li F, Zhao J, Fang R. Toxoplasma gondii α-amylase deletion mutant is a promising vaccine against acute and chronic toxoplasmosis. Microb Biotechnol. 2020;13(6):2057–69. pmid:32959958
- 24. Routy B, Le Chatelier E, Derosa L, Duong CPM, Alou MT, Daillère R, et al. Gut microbiome influences efficacy of PD-1-based immunotherapy against epithelial tumors. Science. 2018;359(6371):91–7. pmid:29097494
- 25. Choi S-I, Shin YC, Lee JS, Yoon YC, Kim JM, Sung M-K. N-Acetylglucosamine and its dimer ameliorate inflammation in murine colitis by strengthening the gut barrier function. Food Funct. 2023;14(18):8533–44. pmid:37655824
- 26. Tominaga M, Matsumori A, Okada I, Yamada T, Kawai C. Beta-blocker treatment of dilated cardiomyopathy. Beneficial effect of carteolol in mice. Circulation. 1991;83(6):2021–8. pmid:1674900
- 27. Khairani S, Fauziah N, Wiraswati HL, Panigoro R, Setyowati EY, Berbudi A. Oral Administration of Piperine as Curative and Prophylaxis Reduces Parasitaemia in Plasmodium berghei ANKA-Infected Mice. J Trop Med. 2022;2022:5721449. pmid:35360190
- 28. Dieleman LA, Palmen MJ, Akol H, Bloemena E, Peña AS, Meuwissen SG, et al. Chronic experimental colitis induced by dextran sulphate sodium (DSS) is characterized by Th1 and Th2 cytokines. Clin Exp Immunol. 1998;114(3):385–91. pmid:9844047
- 29. Lago G, Oruna L, Cremata J, Perez C, Coto G, Lauzan E, et al. Isolation, purification and characterization of hyaluronan from human umbilical cord residues. Carbohydrate Polymers. 2005;62(4):321–6.
- 30. Hwang JS, Kim KH, Park J, Kim SM, Cho H, Lee Y. Glucosamine improves survival in a mouse model of sepsis and attenuates sepsis-induced lung injury and inflammation. J Pharmacol Exp Ther. 2019;294(2):608–22.
- 31. Banerjee A, Herring CA, Chen B, Kim H, Simmons AJ, Southard-Smith AN, et al. Succinate Produced by Intestinal Microbes Promotes Specification of Tuft Cells to Suppress Ileal Inflammation. Gastroenterology. 2020;159(6):2101-2115.e5. pmid:32828819
- 32. Zha X, Liu X, Wei M, Huang H, Cao J, Liu S, et al. Microbiota-derived lysophosphatidylcholine alleviates Alzheimer’s disease pathology via suppressing ferroptosis. Cell Metab. 2025;37(1):169-186.e9. pmid:39510074
- 33. Shendi SS, Selim SM, Sharaf SA, Gouda MA, Sallam HM, Sweed DM, et al. Anti-toxoplasmic effects of celecoxib alone and combined with spiramycin in experimental mice. Acta Trop. 2024;260:107448. pmid:39477047
- 34. Pickard JM, Zeng MY, Caruso R, Núñez G. Gut microbiota: Role in pathogen colonization, immune responses, and inflammatory disease. Immunol Rev. 2017;279(1):70–89. pmid:28856738
- 35. Mejia R, Damania A, Jeun R, Bryan PE, Vargas P, Juarez M, et al. Impact of intestinal parasites on microbiota and cobalamin gene sequences: a pilot study. Parasit Vectors. 2020;13(1):200. pmid:32306993
- 36. Pittman KJ, Knoll LJ. Long-Term Relationships: the Complicated Interplay between the Host and the Developmental Stages of Toxoplasma gondii during Acute and Chronic Infections. Microbiol Mol Biol Rev. 2015;79(4):387–401. pmid:26335719
- 37. Chen J, Zhang C, Yang Z, Wu W, Zou W, Xin Z, et al. Intestinal microbiota imbalance resulted by anti-Toxoplasma gondii immune responses aggravate gut and brain injury. Parasit Vectors. 2024;17(1):284. pmid:38956725
- 38. Yang J, Yang C, Qian J, Li F, Zhao J, Fang R. Toxoplasma gondii α-amylase deletion mutant is a promising vaccine against acute and chronic toxoplasmosis. Microb Biotechnol. 2020;13(6):2057–69. pmid:32959958
- 39. Wang Y, Deng C, Sui M, Wei P, Duan B, Li Z, et al. Acute Toxoplasma gondii Infection Drives Gut Microbiome Dysbiosis and Functional Disruption in Mice as Revealed by Metagenomic Sequencing. Microorganisms. 2025;13(9):2056.
- 40. Toh ZQ, Anzela A, Tang MLK, Licciardi PV. Probiotic therapy as a novel approach for allergic disease. Front Pharmacol. 2012;3:171. pmid:23049509
- 41. Petrof EO, Kojima K, Ropeleski MJ, Musch MW, Tao Y, De Simone C, et al. Probiotics inhibit nuclear factor-kappaB and induce heat shock proteins in colonic epithelial cells through proteasome inhibition. Gastroenterology. 2004;127(5):1474–87. pmid:15521016
- 42. Ma D, Forsythe P, Bienenstock J. Live Lactobacillus rhamnosus [corrected] is essential for the inhibitory effect on tumor necrosis factor alpha-induced interleukin-8 expression. Infect Immun. 2004;72(9):5308–14. pmid:15322027
- 43. Wang Y, Lin J, Cheng Z, Wang T, Chen J, Long M. Bacillus coagulans TL3 Inhibits LPS-Induced Caecum Damage in Rat by Regulating the TLR4/MyD88/NF-κB and Nrf2 Signal Pathways and Modulating Intestinal Microflora. Oxid Med Cell Longev. 2022;2022:5463290. pmid:35178157
- 44. Mazmanian SK, Round JL, Kasper DL. A microbial symbiosis factor prevents intestinal inflammatory disease. Nature. 2008;453(7195):620–5. pmid:18509436
- 45. Xiong M, Sun W. Research progress of probiotics and their protective strategy in the field of inflammatory bowel disease treatment: A review. Medicine (Baltimore). 2024;103(44):e40401. pmid:39495980
- 46. Byndloss MX, Olsan EE, Rivera-Chávez F, Tiffany CR, Cevallos SA, Lokken KL, et al. Microbiota-activated PPAR-γ signaling inhibits dysbiotic Enterobacteriaceae expansion. Science. 2017;357(6351):570–5. pmid:28798125
- 47. VanDussen KL, Funkhouser-Jones LJ, Akey ME, Schaefer DA, Ackman K, Riggs MW. Neonatal mouse gut metabolites influence Cryptosporidium parvum infection in intestinal epithelial cells. mBio. 2020;11(6). pmid:33323514
- 48. van der Ark KCH, Aalvink S, Suarez-Diez M, Schaap PJ, de Vos WM, Belzer C. Model-driven design of a minimal medium for Akkermansia muciniphila confirms mucus adaptation. Microb Biotechnol. 2018;11(3):476–85. pmid:29377524
- 49. Alves I, Santos-Pereira B, de la Cruz N, Campar A, Pinto V, Rodrigues PM, et al. Host-derived mannose glycans trigger a pathogenic γδ T cell/IL-17a axis in autoimmunity. Sci Transl Med. 2023;15(687):eabo1930. pmid:36921032
- 50. Hu X, Sun X, Zhao Y, Iv C, Sun X, Jin M, et al. GlcNac produced by the gut microbiome enhances host influenza resistance by modulating NK cells. Gut Microbes. 2023;15(2):2271620. pmid:37953509
- 51. Duan Y-R, Du Y-H, Liu H-R. Research advances of occludin in vascular endothelial injury. Sheng Li Xue Bao. 2021;73(6):931–9. pmid:34961868