Single-cell transcriptomic analysis of decidual immune cell landscape in the occurrence of adverse pregnancy outcomes induced by Toxoplasma gondii infection.

BACKGROUND: Toxoplasma gondii is an obligate intracellular parasite that can lead to adverse pregnancy outcomes, particularly in early pregnancy. Previous studies have illustrated the landscape of decidual immune cells. However, the landscape of decidual immune cells in the maternal-fetal microenvironment during T. gondii infection remains unknown. METHODS: In this study, we employed single-cell RNA sequencing to analyze the changes in human decidual immune cells following T. gondii infection. The results of scRNA-seq were further validated with flow cytometry, reverse transcription-polymerase chain reaction, western blot, and immunofluorescence staining. RESULTS: Our results showed that the proportion of 17 decidual immune cell clusters and the expression levels of 21 genes were changed after T. gondii infection. Differential gene analysis demonstrated that T. gondii infection induced the differential expression of 279, 312, and 380 genes in decidual NK cells (dNK), decidual macrophages (dMφ), and decidual T cells (dT), respectively. Our results revealed for the first time that several previously unknown molecules in decidual immune cells changed following infection. This result revealed that the function of maternal-fetal immune tolerance declined, whereas the killing ability of decidual immune cells enhanced, eventually contributing to the occurrence of adverse pregnancy outcomes. CONCLUSIONS: This study provides valuable resource for uncovering several novel molecules that play an important role in the occurrence of abnormal pregnancy outcomes induced by T. gondii infection.

A CTCF-binding site in the Mdm1-Il22-Ifng locus shapes cytokine expression profiles and plays a critical role in early Th1 cell fate specification.

Cytokine expression during T cell differentiation is a highly regulated process that involves long-range promoter-enhancer and CTCF-CTCF contacts at cytokine loci. Here, we investigated the impact of dynamic chromatin loop formation within the topologically associating domain (TAD) in regulating the expression of interferon gamma (IFN-γ) and interleukin-22 (IL-22); these cytokine loci are closely located in the genome and are associated with complex enhancer landscapes, which are selectively active in type 1 and type 3 lymphocytes. In situ Hi-C analyses revealed inducible TADs that insulated Ifng and Il22 enhancers during Th1 cell differentiation. Targeted deletion of a 17 bp boundary motif of these TADs imbalanced Th1- and Th17-associated immunity, both in vitro and in vivo, upon Toxoplasma gondii infection. In contrast, this boundary element was dispensable for cytokine regulation in natural killer cells. Our findings suggest that precise cytokine regulation relies on lineage- and developmental stage-specific interactions of 3D chromatin architectures and enhancer landscapes.

Fam96a is essential for the host control of Toxoplasma gondii infection by fine-tuning macrophage polarization via an iron-dependent mechanism.

BACKGROUND: Toxoplasmosis affects a quarter of the world's population. Toxoplasma gondii (T.gondii) is an intracellular parasitic protozoa. Macrophages are necessary for proliferation and spread of T.gondii by regulating immunity and metabolism. Family with sequence similarity 96A (Fam96a; formally named Ciao2a) is an evolutionarily conserved protein that is highly expressed in macrophages, but whether it play a role in control of T. gondii infection is unknown. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we utilized myeloid cell-specific knockout mice to test its role in anti-T. gondii immunity. The results showed that myeloid cell-specific deletion of Fam96a led to exacerbate both acute and chronic toxoplasmosis after exposure to T. gondii. This was related to a defectively reprogrammed polarization in Fam96a-deficient macrophages inhibited the induction of immune effector molecules, including iNOS, by suppressing interferon/STAT1 signaling. Fam96a regulated macrophage polarization process was in part dependent on its ability to fine-tuning intracellular iron (Fe) homeostasis in response to inflammatory stimuli. In addition, Fam96a regulated the mitochondrial oxidative phosphorylation or related events that involved in control of T. gondii. CONCLUSIONS/SIGNIFICANCE: All these findings suggest that Fam96a ablation in macrophages disrupts iron homeostasis and inhibits immune effector molecules, which may aggravate both acute and chronic toxoplasmosis. It highlights that Fam96a may autonomously act as a critical gatekeeper of T. gondii control in macrophages.

Observing astrocyte polarization in brains from mouse chronically infected with Toxoplasma gondii.

Toxoplasma gondii (T. gondii) is a protozoan parasite that infects approximately one-third of the global human population, often leading to chronic infection. While acute T. gondii infection can cause neural damage in the central nervous system and result in toxoplasmic encephalitis, the consequences of T. gondii chronic infection (TCI) are generally asymptomatic. However, emerging evidence suggests that TCI may be linked to behavioral changes or mental disorders in hosts. Astrocyte polarization, particularly the A1 subtype associated with neuronal apoptosis, has been identified in various neurodegenerative diseases. Nevertheless, the role of astrocyte polarization in TCI still needs to be better understood. This study aimed to establish a mouse model of chronic TCI and examine the transcription and expression levels of glial fibrillary acidic protein (GFAP), C3, C1q, IL-1α, and TNF-α in the brain tissues of the mice. Quantitative real-time PCR (qRT-PCR), enzyme-linked immunosorbent assay, and Western blotting were employed to assess these levels. Additionally, the expression level of the A1 astrocyte-specific marker C3 was evaluated using indirect fluorescent assay (IFA). In mice with TCI, the transcriptional and expression levels of the inflammatory factors C1q, IL-1α, and TNF-α followed an up-down-up pattern, although they remained elevated compared to the control group. These findings suggest a potential association between astrocyte polarization towards the A1 subtype and synchronized changes in these three inflammatory mediators. Furthermore, immunofluorescence assay (IFA) revealed a significant increase in the A1 astrocytes (GFAP+C3+) proportion in TCI mice. This study provides evidence that TCI can induce astrocyte polarization, a biological process that may be influenced by changes in the levels of three inflammatory factors: C1q, IL-1α, and TNF-α. Additionally, the release of neurotoxic substances by A1 astrocytes may be associated with the development of TCI.

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

BACKGROUND: Toxoplasma 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. METHODS: Quantitative 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). RESULTS: We 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. CONCLUSION: In 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.

Phosphoproteomic analysis reveals changes in A-Raf-related protein phosphorylation in response to Toxoplasma gondii infection in porcine macrophages.

BACKGROUND: Toxoplasma gondii is an obligate intracellular protozoan parasite that causes severe threats to humans and livestock. Macrophages are the cell type preferentially infected by T. gondii in vivo. Protein phosphorylation is an important posttranslational modification involved in diverse cellular functions. A rapidly accelerated fibrosarcoma kinase (A-Raf) is a member of the Raf family of serine/threonine protein kinases that is necessary for MAPK activation. Our previous research found that knockout of A-Raf could reduce T. gondii-induced apoptosis in porcine alveolar macrophages (3D4/21 cells). However, limited information is available on protein phosphorylation variations and the role of A-Raf in macrophages infected with T. gondii. METHODS: We used immobilized metal affinity chromatography (IMAC) in combination with liquid chromatography tandem mass spectrometry (LC-MS/MS) to profile changes in phosphorylation in T. gondii-infected 3D4/21 and 3D4/21-ΔAraf cells. RESULTS: A total of 1647 differentially expressed phosphorylated proteins (DEPPs) with 3876 differentially phosphorylated sites (DPSs) were identified in T. gondii-infected 3D4/21 cells (p3T group) when compared with uninfected 3D4/21 cells (pho3 group), and 959 DEPPs with 1540 DPSs were identified in the p3T group compared with infected 3D4/21-ΔAraf cells (p3KT group). Venn analysis revealed 552 DPSs corresponding to 406 DEPPs with the same phosphorylated sites when comparing p3T/pho3 versus p3T/p3KT, which were identified as DPSs and DEPPs that were directly or indirectly related to A-Raf. CONCLUSIONS: Our results revealed distinct responses of macrophages to T. gondii infection and the potential roles of A-Raf in fighting infection via phosphorylation of crucial proteins.

IL-12 Mediates T-bet-Expressing Myeloid Cell-Dependent Host Resistance against Toxoplasma gondii.

To defend against intracellular pathogens such as Toxoplasma gondii, the host generates a robust type 1 immune response. Specifically, host defense against T. gondii is defined by an IL-12-dependent IFN-γ response that is critical for host resistance. Previously, we demonstrated that host resistance is mediated by T-bet-dependent ILC-derived IFN-γ by maintaining IRF8+ conventional type 1 dendritic cells during parasitic infection. Therefore, we hypothesized that innate lymphoid cells are indispensable for host survival. Surprisingly, we observed that T-bet-deficient mice succumb to infection quicker than do mice lacking lymphocytes, suggesting an unknown T-bet-dependent-mediated host defense pathway. Analysis of parasite-mediated inflammatory myeloid cells revealed a novel subpopulation of T-bet+ myeloid cells (TMCs). Our results reveal that TMCs have the largest intracellular parasite burden compared with other professional phagocytes, suggesting they are associated with active killing of T. gondii. Mechanistically, we established that IL-12 is necessary for the induction of inflammatory TMCs during infection and these cells are linked to a role in host survival.

Toxoplasma gondii infection and testosterone alteration: A systematic review and meta-analyses.

BACKGROUND: Toxoplasma gondii (T. gondii) is a worldwide distributed protozoan parasite which has infected a wide range of warm-blooded animals and humans. The most common form of T. gondii infection is asymptomatic (latent); nevertheless, latent toxoplasmosis can induce various alterations of sex hormones, especially testosterone, in infected humans and animals. On the other hand, testosterone is involved in behavioral traits and reproductive functions in both sexes. Hence, the purpose of this systematic review is to summarize the available evidence regarding the association between T. gondii infection and testosterone alteration. METHODS: In the setting of a systematic review, an electronic search (any date to 10 January 2023) without language restrictions was performed using Science Direct, Web of Science, PubMed, Scopus, and Google Scholar. The PRISMA guidelines were followed. Following the initial search, a total of 12,306 titles and abstracts were screened initially; 12,281 were excluded due to the lack of eligibility criteria or duplication. Finally, 24 articles met the included criteria. A mean±standard deviation (SD) was calculated to assess the difference of testosterone between T. gondii positive and T. gondii negative humans. The possibility of publication bias was assessed using Egger's regression. P-value < 0.05 was considered statistically significant. RESULTS: This systematic review identified 24 articles (18 studies in humans and six studies in animals). Most human studies (13 out of 19) reported an increased level of testosterone following latent toxoplasmosis in males, while three studies reported decreased levels and two studies reported an insignificant change. Eleven articles (seven datasets in males and seven datasets in females) were eligible to be included in the data synthesis. Based on the random-effects model, the pooled mean± SD of testosterone in T. gondii positive than T. gondii negative was increased by 0.73 and 0.55 units in males and females, respectively. The Egger's regression did not detect a statistically significant publication bias in males and females (p = value = 0.95 and 0.71), respectively. Three studies in male animals (rats, mice, and spotted hyenas) and two studies in female animals (mice and spotted hyenas) reported a decline in testosterone in infected compared with non-infected animals. While, one study in female rats reported no significant changes of testosterone in infected than non-infected animals. Moreover, two studies in male rats reported an increased level of testosterone in infected than non-infected animals. CONCLUSIONS: This study provides new insights about the association between T. gondii infection and testosterone alteration and identifies relevant data gaps that can inform and encourage further studies. The consequence of increased testosterone levels following T. gondii infection could partly be associated with increased sexual behavior and sexual transmission of the parasite. On the other hand, declining testosterone levels following T. gondii infection may be associated with male reproductive impairments, which were observed in T. gondii-infected humans and animals. Furthermore, these findings suggest the great need for more epidemiological and experimental investigations in depth to understand the relationship between T. gondii infection and testosterone alteration alongside with future consequences of testosterone alteration.

Toxoplasma gondii modulates the host cell cycle, chromosome segregation, and cytokinesis irrespective of cell type or species origin.

BACKGROUND: Toxoplasma gondii is an apicomplexan intracellular obligate parasite and the etiological agent of toxoplasmosis in humans, domestic animals and wildlife, causing miscarriages and negatively impacting offspring. During its intracellular development, it relies on nutrients from the host cell, controlling several pathways and the cytoskeleton. T. gondii has been proven to control the host cell cycle, mitosis and cytokinesis, depending on the time of infection and the origin of the host cell. However, no data from parallel infection studies have been collected. Given that T. gondii can infect virtually any nucleated cell, including those of humans and animals, understanding the mechanism by which it infects or develops inside the host cell is essential for disease prevention. Therefore, we aimed here to reveal whether this modulation is dependent on a specific cell type or host cell species. METHODS: We used only primary cells from humans and bovines at a maximum of four passages to ensure that all cells were counted with appropriate cell cycle checkpoint control. The cell cycle progression was analysed using fluorescence-activated cell sorting (FACS)-based DNA quantification, and its regulation was followed by the quantification of cyclin B1 (mitosis checkpoint protein). The results demonstrated that all studied host cells except bovine colonic epithelial cells (BCEC) were arrested in the S-phase, and none of them were affected in cyclin B1 expression. Additionally, we used an immunofluorescence assay to track mitosis and cytokinesis in uninfected and T. gondii-infected cells. RESULTS: The results demonstrated that all studied host cell except bovine colonic epithelial cells (BCEC) were arrested in the S-phase, and none of them were affected in cyclin B1 expression. Our findings showed that the analysed cells developed chromosome segregation problems and failed to complete cytokinesis. Also, the number of centrosomes per mitotic pole was increased after infection in all cell types. Therefore, our data suggest that T. gondii modulates the host cell cycle, chromosome segregation and cytokinesis during infection or development regardless of the host cell origin or type.

Toxoplasma gondii Infections and Associated Factors in Female Children and Adolescents, Germany.

In a representative sample of female children and adolescents in Germany, Toxoplasma gondii seroprevalence was 6.3% (95% CI 4.7%-8.0%). With each year of life, the chance of being seropositive increased by 1.2, indicating a strong force of infection. Social status and municipality size were found to be associated with seropositivity.

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.

BACKGROUND: Toxoplasma 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. METHODS: Quantitative 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. RESULTS: In 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. CONCLUSIONS: T. 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.

The acyl-CoA synthetase TgACS1 allows neutral lipid metabolism and extracellular motility in Toxoplasma gondii through relocation via its peroxisomal targeting sequence (PTS) under low nutrient conditions.

Apicomplexa parasites cause major diseases such as toxoplasmosis and malaria that have major health and economic burdens. These unicellular pathogens are obligate intracellular parasites that heavily depend on lipid metabolism for the survival within their hosts. Their lipid synthesis relies on an essential combination of fatty acids (FAs) obtained from both de novo synthesis and scavenging from the host. The constant flux of scavenged FA needs to be channeled toward parasite lipid storage, and these FA storages are timely mobilized during parasite division. In eukaryotes, the utilization of FA relies on their obligate metabolic activation mediated by acyl-co-enzyme A (CoA) synthases (ACSs), which catalyze the thioesterification of FA to a CoA. Besides the essential functions of FA for parasite survival, the presence and roles of ACS are yet to be determined in Apicomplexa. Here, we identified TgACS1 as a Toxoplasma gondii cytosolic ACS that is involved in FA mobilization in the parasite specifically during low host nutrient conditions, especially in extracellular stages where it adopts a different localization. Heterologous complementation of yeast ACS mutants confirmed TgACS1 as being an Acyl-CoA synthetase of the bubble gum family that is most likely involved in ß-oxidation processes. We further demonstrate that TgACS1 is critical for gliding motility of extracellular parasite facing low nutrient conditions, by relocating to peroxisomal-like area.IMPORTANCEToxoplasma gondii, causing human toxoplasmosis, is an Apicomplexa parasite and model within this phylum that hosts major infectious agents, such as Plasmodium spp., responsible for malaria. The diseases caused by apicomplexans are responsible for major social and economic burdens affecting hundreds of millions of people, like toxoplasmosis chronically present in about one-third of the world's population. Lack of efficient vaccines, rapid emergence of resistance to existing treatments, and toxic side effects of current treatments all argue for the urgent need to develop new therapeutic tools to combat these diseases. Understanding the key metabolic pathways sustaining host-intracellular parasite interactions is pivotal to develop new efficient ways to kill these parasites. Current consensus supports parasite lipid synthesis and trafficking as pertinent target for novel treatments. Many processes of this essential lipid metabolism in the parasite are not fully understood. The capacity for the parasites to sense and metabolically adapt to the host physiological conditions has only recently been unraveled. Our results clearly indicate the role of acyl-co-enzyme A (CoA) synthetases for the essential metabolic activation of fatty acid (FA) used to maintain parasite propagation and survival. The significance of our research is (i) the identification of seven of these enzymes that localize at different cellular areas in T. gondii parasites; (ii) using lipidomic approaches, we show that TgACS1 mobilizes FA under low host nutrient content; (iii) yeast complementation showed that acyl-CoA synthase 1 (ACS1) is an ACS that is likely involved in peroxisomal ß-oxidation; (iv) the importance of the peroxisomal targeting sequence for correct localization of TgACS1 to a peroxisomal-like compartment in extracellular parasites; and lastly, (v) that TgACS1 has a crucial role in energy production and extracellular parasite motility.

Multicenter evaluation of the Toxoplasma gondii Real-TM (Sacace) kit performance for the molecular diagnosis of toxoplasmosis.

The molecular detection of Toxoplasma gondii DNA is a key tool for the diagnosis of disseminated and congenital toxoplasmosis. This multicentric study from the Molecular Biology Pole of the French National Reference Center for toxoplasmosis aimed to evaluate Toxoplasma gondii Real-TM PCR kit (Sacace). The study compared the analytical and clinical performances of this PCR assay with the reference PCRs used in proficient laboratories. PCR efficiencies varied from 90% to 112%; linearity zone extended over four log units (R2 > 0.99) and limit of detection varied from 0.01 to ≤1 Tg/mL depending on the center. Determined on 173 cryopreserved DNAs from a large range of clinical specimens, clinical sensitivity was 100% [106/106; 95 confidence interval (CI): 96.5%-100%] and specificity was 100% (67/67; 95 CI: 94.6%-100%). The study revealed two potential limitations of the Sacace PCR assay: the first was the inconsistency of the internal control (IC) when added to the PCR mixture. This point was not found under routine conditions when the IC was added during the extraction step. The second is a lack of practicality, as the mixture is distributed over several vials, requiring numerous pipetting operations. Overall, this study provides useful information for the molecular diagnosis of toxoplasmosis; the analytical and clinical performances of the Sacace PCR kit were satisfactory, the kit having sensitivity and specificity similar to those of expert center methods and being able to detect low parasite loads, at levels where multiplicative analysis gives inconsistently positive results. Finally, the study recommends multiplicative analysis in particular for amniotic fluids, aqueous humor, and other single specimens.

Toxoplasma gondii infection-induced host cellular DNA damage is strain-dependent and leads to the activation of the ATM-dependent homologous recombination pathway.

Toxoplasma gondii is a globally occurring apicomplexan parasite that infects humans and animals. Globally, different typical and atypical haplotypes of T. gondii induce varying pathologies in hosts. As an obligate intracellular protozoon, T. gondii was shown to interfere with host cell cycle progression, leading to mitotic spindle alteration, chromosome segregation errors and cytokinesis failure which all may reflect chromosomal instability. Referring to strain-dependent virulence, we here studied the potential of different T. gondii strains (RH, Me49 and NED) to drive DNA damage in primary endothelial host cells. Utilizing microscopic analyses, comet assays and γ-H2AX quantification, we demonstrated a strain-dependent induction of binucleated host cells, DNA damage and DNA double strand breaks, respectively, in T. gondii-infected cells with the RH strain driving the most prominent effects. Interestingly, only the NED strain significantly triggered micronuclei formation in T. gondii-infected cells. Focusing on the RH strain, we furthermore demonstrated that T. gondii-infected primary host cells showed a DNA damage response by activating the ATM-dependent homologous recombination (HR) pathway. In contrast, key molecules of the nonhomologous DNA end joining (NHEJ) pathway were either not affected or downregulated in RH-infected host cells, suggesting that this pathway is not activated by infection. In conclusion, current finding suggests that T. gondii infection affects the host cell genome integrity in a strain-dependent manner by causing DNA damage and chromosomal instability.

Is GRA6 Gene a Suitable Marker for Molecular Typing of Toxoplasma Gondii? A Scoping Systematic Review.

Toxoplasmosis is a zoonotic disease with a worldwide prevalence that is caused by Toxoplasma gondii. This study aimed to summarize available data on genotyping T. gondii strains based on the GRA6 gene marker in different hosts around the world. We conducted a comprehensive literature search using five international databases (PubMed, Scopus, Science Direct, Web of Science, and Google Scholar) from inception until December 2021. We identified 32 papers eligible for inclusion in this systematic review. The majority of studies (50%) were carried out in Iran (n = 16) to identify T. gondii genotypes based on the GRA6 gene. Other countries with reported studies include China, Japan, Sweden, and Italy (n = 2 each). Out of 3,434 samples collected from various hosts, most studies (n = 11) focused on human samples (34.4%), followed by ovine (n = 7), pig (n = 4), goat (n = 3) and soil and cattle (n = 2).Using various molecular methods such as conventional PCR, nested-PCR, real-time PCR, microsatellite analysis, and Restriction Fragment Length Polymorphism (RFLP), we found DNA positive results in 805 out of 3,434 samples. Of these, 285 (35.40%), 207 (25.71%), 182 (22.60%), 65 (8.07%), and 18 (2.23%) were infected with types I, II, III, mix I, II, III, and mix II, III, respectively. Our data demonstrate that the GRA6 gene marker has sufficient polymorphism to detect three types of T. gondii genotypes in various hosts. Identifying the specific genotype could be valuable in developing new strategies for treatment, vaccination, diagnosis, control, and prevention of T. gondii infection.

Anti-Toxoplasma gondii effect of tylosin in vitro and in vivo.

BACKGROUND: Toxoplasma gondii is an important protozoan pathogen with medical and veterinary importance worldwide. Drugs currently used for treatment of toxoplasmosis are less effective and sometimes cause serious side effects. There is an urgent need for the development of more effective drugs with relatively low toxicity. METHODS: The effect of tylosin on the viability of host cells was measured using CCK8 assays. To assess the inhibition of tylosin on T. gondii proliferation, a real-time PCR targeting the B1 gene was developed for T. gondii detection and quantification. Total RNA was extracted from parasites treated with tylosin and then subjected to transcriptome analysis by RNA sequencing (RNA-seq). Finally, murine infection models of toxoplasmosis were used to evaluate the protective efficacy of tylosin against T. gondii virulent RH strain or avirulent ME49 strain. RESULTS: We found that tylosin displayed low host toxicity, and its 50% inhibitory concentration was 175.3 µM. Tylsoin also inhibited intracellular T. gondii tachyzoite proliferation, with a 50% effective concentration of 9.759 µM. Transcriptome analysis showed that tylosin remarkably perturbed the gene expression of T. gondii, and genes involved in "ribosome biogenesis (GO:0042254)" and "ribosome (GO:0005840)" were significantly dys-regulated. In a murine model, tylosin treatment alone (100 mg/kg, i.p.) or in combination with sulfadiazine sodium (200 mg/kg, i.g.) significantly prolonged the survival time and raised the survival rate of animals infected with T. gondii virulent RH or avirulent ME49 strain. Meanwhile, treatment with tylosin significantly decreased the parasite burdens in multiple organs and decreased the spleen index of mice with acute toxoplasmosis. CONCLUSIONS: Our findings suggest that tylosin exhibited potency against T. gondii both in vitro and in vivo, which offers promise for treatment of human toxoplasmosis.

Endogenous IL-27 during toxoplasmosis limits early monocyte responses and their inflammatory activation by pathological T cells.

Mice that lack the genes for IL-27, or the IL-27 receptor, and infected with Toxoplasma gondii develop T cell-mediated pathology. Here, studies were performed to determine the impact of endogenous IL-27 on the immune response to T. gondii in wild-type (WT) mice. Analysis of infected mice revealed the early production of IL-27p28 by a subset of Ly6Chi, inflammatory monocytes, and sustained IL-27p28 production at sites of acute and chronic infection. Administration of anti-IL-27p28 prior to infection resulted in an early (day 5) increase in levels of macrophage and granulocyte activation, as well as enhanced effector T cell responses, as measured by both cellularity, cytokine production, and transcriptional profiling. This enhanced acute response led to immune pathology, while blockade during the chronic phase of infection resulted in enhanced T cell responses but no systemic pathology. In the absence of IL-27, the enhanced monocyte responses observed at day 10 were a secondary consequence of activated CD4+ T cells. Thus, in WT mice, IL-27 has distinct suppressive effects that impact innate and adaptive immunity during different phases of this infection. IMPORTANCE: The molecule IL-27 is critical in limiting the immune response to the parasite Toxoplasma gondii. In the absence of IL-27, a lethal, overactive immune response develops during infection. However, when exactly in the course of infection this molecule is needed was unclear. By selectively inhibiting IL-27 during this parasitic infection, we discovered that IL-27 was only needed during, but not prior to, infection. Additionally, IL-27 is only needed in the active areas in which the parasite is replicating. Finally, our work found that a previously unstudied cell type, monocytes, was regulated by IL-27, which contributes further to our understanding of the regulatory networks established by this molecule.

Anti-Toxoplasma In Vitro and In Vivo Activity of Pyrus boissieriana Arbutin-Rich Fraction.

PURPOSE: Pyrus boissieriana is a rich source of arbutin and has been used in herbal medicine to treat infectious diseases. This study aimed to investigate the effect of the arbutin-rich fraction of Pyrus boissieriana aerial parts on Toxoplasma gondii In Vitro and In Vivo. METHODS: An arbutin-rich fraction of P. boissieriana was prepared beforehand. Flow cytometry was used to evaluate the effect of different concentrations (1-512 µg/ml) of the P. boissieriana arbutin-rich fraction on Toxoplasma tachyzoites (RH strain). The cytotoxicity of the concentrations on the macrophage J774 cell line was also investigated by MTT assay. For In Vivo investigation, 4-6-week-old female mice infected with the RH strain of T. gondii were treated with different doses (16, 32, 64, 256, and 512 mg/kg) of the fraction using gavage. RESULTS: The highest and lowest lethality of the tachyzoites were 89.6% and 25.9% related to the concentrations of 512 µg/ml and 1 µg/ml, respectively, with an IC50 value of 18.1 µg/ml ± 0.37. The cytotoxicity test showed an IC50 value of 984.3 µg/ml ± 0.76 after 48 h incubation. The mean survival of mice at the lowest treated dose (16 mg/kg) was 6.6 days, and it was 15 days at the highest dose (512 mg/kg). The concentrations of 512, 256, 128, and 64 mg/kg of the fraction compared to the negative control (6.2 days mean survival) significantly increased the survival time of mice (P < 0.001, P = 0.009, P = 0.018, and P = 0.021, respectively). CONCLUSION: The results showed that the arbutin-rich fraction of P. boissieriana is effective against T. gondii In Vitro and In Vivo and may be a reliable alternative to conventional treatment for toxoplasmosis, although further studies are necessary.

In vitro production of cat-restricted Toxoplasma pre-sexual stages.

Sexual reproduction of Toxoplasma gondii, confined to the felid gut, remains largely uncharted owing to ethical concerns regarding the use of cats as model organisms. Chromatin modifiers dictate the developmental fate of the parasite during its multistage life cycle, but their targeting to stage-specific cistromes is poorly described1,2. Here we found that the transcription factors AP2XII-1 and AP2XI-2 operate during the tachyzoite stage, a hallmark of acute toxoplasmosis, to silence genes necessary for merozoites, a developmental stage critical for subsequent sexual commitment and transmission to the next host, including humans. Their conditional and simultaneous depletion leads to a marked change in the transcriptional program, promoting a full transition from tachyzoites to merozoites. These in vitro-cultured pre-gametes have unique protein markers and undergo typical asexual endopolygenic division cycles. In tachyzoites, AP2XII-1 and AP2XI-2 bind DNA as heterodimers at merozoite promoters and recruit MORC and HDAC3 (ref. 1), thereby limiting chromatin accessibility and transcription. Consequently, the commitment to merogony stems from a profound epigenetic rewiring orchestrated by AP2XII-1 and AP2XI-2. Successful production of merozoites in vitro paves the way for future studies on Toxoplasma sexual development without the need for cat infections and holds promise for the development of therapies to prevent parasite transmission.

Toxoplasma gondii Causes Adverse Pregnancy Outcomes by Damaging Uterine Tissue-Resident NK Cells That Secrete Growth-Promoting Factors.

Vertical transmission of the intracellular parasite, Toxoplasma gondii can lead to adverse pregnancy outcomes especially when infection occurs in early pregnancy. Decidual natural killer (dNK) cells accumulate at the maternal-fetal interface in large numbers during early pregnancy. Their nutritional roles during infection with T. gondii remain poorly defined. In the present study, we demonstrated that a functional deficiency of the uterine tissue-resident NK (trNK) cells, a subset of dNK cells, contributes to the adverse pregnancy outcomes induced by T. gondii in early pregnancy. Adverse pregnancy outcomes could be ameliorated by adoptive transfer of trNK cells. Moreover, fetal growth restriction could be improved after supplementation of growth-promoting factors. In addition to the widely recognized disturbance of the immune balance at the interface between the mother and the fetus, our study reveals a novel mechanism in T. gondii that contributes to the adverse pregnancy outcomes.