Gondii-infected Mice Research Articles

Cellular Senescence Contributes to Colonic Barrier Integrity Impairment Induced by Toxoplasma gondii Infection.

Toxoplasma gondii (T. gondii) induces gut barrier integrity impairment, which is crucial to the establishment of long-term infection in hosts. Cellular senescence is an imperative event that drives disease progression. Several studies have indicated that T. gondii induces oxidative stress and cell cycle blockade in the tissues of hosts, suggesting cellular senescence induced by the parasite. Here, we explored whether cell senescence is involved in T. gondii-mediated colonic barrier integrity damage in mice. C57BL/6J mice were infected with 10 cysts of T. gondii. Senolytic therapy (dasatinib and quercetin, DQ, a combination therapy for reducing senescent cells) was given by oral gavage 4weeks post-infection. Alcian blue staining, immunofluorescence, western blot, quantitative PCR (qPCR), and enzyme-linked immunosorbent assay (ELISA) were employed to evaluate the thickness of the colonic mucus layer, the expression profiles of genes and proteins related to tight junction function and cellular senescence in the colonic tissues, and the levels of serum lipopolysaccharides (LPS), respectively. T. gondii-infected mice exhibited deteriorated secreted mucus, shortened length, decreased expression of zonula occludens-1 (ZO-1) and occludin in the colon, accompanied by elevated levels of serum LPS. Moreover, the infection upregulated cell senescence-related markers (p16INK4A, p21CIP1) while inhibiting Lamin B1 expression. In addition, the expression levels of senescence-associated secretory phenotypes (SASPs), including IL-1β, TNF-α, IL-6, MMP9 and CXCL10, were upregulated post-infection. Notably, reducing cell senescence with DQ administration, significantly ameliorated the colonic pathological alterations induced by T. gondii infection. This study uncovers for the first time that cellular senescence contributes to the colonic barrier integrity damage induced by chronic T. gondii infection. Importantly, we provide evidence that senolytic therapy exerts a therapeutic effect on the intestinal pathological lesions.

Evaluation of the efficicacy of myrcene in the treatment of Eimeria tenella and Toxoplasma gondii infection

Protozoan parasites such as Eimeria tenella and Toxoplasma gondii pose significant health challenges in livestock and humans. The limited treatment options and rising drug resistance underscore the urgent need for new therapies. This study investigates myrcene, a monoterpene hydrocarbon classified for its antiprotozoal potential against E. tenella and T. gondii infections. Initially, we examined its effect on the sporulation process of E. tenella oocysts in vitro and its anti-E. tenella activity in vivo. Myrcene significantly reduced the sporulation rate of E. tenella oocysts at 3 and 4 mg/kg. In vivo experiments demonstrated that treatment with 4 mg/kg myrcene significantly reduced E. tenella load and oocyst output, as well as cecal lesion and weight loss caused by E. tenella infection, showing moderate anti-E. tenella activity, with an Anticoccidial Index (ACI) of 161.4. Furthermore, we investigated the anti-T. gondii activity of myrcene both in vitro and in vivo. In vitro studies showed that treatment with myrcene effectively inhibited the invasion rate and intracellular proliferation ability of T. gondii tachyzoite in DF-1 cells in a dose-dependent manner. In vivo administration prolonged the survival time in T. gondii-infected mice, suggesting notable protective effects. Additionally, it mitigated T. gondii-induced hepatosplenic toxicity by reducing parasite load in the liver and spleen, and ameliorating liver function as evidenced by decreased serum transaminase levels. In conclusion, the findings demonstrate promising anti-E. tenella and anti-T. gondii activity exhibited by myrcene warranting further exploration into its mechanisms and potential therapeutic applications.

Evaluation of protective efficacy of recombinant Toxoplasma gondii DDX39 protein vaccine against acute and chronic T. gondii infection in mice

Toxoplasma gondii, a pervasive parasite responsible for toxoplasmosis, poses significant health risks to humans and animals. In this study, we investigated the immunogenicity and protective efficacy of the recombinant T. gondii DDX39 protein formulated with ISA201 adjuvant (rTgDDX39) as a candidate vaccine against toxoplasmosis. The full-length of TgDDX39 gene was successfully amplified, cloned into the pET-30a vector, and expressed in BL21 (DE3) competent cells, which was purified and identified as a 57.1 kDa protein via sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Western blot analysis confirmed that rTgDDX39 was specifically recognized by serum from T. gondii-infected mice. Furthermore, immunization of rats with rTgDDX39 generated antiserum that could specifically recognize the native TgDDX39 protein in T. gondii tachyzoite lysates. Immunofluorescence assay revealed that TgDDX39 was primarily located in the nucleus and perinuclear region of tachyzoites. Our vaccination strategy significantly increased T cell proliferation, with CD4+T cells rising by 21.9% and CD8+T cells by 57.8% by the sixth week compared to the adjuvant control group. Additionally, high titers of anti-rTgDDX39 IgG antibodies were detected in vaccinated mice, with a notable induction of IgG1 and IgG2a isotypes, and IgG1/IgG2a > 1 suggests a Th2-biased immune response.Furthermore, in vitro and in vivo assays demonstrated that polyclonal antibodies raised against rTgDDX39 could inhibit the proliferation of T. gondii RH tachyzoites, highlighting the potential of these antibodies to neutralize this parasite effectively. This study provides compelling evidence of the immunogenicity and protective efficacy of rTgDDX39, supporting its potential as a potential candidate vaccine against toxoplasmosis. The protective efficacy of the vaccine was evaluated in mice challenged with acute (RH) and chronic (PRU) strains of T. gondii, showing a survival time extended to 17 days in the acute model, compared to 13.5 and 14 days in the control groups, and a significant 34% reduction in cyst burden in the chronic model. Additionally, the survival rate in the PRU-infected mice increased from 15 to 20% in the control groups to 45% in the vaccinated group. In vitro and in vivo assays demonstrated that polyclonal antibodies raised against rTgDDX39 could inhibit the proliferation of T. gondii RH tachyzoites, highlighting the potential of these antibodies to neutralize the parasite effectively. This study provides compelling evidence of the immunogenicity and protective efficacy of rTgDDX39, supporting its potential as a candidate vaccine against toxoplasmosis.

Toxoplasma gondii Infection of Alzheimer's Disease Mice Reduces Brain Amyloid Density Globally and Regionally.

Toxoplasma gondii infection of Alzheimer's disease model mice decreases amyloid β plaques. We aimed to determine if there is a brain regional difference in amyloid β reduction in the brains of T. gondii-infected compared to control mice. Three-month-old 5xFAD (AD model) mice were injected with T. gondii or with phosphate-buffered saline as a control. Intact brains were harvested at 6 weeks postinfection, optically cleared using iDISCO+, and brain-wide amyloid burden was visualized using volumetric light-sheet imaging. Amyloid signal was quantified across each brain and computationally mapped to the Allen Institute Brain Reference Atlas to determine amyloid density in each region. A brain-wide analysis of amyloid in control and T. gondii-infected 5xFAD mice revealed that T. gondii infection decreased amyloid burden in the brain globally as well as in the cortex and hippocampus, and many daughter regions. Daughter regions that showed reduced amyloid burden included the prelimbic cortex, visual cortex, and retrosplenial cortex. The olfactory tubercle, a region known to have increased monocytes following T. gondii infection, also showed reduced amyloid after infection. T. gondii infection of AD mice reduces amyloid burden in a brain region-specific manner that overlaps with known regions of T. gondii infection and peripheral immune cell infiltration.

Gut microbiota mediates anxiety-like behaviors induced by chronic infection of Toxoplasma gondii in mice

ABSTRACT Background Chronic infection with the neurotropic parasite Toxoplasma gondii (T. gondii) can cause anxiety and gut microbiota dysbiosis in hosts. However, the potential role of gut microbiota in anxiety induced by the parasite remains unclear. Methods C57BL/6J mice were infected with 10 cysts of T. gondii. Antibiotic depletion of gut microbiota and fecal microbiota transplantation experiments were utilized to investigate the causal relationship between gut microbiota and anxiety. Anxiety-like behaviors were examined by the elevated plus maze test and the open field test; blood, feces, colon and amygdala were collected to evaluate the profiles of serum endotoxin (Lipopolysaccharide, LPS) and serotonin (5-hydroxytryptamine, 5-HT), gut microbiota composition, metabolomics, global transcriptome and neuroinflammation in the amygdala. Furthermore, the effects of Diethyl butylmalonate (DBM, an inhibitor of mitochondrial succinate transporter, which causes the accumulation of endogenous succinate) on the disorders of the gut-brain axis were evaluated. Results Here, we found that T. gondii chronic infection induced anxiety-like behaviors and disturbed the composition of the gut microbiota in mice. In the amygdala, T. gondii infection triggered the microglial activation and neuroinflammation. In the colon, T. gondii infection caused the intestinal dyshomeostasis including elevated colonic inflammation, enhanced bacterial endotoxin translocation to blood and compromised intestinal barrier. In the serum, T. gondii infection increased the LPS levels and decreased the 5-HT levels. Interestingly, antibiotics ablation of gut microbiota alleviated the anxiety-like behaviors induced by T. gondii infection. More importantly, transplantation of the fecal microbiota from T. gondii-infected mice resulted in anxiety and the transcriptomic alteration in the amygdala of the antibiotic-pretreated mice. Notably, the decreased abundance of succinate-producing bacteria and the decreased production of succinate were observed in the feces of the T. gondii-infected mice. Moreover, DBM administration ameliorated the anxiety and gut barrier impairment induced by T. gondii infection. Conclusions The present study uncovers a novel role of gut microbiota in mediating the anxiety-like behaviors induced by chronic T. gondii infection. Moreover, we show that DBM supplementation has a beneficial effect on anxiety. Overall, these findings provide new insights into the treatment of T. gondii-related mental disorders.

CCR2-dependent placental migration of inflammatory monocytes suppresses abnormal pregnancies caused by Toxoplasma gondii infection.

Toxoplasma gondii (T. gondii) is a zoonotic protozoan parasite that causes congenital toxoplasmosis, including fetal death, abortion, stillbirth, morphological abnormalities, and premature birth. Primary T. gondii infection in pregnant women results in congenital toxoplasmosis. C-C chemokine receptor (CCR) 2 is reportedly a critical host defense factor against T. gondii infection. However, details of the role of CCR2 in the host immune response to T. gondii in congenital toxoplasmosis remain unclear. Here, we infected pregnant CCR2-deficient mice with T. gondii, resulting in stillbirth, embryonic resorption, fetal morphological abnormalities, and preterm delivery at significantly higher rates than those in pregnant wild-type (WT) mice. Consistent with the severity of abnormal pregnancy, a large area of placental hemorrhage and a large number of T. gondii infections around the hemorrhagic area were observed in the placentas of CCR2-deficient mice. In addition, the accumulation of inflammatory monocytes in the placenta was reduced in CCR2-deficient mice during infection. We further confirmed that the adoptive transfer of inflammatory monocytes collected from WT mice into T. gondii-infected pregnant CCR2-deficient mice effectively suppressed placental damage and abnormal pregnancy. Collectively, CCR2 contributes to pregnancy maintenance by regulating the migration of inflammatory monocytes into the placenta of T. gondii-infected pregnant mice.

The role of microRNA-142a in Toxoplasma gondii infection-induced downregulation of Foxp3: implications for adverse pregnancy outcomes

BackgroundToxoplasma gondii (T. gondii) is capable of infecting nearly all warm-blooded animals and approximately 30% of the global population. Though most infections are subclinical in immunocompetent individuals, congenital contraction can lead to severe consequences such as spontaneous abortion, stillbirth, and a range of cranio-cerebral and/or ocular abnormalities. Previous studies reported that T. gondii-infected pregnancy mice unveiled a deficit in both the amount and suppressive functions of regulatory T (Treg) cells, accompanied with reduced levels of forkhead box p3 (Foxp3). Recently, accumulative studies have demonstrated that microRNAs (miRNAs) are, to some extent, relevant to T. gondii infection. However, the link between alterations in miRNAs and downregulation of Foxp3 triggered by T. gondii has been only sporadically studied.MethodsQuantitative reverse transcription polymerase chain reaction (RT-qPCR), protein blotting and immunofluorescence were employed to evaluate the impact of T. gondii infection and antigens on miRNA transcription and Foxp3 expression. Dual-luciferase reporter gene assays were performed to examine the fluorescence activity in EL4 cells, which were transfected with recombinant plasmids containing full-length/truncated/mutant microRNA-142a-3p (miR-142a) promoter sequence or wild type/mutant of Foxp3 3’ untranslated region (3’ UTR).ResultsWe found a pronounced increase in miR-142a transcription, concurrent with a decrease in Foxp3 expression in T. gondii-infected mouse placental tissue. Similarly, comparable findings have been experimentally confirmed through the treatment of EL4 cells with T. gondii antigens (TgAg) in vitro. Simultaneously, miR-142a mimics attenuated Foxp3 expression, whereas its inhibitors markedly augmented Foxp3 expression. miR-142a promoter activity was elevated upon the stimulation of T. gondii antigens, which mitigated co-transfection of mutant miR-142a promoter lacking P53 target sites. miR-142a mimics deceased the fluorescence activity of Foxp3 3’ untranslated region (3’ UTR), but it did not affect the fluorescence activity upon the co-transfection of mutant Foxp3 3’ UTR lacking miR-142a target site.ConclusionIn both in vivo and in vitro studies, a negative correlation was discovered between Foxp3 expression and miR-142a transcription. TgAg enhanced miR-142a promoter activity to facilitate miR-142a transcription through a P53-dependent mechanism. Furthermore, miR-142a directly targeted Foxp3 3’ UTR, resulting in the downregulation of Foxp3 expression. Therefore, harnessing miR-142a may be a possible therapeutic approach for adverse pregnancy caused by immune imbalances, particularly those induced by T. gondii infection.

Linalool-zinc oxide nanocomposite controls Toxoplasma gondii infection through inhibiting inflammation, oxidative stress, and pathogenicity.

The present in vitro and in vivo study aimed to fabricate and characterize linalool-zinc oxide nanoparticles (Lin-ZNP) and evaluate their effectiveness against Toxoplasma gondii infection in terms of inflammation, oxidative stress, and pathogenicity. Lin-ZNP was synthesized using an ethanolic solution of polyvinyl alcohol. The anti-Toxoplasma and cytotoxicity activities of Lin-ZNP were investigated, along with its effects on nitric oxide (NO) production, caspase-3 activity, and pro-inflammatory genes. After treating T. gondii-infected mice with Lin-ZNP for 14 days, the number and size of tissue cysts, antioxidant potential, pro-inflammatory cytokines, and T. gondii pathogenicity-related genes were evaluated by real-time polymerase chain reaction and Western blot analysis. The Lin-ZNP composite showed a reduced tendency with an average size of 105 nm. Lin-ZNP significantly reduced the viability of tachyzoites. The obtained selectivity index higher than 10, indicating high specificity for parasites with low cytotoxicity to normal cells. The Lin-ZNP significantly (p < 0.05) increased the production of NO, caspase-3 activity, and the expression levels of pro-inflammatory genes. Lin-ZNP significantly (p < 0.001) decreased the size and number of tissue cysts and caused a significant reduction in the level of malondialdehyde and a considerable increase (p < 0.001) in antioxidant enzymes and their expression genes. Lin-ZNP significantly downregulated both mRNA and protein expression of the inflammation-related markers associated with the TLRs/NF-κB pathway. The expression levels of the T. gondii pathogenicity-related genes were significantly downregulated (p < 0.05). The recent survey indicated that Lin-ZNP manages T. gondii infection by its antioxidant activity and inhibiting the TLRs/NF-κB pathway without toxicity in mice.

Inhibition of Foxp3 expression in the placenta of mice infected intraperitoneally by toxoplasma gondii tachyzoites: insights into the PPARγ/miR-7b-5p/Sp1 signaling pathway

BackgroundToxoplasma gondii, an obligate intracellular parasitic protozoa, infects approximately 30% of the global population. Contracting T. gondii at the primary infection of the mother can result in neonatal microcephaly, chorioretinitis, hydrocephalus, or mortality. Our previous study indicated that pregnant mice infected with T. gondii displayed a decrease in both the number and the suppressive ability of regulatory T cells, accompanied by the reduced Forkhead box P3 (Foxp3). Numerous studies have proved that microRNAs (miRNAs) are implicated in T. gondii infection, but there is meager evidence on the relationship between alterations of miRNAs and downregulation of Foxp3 induced by T. gondii.MethodsQuantitative reverse transcription polymerase chain reaction was utilized to detect the transcriptions of miRNAs and Foxp3. Protein blotting and immunofluorescence were used to detect the expressions of Foxp3 and related transcription factors. The structure of mouse placenta was observed by hematoxylin and eosin (HE) staining.To examine the activity of miR-7b promoter and whether miR-7b-5p targets Sp1 to suppress Foxp3 expression, we constructed recombinant plasmids containing the full-length/truncated/mutant miR-7b promoter sequence or wildtype/mutant of Sp1 3' untranslated region (3' UTR) to detect the fluorescence activity in EL4 cells.ResultsIn T. gondii-infected mice, miR-7b transcription was significantly elevated, while Foxp3 expression was decreased in the placenta. In vitro, miR-7b mimics downregulated Foxp3 expression, whereas its inhibitors significantly upregulated Foxp3 expression. miR-7b promoter activity was elevated upon the stimulation of T. gondii antigens, which was mitigated by co-transfection of mutant miR-7b promoter lacking peroxisome proliferator-activated receptor γ (PPARγ) target sites. Additionally, miR-7b mimics diminished Sp1 expression, while miR-7b inhibitors elevated its expression. miR-7b mimics deceased the fluorescence activity of Sp1 3' untranslated region (3' UTR), but it failed to impact the fluorescence activity upon the co-transfection of mutant Sp1 3' UTR lacking miR-7b target site.ConclusionsT. gondii infection and antigens promote miR-7b transcription but inhibit Foxp3 protein and gene levels. T. gondii antigens promote miR-7b promoter activity by a PPARγ-dependent mechanism. miR-7b directly binds to Sp1 3' UTR to repress Sp1 expression. Understanding the regulatory functions by which T. gondii-induced miR-7b suppresses Foxp3 expression can provide new perspectives for the possible therapeutic avenue of T. gondii-induced adverse pregnancy outcomes.Graphical

Immunomodulatory effect of propolis Trigona spp. in mice infected with Toxoplasma gondii.

Toxoplasmosis is a zoonotic disease caused by Toxoplasma gondii. Infection with virulent strains of T. gondii induces excessive production of pro-inflammatory cytokines and increases fatality. Propolis has potential as an anti-inflammatory and immunomodulator. This study aimed to evaluate the pro-inflammatory cytokines response of Interleukin-2 (IL-2) and IL-17, and the anti-inflammatory cytokine IL-10 in mice infected with T. gondii RH strain and treated with ethanol extract of Trigona bee propolis (EEP) from Lombok, West Nusa Tenggara (NTB), Indonesia. A total of 36 mice were divided into six groups with six mice per group. Group 1 was considered as a negative control; mice were not infected and not treated while in groups 2 to 6, mice were infected with 100 T. gondii (tachyzoites). Mice in group 2 were infected but not treated whereas in groups 3, 4, 5, and 6, respectively, were treated orally with Cotrimoxazole 40 mg/kg body weight, EEP 1 mg, EEP 2 mg, and EEP 4 mg, the day after infection for 3 days. All mice under study were euthanized following ethical considerations with prior approvals, on the fifth day post-infection and spleens were taken to observe cytokines using flow cytometry. Statistical analysis was performed with one-way ANOVA and Duncan Multiple Range Test (you may add any statistical tool/software if used?? Like R version?, SPSS, version? If you used any of these/others)? EEP (1-4 mg) increased CD4+ T cells, CD8+ T cells, and CD11b+ cells in infected mice, though not significantly (p > 0.05), unlike cotrimoxazole, which showed significant increases (p < 0.05). EEP at 2 mg and 4 mg (G5 and G6) reduced IL-2 and IL-17 in CD4+ T cells, and at 4 mg (G6) in CD8+ T cells. IL-10 expression increased in CD11b+ cells at 1 mg and in CD4+ T cells at 4 mg (G6). Propolis ethanol extract from Trigona spp. bees acts as an immunomodulator and anti-inflammatory in T. gondii-infected mice, supporting its potential as an anti-toxoplasmosis agent.

Vascular Endothelial Growth Factor-C Treatment Enhances Cerebrospinal Fluid Outflow during Toxoplasma gondii Brain Infection but Does Not Improve Cerebral Edema

Cerebral edema frequently develops in the setting of brain infection and can contribute to elevated intracranial pressure, a medical emergency. How excess fluid is cleared from the brain is not well understood. Previous studies have shown that interstitial fluid is transported out of the brain along perivascular channels that collect into the cerebrospinal fluid (CSF)-filled subarachnoid space. CSF is then removed from the central nervous system through venous and lymphatic routes. The current study tested the hypothesis that increasing lymphatic drainage of CSF would promote clearance of cerebral edema fluid during infection with the neurotropic parasite Toxoplasma gondii. Fluorescent microscopy and magnetic resonance imaging was used to show that C57BL/6 mice develop vasogenic edema 4 to 5 weeks after infection with T. gondii. Tracer experiments were used to evaluate how brain infection affects meningeal lymphatic function, which demonstrated a decreased rate in CSF outflow in T. gondii-infected mice. Next, mice were treated with a vascular endothelial growth factor (VEGF)-C-expressing viral vector, which induced meningeal lymphangiogenesis and improved CSF outflow in chronically infected mice. No difference in cerebral edema was observed between mice that received VEGF-C and those that rececived sham treatment. Therefore, although VEGF-C treatment can improve lymphatic outflow in mice infected with T. gondii, this effect does not lead to increased clearance of edema fluid from the brains of these mice.

Anti- Toxoplasma gondii Properties of Ginseng polysaccharides and saponins.

New anti- Toxoplasma gondii agents are in demand due to the emergence of high toxicity. Ginseng polysaccharides and saponins can be used to treat the replication of Toxoplasma gondii in an attempt to determine whether the medicinal uses of ginseng are supported by pharmacological effects. Anti- Toxoplasma gondii activities of ginseng polysaccharides and saponins were examined in vitro and in vivo. The findings are the survival time and rate of Toxoplasma gondii infected mice after the intake of the total polysaccharides and saponins increased compared to untreated control mice. The survival rate of mice treated with ginseng saponins was the highest at 83.3%, the phenomenon of splenomegaly of mice was decreased especially ( p < 0.05) treated with ginseng polysaccharides. Accordingly, ginseng polysaccharides and saponins have a potential application in anti-Toxoplasma gondii treatments.

Brain-localized CD4 and CD8 T cells perform correlated random walks and not Levy walks.

Background. For survival of the organism, T cells must efficiently control pathogens invading different peripheral tissues. Whether or not such control is achieved by utilizing different movement strategies in different tissues remains poorly understood. Liver-localized CD8 T cells perform correlated random walks --- a type of a Brownian walk -- in liver sinusoids but in some condition these T cells may also perform Levy flights -- rapid and large displacements by floating with the blood flow. CD8 T cells in lymph nodes or skin also undergo Brownian walks. A recent study suggested that brain-localized CD8 T cells, specific to Toxoplasma gondii, perform generalized Levy walks -- a walk type in which T cells alternate pausing and displacing long distances --- which may indicate that brain is a unique organ where T cells exhibit movement strategies different from other tissues. Methods. We quantified movement patterns of brain-localized Plasmodium berghei-specific CD4 and CD8 T cells by using well-established statistical and computational methods. Results. We found that T cells change their movement pattern with time since infection and that CD4 T cells move faster and turn less than CD8 T cells. Importantly, both CD4 and CD8 T cells move in the brain by correlated random walks without long displacements challenging previous observations. We have also re-analyzed the movement data of brain-localized CD8 T cells in T. gondii-infected mice and found no evidence of Levy walks. We hypothesize that the previous conclusion of Levy walks of T. gondii-specific CD8 T cells in the brain was reached due to missing time-frames in the data that create an impression of large movement lengths between assumed-to-be-sequential movements. Conclusion. Our results suggests that movement strategies of CD8 T cells are largely similar between LNs, liver, and the brain and consistent with correlated random walks and not Levy walks.

Toxoplasma gondii Induces Pyroptosis in Human Placental Trophoblast and Amniotic Cells by Inducing ROS Production and Activation of Cathepsin B and NLRP1/NLRP3/NLRC4/AIM2 Inflammasome

Toxoplasma gondii infection in pregnant women may cause fetal anomalies; however, the underlying mechanisms remain unclear. The current study investigated whether T. gondii induces pyroptosis in human placental cells and the underlying mechanisms. Human placental trophoblast (BeWo and HTR-8/SVneo) and amniotic (WISH) cells were infected with T. gondii, and then reactive oxygen species (ROS) production, cathepsin B (CatB) release, inflammasome activation, and pyroptosis induction were evaluated. The molecular mechanisms of these effects were investigated by treating the cells with ROS scavengers, a CatB inhibitor, or inflammasome-specific siRNA. T. gondii infection induced ROS generation and CatB release into the cytosol in placental cells but decreased mitochondrial membrane potential. T. gondii-infected human placental cells and villi exhibited NLRP1, NLRP3, NLRC4, and AIM2 inflammasome activation and subsequent pyroptosis induction, as evidenced by increased expression of ASC, cleaved caspase-1, and mature IL-1β and gasdermin D cleavage. In addition to inflammasome activation and pyroptosis induction, adverse pregnancy outcome was shown in a T. gondii-infected pregnant mouse model. Administration of ROS scavengers, CatB inhibitor, or inflammasome-specific siRNA into T. gondii-infected cells reversed these effects. Collectively, these findings show that T. gondii induces NLRP1/NLRP3/NLRC4/AIM2 inflammasome-dependent caspase-1-mediated pyroptosis via induction of ROS production and CatB activation in placental cells. This mechanism may play an important role in inducing cell injury in congenital toxoplasmosis.

CRISPR/Cas12a combined with RPA for detection of T. gondii in mouse whole blood

BackgroundToxoplasma gondii is an opportunistic protozoan that is ubiquitous in humans and animals. It can invade any human organ and cause severe diseases, including toxoplasma ophthalmopathy, meningoencephalitis, and liver necrosis. Porcine toxoplasmosis is prevalent in China. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and Cas (CRISPR-Associated Protein) systems are widely used for gene editing and pathogen detection. CRISPR-based diagnostics are molecular assays that have been developed to detect parasites with high sensitivity and specificity.MethodsThis study aimed to establish a combined CRISPR/Cas12a and RPA rapid detection method for T. gondii by targeting the B1 gene and 529 bp repeat element (529 RE). The detection results could be visualized by the fluorescence or lateral flow strips (LFS). The sensitivity and specificity of the method were evaluated, and T. gondii-infected mouse blood was used for detection.ResultsThe results indicated that the established method for T. gondii detection was satisfactory, with a detection limit of 1.5 cp/μl for the two loci. Moreover, the B1 gene could detect 1 tachyzoite per reaction, and the 529 RE could detect 0.1 tachyzoite per reaction, consistently with the highly sensitive nested polymerase chain reaction (PCR) results. The method was suitable for strains, including RH, and did not cross-react with other protozoa DNA with similar habits. The T. gondii-infected mouse blood samples were all positive for T. gondii at 1, 3, and 5 days post infection (dpi).ConclusionsThis study established a rapid, sensitive, and time-saving DNA detection method for T. gondii that has the potential to be an alternative tool for T. gondii detection in the field.Graphical abstract

ScDual-Seq of Toxoplasma gondii-infected mouse BMDCs reveals heterogeneity and differential infection dynamics.

Dendritic cells and macrophages are integral parts of the innate immune system and gatekeepers against infection. The protozoan pathogen, Toxoplasma gondii, is known to hijack host immune cells and modulate their immune response, making it a compelling model to study host-pathogen interactions. Here we utilize single cell Dual RNA-seq to parse out heterogeneous transcription of mouse bone marrow-derived dendritic cells (BMDCs) infected with two distinct genotypes of T. gondii parasites, over multiple time points post infection. We show that the BMDCs elicit differential responses towards T. gondii infection and that the two parasite lineages distinctly manipulate subpopulations of infected BMDCs. Co-expression networks define host and parasite genes, with implications for modulation of host immunity. Integrative analysis validates previously established immune pathways and additionally, suggests novel candidate genes involved in host-pathogen interactions. Altogether, this study provides a comprehensive resource for characterizing host-pathogen interplay at high-resolution.

Dimethyl itaconate ameliorates the deficits of goal-directed behavior in Toxoplasma gondii infected mice.

The neurotrophic parasite Toxoplasma gondii (T. gondii) has been implicated as a risk factor for neurodegenerative diseases. However, there is only limited information concerning its underlying mechanism and therapeutic strategy. Here, we investigated the effects of T. gondii chronic infection on the goal-directed cognitive behavior in mice. Moreover, we evaluated the preventive and therapeutic effect of dimethyl itaconate on the behavior deficits induced by the parasite. The infection model was established by orally infecting the cysts of T. gondii. Dimethyl itaconate was intraperitoneally administered before or after the infection. Y-maze and temporal order memory (TOM) tests were used to evaluate the prefrontal cortex-dependent behavior performance. Golgi staining, transmission electron microscopy, indirect immunofluorescence, western blot, and RNA sequencing were utilized to determine the pathological changes in the prefrontal cortex of mice. We showed that T. gondii infection impaired the prefrontal cortex-dependent goal-directed behavior. The infection significantly downregulated the expression of the genes associated with synaptic transmission, plasticity, and cognitive behavior in the prefrontal cortex of mice. On the contrary, the infection robustly upregulated the expression of activation makers of microglia and astrocytes. In addition, the metabolic phenotype of the prefrontal cortex post infection was characterized by the enhancement of glycolysis and fatty acid oxidation, the blockage of the Krebs cycle, and the disorder of aconitate decarboxylase 1 (ACOD1)-itaconate axis. Notably, the administration of dimethyl itaconate significantly prevented and treated the cognitive impairment induced by T. gondii, which was evidenced by the improvement of behavioral deficits, synaptic ultrastructure lesion and neuroinflammation. The present study demonstrates that T. gondii infection induces the deficits of the goal-directed behavior, which is associated with neuroinflammation, the impairment of synaptic ultrastructure, and the metabolic shifts in the prefrontal cortex of mice. Moreover, we report that dimethyl itaconate has the potential to prevent and treat the behavior deficits.

Novel insights on the therapeutic effect of levamisole on the chronic toxoplasmosis in mice model.

Seventy-five Me49 Toxoplasma gondii infected Swiss albino mice were divided into five groups; (GI): noninfected control group; (GII): infected untreated control group; (GIII): infected- LEVA treated group; (GIV): infected and received combination of spiramycin and LEVA and (GV): infected-spiramycin treated group. The impact was assessed through brain cyst count by Quantitative Real-Time Polymerase Chain Reaction (PCR), interferon gamma (IFN-γ) assay, histopathological study, and total blood counts. The progression of chronic toxoplasmosis could only be partially controlled by using either levamisole or spiramycin as a separate drug. The combined spiramycin and levamisole treatment significantly decreased the burden of Toxoplasma brain cyst, increased IFN-γ level, total blood parameters and improved the histopathological features especially at the late stage of infection. Levamisole effectively modulated Toxoplasma-induced immune responses, resulting in chronic toxoplasmosis remission. Further clinical trials will be needed to study the effect of these combination in HIV/AIDS (human immunodeficiency virus) patients with toxoplasmosis.

CD8 +T cell recognition of neuronal cysts during chronic Toxoplasma gondiiinfection

Abstract The parasite Toxoplasma gondiiestablishes persistent infection within neurons by forming cysts that harbor latent form bradyzoites. The ability to form cysts is dependent on the parasite transcription factor BFD1 and, given the presence of a thick cyst wall, it was previously thought that cysts were protected from immune recognition. However, the generation of a BAG1-OVA parasite line, in which OVA protein expression is restricted to bradyzoites, revealed that in vivo infection elicited the activation and expansion of OVA-specific OT-I CD8 +T cells. To determine whether endogenous CD8 +T cells can recognize natural cyst-derived antigens and potentially participate in cyst control, in vitro T cell recognition assays were performed using a BFD1-inducible line of parasites that form in vitro cysts. In these assays, CD8 +T cells isolated from T. gondii-infected mice recognized both infected murine fibroblasts and neurons containing cysts. These studies indicate that cyst-specific T cells are present during infection and future studies will explore if any parasite antigens transported beyond the cyst wall can be presented by neurons for T cell recognition. Supported by NIH grant R01 AI157247-01

Potent anti-Toxoplasma gondii activity of 4-chlorophenylthioacetone-derived thiosemicarbazones: Involvement of CCR2 and CCR5 receptors and 5-lipoxygenase in the mode of action

Toxoplasmosis is a disease requiring therapeutic innovation, and thiosemicarbazones with antimicrobial activity are candidates to control Toxoplasma gondii infection. Here, the anti-T. gondii activities of (E)-2-(1-(4-chlorophenylthio)propan-2-ylidene)-hydrazinecarbothioamides (Ca and Cb) were investigated. T. gondii-infected macrophages (MOs) or glial cells treated with Ca or Cb showed a decrease in the number of intracellular parasites. A deficiency in the chemokine receptor CCR2, but not CCR5, partially reduced anti-T. gondii activity induced by Ca or Cb. In contrast, a deficiency in 5-lipoxygenase (5-LO) activity potentiated anti-T. gondii activities induced by these compounds. In vivo treatment with Ca increased the survival of T. gondii-infected wild-type mice, and this was associated with increased IFN-γ and IL-12 production. A deficiency in CCR5 or CCR2 abolished the protective effect of Ca treatment in vivo, while a deficiency in 5-LO increased Cb anti-T. gondii effects. Collectively, our data suggest that Ca and Cb are potential therapeutic candidates for the treatment of toxoplasmosis.