Involvement of extracellular vesicles in the interaction of hosts and Toxoplasma gondii.

Toxoplasma gondii, the causative agent of toxoplasmosis, is widely distributed. This protozoan parasite is one of the best adapted, being able to infect innumerous species of animals and different types of cells. This chapter reviews current literature on extracellular vesicles secreted by T. gondii and by its hosts. The topics describe the life cycle and transmission (1); toxoplasmosis epidemiology (2); laboratorial diagnosis approach (3); The T. gondii interaction with extracellular vesicles and miRNAs (4); and the perspectives on T. gondii infection. Each topic emphases the host immune responses to the parasite antigens and the interaction with the extracellular vesicles and miRNAs.

Toxoplasma-induced behavior changes - is microbial dysbiosis the missing link?

Toxoplasma gondii (T. gondii) is one of the most successful intracellular protozoa in that it can infect the majority of mammalian cell types during the acute phase of infection. Furthermore, it is able to establish a chronic infection for the host's entire lifespan by developing an encysted parasite form, primarily in the muscles and brain of the host, to avoid the host immune system. The infection affects one third of the world population and poses an increased risk for people with a suppressed immune system. Despite the dormant characteristics of chronic T. gondii infection, there is much evidence suggesting that this infection leads to specific behavior changes in both humans and rodents. Although numerous hypotheses have been put forth, the exact mechanisms underlying these behavior changes have yet to be understood. In recent years, several studies revealed a strong connection between the gut microbiome and the different organ systems that are affected in T. gondii infection. While it is widely studied and accepted that acute T. gondii infection can lead to a dramatic disruption of the host's normal, well-balanced microbial ecosystem (microbial dysbiosis), changes in the gut microbiome during the chronic stage of infection has not been well characterized. This review is intended to briefly inspect the different hypotheses that attempt to explain the behavior changes during T. gondii infection. Furthermore, this review proposes to consider the potential link between gut microbial dysbiosis, and behavior changes in T. gondii infection as a novel way to describe the underlying mechanism.

Metagenomic analysis of atheroma plaques for identification of microorganisms indicates presence of Toxoplasma gondii as a possible etiological agent.

BACKGROUND: Cardiovascular diseases (CVDs) are the leading cause of death worldwide. Vital organs like the heart are affected by the occlusion of blood vessels due to atherosclerotic plaque formation. However, the role of infectious agents has always been an essential subject of investigation. This study investigated the presence of microorganisms, including nanobacteria, in atherosclerotic plaques removed from human carotid arteries by microbiological and metagenomic examination. METHODS: Atheroma plaque samples were obtained from 20 patients with carotid artery stenosis who had atherectomy by surgery or percutaneous intervention. Nanobacteria were grown by culturing homogenates of the atheroma plaques. Whole genome sequencing was done for samples. Because of the high percentage of Toxoplasma gondii (T. gondii) DNA, PCR investigation was applied to detect T. gondii DNA in the samples. RESULTS: A molecular analysis of nanobacteria revealed them to be made of human proteins, supporting the theory that they are not living organisms. According to sequencing results, samples showed that more than 50 % of the metagenomic sequences belonged to Toxoplasma gondii. PCR investigation indicated that T. gondii DNA was positive in 8 (40 %) of 20 plaques. CONCLUSIONS: Further evidence regarding the role of T. gondii in the etiology of plaque formation may help determine the strategy for prevention and treatment of infections in preventing atheroma plaque formation in the future.

Trem2/Syk/PI3K axis contributes to the host protection against Toxoplasma gondii-induced adverse pregnancy outcomes via modulating decidual macrophages.

Decidual macrophages residing at the maternal-fetal interface have been recognized as pivotal factors for maintaining normal pregnancy; however, they are also key target cells of Toxoplasma gondii (T. gondii) in the pathology of T. gondii-induced adverse pregnancy. Trem2, as a functional receptor on macrophage surface, recognizes and binds various kinds of pathogens. The role and underlying mechanism of Trem2 in T. gondii infection remain elusive. In the present study, we found that T. gondii infection downregulated Trem2 expression and that Trem2-/- mice exhibited more severe adverse pregnancy outcomes than wildtype mice. We also demonstrated that T. gondii infection resulted in increased decidual macrophages, which were significantly reduced in the Trem2-/- pregnant mouse model as compared to wildtype control animals. We further described the inhibited proliferation, migration, and invasion functions of trophoblast cell by T. gondii antigens through macrophages as an "intermediate bridge", while this inhibition can be rescued by Trem2 agonist HSP60. Concurrently, Trem2 deficiency in bone marrow-derived macrophages (BMDMs) heightened the inhibitory effect of TgAg on the migration and invasion of trophoblast cells, accompanied by higher pro-inflammatory factors (IL-1ß, IL-6 and TNF-α) but a lower chemokine (CXCL1) in T. gondii antigens-treated BMDMs. Furthermore, compelling evidence from animal models and in vitro cell experiments suggests that T. gondii inhibits the Trem2-Syk-PI3K signaling pathway, leading to impaired function of decidual macrophages. Therefore, our findings highlight Trem2 signaling as an essential pathway by which decidual macrophages respond to T. gondii infection, suggesting Trem2 as a crucial sensor of decidual macrophages and potential therapeutic target in the pathology of T. gondii-induced adverse pregnancy.

Live-attenuated PruΔgra72 strain of Toxoplasma gondii induces strong protective immunity against acute and chronic toxoplasmosis in mice.

BACKGROUND: Toxoplasma gondii is an intracellular opportunistic pathogenic protozoan that poses serious threats, particularly in immunocompromised individuals. In the absence of a robust prophylactic measure, the mitigation and management of toxoplasmosis present formidable challenges to public health. We recently found that GRA72 plays an important role in parasitophorous vacuole (PV) morphology, growth and virulence of T. gondii. However, whether gra72-deficient strain can be used as a vaccine remains unknown. METHODS: We first examined the attenuated virulence of gra72 gene knockout strain (PruΔgra72) and the parasite load in organs of the infected mice. Subsequently, we evaluated the immune-protective effects of the PruΔgra72 vaccination against challenge with various types of T. gondii tachyzoites and Pru cysts. Furthermore, levels of antibodies and cytokines induced by PruΔgra72 vaccination were examined. Statistical analysis was conducted by Student's t-test or Mantel-Cox log-rank test based on data obtained from three independent experiments with GraphPad Prism 8.0. RESULTS: We found that PruΔgra72 strain exhibited a significantly attenuated virulence even at the highest dose of 5 × 107 tachyzoites in Kunming mice model. The significant decrease of brain cyst burden and parasite load in the organs of the PruΔgra72-infected mice suggested its potentiality as a live-attenuated vaccine. Hence, we explored the protective immunity of PruΔgra72 vaccination against toxoplasmosis. Results showed that vaccination with 5 × 106 PruΔgra72 tachyzoites triggered a strong and sustained Th1-biased immune response, marked by significantly increased levels of anti-T. gondii IgG antibodies, and significantly higher levels of Th1 type cytokines (IL-2, IL-12 and IFN-γ) compared to that of Th2 type (IL-4 and IL-10). Vaccination with 5 × 106 PruΔgra72 tachyzoites in mice conferred long-term protection against T. gondii infection by less virulent tachyzoites (ToxoDB#9 PYS and Pru strains) and Pru cysts, provided partial protection against acute infection by high virulent Type I RH tachyzoites and significantly decreased brain cyst burden of chronically infected mice. CONCLUSIONS: The avirulent PruΔgra72 induced strong protective immunity against acute and chronic T. gondii infection and is a promising candidate for developing a safe and effective live-attenuated vaccine against T. gondii infection.

Trends of Toxoplasma gondii and common transfusable venereal infections among blood donors in Menoufia Province, Egypt.

Blood transfusion has a hazard of transmission of many pathogens, including Toxoplasma gondii (T. gondii) and other venereal infections. It is crucial to conduct epidemiological surveillance to detect the prevalence of these pathogens. The study aimed to assess the seroprevalence of T. gondii and common transfusable venereal infections among healthy blood donors in Menoufia Province, Egypt, and identify associated risk factors. Four hundred twenty individuals were recruited between January and April 2023 for cross-sectional descriptive research from the blood banks of Menoufia University medical hospitals. Collected blood samples were screened for anti-T. gondii IgM and IgG, HBsAg, anti-HCV antibodies, HIV p24 antigen and anti-HIV antibodies, and anti-Treponema pallidum antibodies. 46 (11.0%) and 22 donors (5.2%) individuals tested positive for anti-T. gondii IgG with a 95% CI (8.3-14.6) and IgM with a 95% CI (3.5-8.1), respectively, while one patient (0.2%) was positive for both antibodies. Regarding venereal infections, 12 (2.9%) were positive for HBV, 6 (1.4%) were positive for HCV, 7 (1.7%) were positive for HIV, and none of the tested population showed positivity for syphilis. Female gender, consumption of raw meat, agricultural environment, poor awareness about T. gondii, and blood group type (especially AB and O groups) were identified as independent risk factors for T. gondii infection. The study highlights the importance of testing blood donors for T. gondii and common transfusable venereal illnesses. Starting health education programs and preventative measures, such as suitable meat handling and cleanliness practices, is critical for minimizing the occurrence of these illnesses. Larger-scale additional study is advised to confirm these results and provide guidance for public health initiatives.

Inflammatory response and parasite regulation in acute toxoplasmosis: the role of P2X7 receptor in controlling virulent atypical genotype strain of Toxoplasma gondii.

Toxoplasmosis is a globally significant disease that poses a severe threat to immunocompromised individuals, especially in Brazil, where a high prevalence of virulent and atypical strains of Toxoplasma gondii is observed. In 1998, the EGS strain, exhibiting a unique infection phenotype, was isolated in Brazil, adding to the complexity of strain diversity. The P2X7 receptor is critical in inflammation and controlling intracellular microorganisms such as T. gondii. However, its genetic variability can result in receptor dysfunction, potentially worsening susceptibility. This study investigates the role of the P2X7 receptor during acute infection induced by the EGS atypical strain, offering insight into the mechanisms of T. gondii infection in this context. We infected the female C57BL/6 (WT) or P2X7 knockout (P2X7-/-) by gavage. The EGS infection causes intestinal inflammation. The P2X7-/- mice presented higher parasite load in the intestine, spleen, and liver. The absence of the P2X7 receptor disrupts inflammatory cell balance by reducing NLRP3, IL-1ß, and Foxp3 expression while increasing IFN-γ expression and production in the intestine. In the liver, P2X7-/- animals demonstrate diminished inflammatory infiltrate within the portal and lobular regions concurrent with an enlargement of the spleen. In conclusion, the infection of mice with the EGS strain elicited immune alterations, leading to acute inflammation and cytokine dysregulation, while the P2X7 receptor conferred protection against parasitic proliferation across multiple organs.

Anti-Toxoplasma gondii effects of XYP1-derived peptides and regulatory mechanisms of XYP1.

BACKGROUND: Toxoplasmosis, caused by Toxoplasma gondii , poses serious health issues for humans and animals. Individuals with impaired immune systems are more susceptible to severe toxoplasmosis. Pregnant women infected by T. gondii can face the possibility of birth defects and miscarriages. While pyrimethamine and sulfadiazine are commonly used drugs in clinical practice, concerns over their side effects and resistance are on the rise. A spider peptide XYP1 isolated from Lycosa coelestis had potent anti-T. gondii effects, but it had a high synthesis cost and strong cytotoxicity. METHODS: This study intended to modify XYP1 for producing derived peptides via amino acid truncation and substitution. The anti-T. gondii effect was evaluated by trypan blue staining assay and killing experiment of RH strain tachyzoites. The CCK8 and hemolysis assays were used to compare their safeties. The morphological changes of T. gondii were observed by scanning electron microscope and transmission electron microscope. In addition, the mechanism of XYP1 against T. gondii through RNA-sequencing was further explored. RESULTS: In vivo and in vitro experiments revealed that XYP1-18 and XYP1-18-1 had excellent anti-T. gondii activity with lower cytotoxicity and hemolysis activity than XYP1. XYP1, XYP1-18, and XYP1-18-1 were able to disrupt the surface membrane integrity of T. gondii tachyzoites, forming pores and causing the disruption of organelles. Furthermore, RNA-sequencing analysis indicated that XYP1 could stimulate the host immune response to effectively eliminate T. gondii and lessen the host's inflammatory reaction. CONCLUSIONS: XYP1-18 had lower cytotoxicity and hemolysis activity than XYP1, as well as significantly extending the survival time of the mice. XYP1 played a role in host inflammation and immune responses, revealing its potential mechanism. Our research provided valuable insights into the development and application of peptide-based drugs, offering novel strategies and directions for treating toxoplasmosis.

Enzymatically enhanced ultrastructure expansion microscopy unlocks expansion of in vitro Toxoplasma gondii cysts.

Expansion microscopy (ExM) is an innovative approach to achieve super-resolution images without using super-resolution microscopes, based on the physical expansion of the sample. The advent of ExM has unlocked the detail of super-resolution images for a broader scientific circle, lowering the cost and entry skill requirements for the field. One of its branches, ultrastructure expansion microscopy (U-ExM), has become popular among research groups studying apicomplexan parasites, including the acute stage of Toxoplasma gondii infection. Here, we show that the chronic cyst-forming stage of Toxoplasma, however, resists U-ExM expansion, impeding precise protein localization. We then solve the in vitro cyst's resistance to denaturation required for successful U-ExM. As the cyst's main structural protein CST1 contains a mucin domain, we added an enzymatic digestion step using the pan-mucinase StcE prior to the expansion protocol. This allowed full expansion of the cysts in fibroblasts and primary neuronal cell culture without disrupting immunofluorescence analysis of parasite proteins. Using StcE-enhanced U-ExM, we clarified the localization of the GRA2 protein, which is important for establishing a normal cyst, observing GRA2 granules spanning across the CST1 cyst wall. The StcE-U-ExM protocol allows accurate pinpointing of proteins in the bradyzoite cyst, which will greatly facilitate investigation of the underlying biology of cyst formation and its vulnerabilities. IMPORTANCE: Toxoplasma gondii is an intracellular parasite capable of establishing long-term chronic infection in nearly all warm-blooded animals. During the chronic stage, parasites encapsulate to form cysts predominantly in neurons and skeletal muscle. Current anti-Toxoplasma drugs do not eradicate chronic parasites, leaving a reservoir of infection. The cyst is critical for disease transmission and pathology, yet it is harder to study, with the function of many chronic-stage proteins still unknown. Ultrastructure expansion microscopy, a new method to overcome the light microscopy's diffraction limit by physically expanding the sample, allowed in-depth studies of acute Toxoplasma infection. We show that Toxoplasma cysts resist expansion using standard protocol, but an additional enzymatic digestion with the mucinase StcE allows full expansion. This protocol offers new avenues for examining the chronic stage, including precise spatial organization of cyst-specific proteins, linking these locations to morphological structures, and detailed investigations of components of the durable cyst wall.

A Toxoplasma gondii thioredoxin with cell adhesion and antioxidant function.

Background: Toxoplasma gondii (T. gondii) is a widespread, zoonotic protozoan intracellular parasite with a complex life cycle, which can cause toxoplasmosis, a potentially serious disease. During the invasion process, T. gondii proteins first bind to the relevant host cell receptors, such as glycosaminoglycan molecule (GAG-binding motif), which is one of the main receptors for parasites or virus to infect host cells. However, research on TGME49_216510 (T. gondii Trx21), a protein from Toxoplasma gondii, is limited. Methods: Bioinformatics analysis of the Trx21 protein was performed firstly. And specific primers were then designed using the conserved domain and GAG-binding motif to amplify, express, and purify a fragment of the Trx21 protein. The purified Trx21-GST protein was used for antioxidant and cell adhesion experiments. Simultaneously, mice were immunized with Trx21-His to generate specific polyclonal antibodies for subcellular localization analysis. Results: The Trx21 protein, consisting of 774 amino acids, included a transmembrane region, three GAG-binding motifs, and a Thioredoxin-like domain. The recombinant Trx21-His protein had a molecular mass of about 31 kDa, while the Trx21-GST protein had a molecular mass of about 55 kDa, which was analyzed by SDS-PAGE and Western blot. Subcellular localization analysis by IFA revealed that Trx21 is predominantly distributed in the cytoplasm of T. gondii. Furthermore, Trx21 exhibited a protective effect on supercoiled DNA against metal-catalyzed oxidation (MCO) and demonstrated adhesion abilities to Vero cells. Conclusions: These results indicate that Trx21 plays an important role in host cell interaction and oxidative damage.

Development of an indirect ELISA for detecting Toxoplasma gondii IgG antibodies based on a recombinant TgIMP1 protein.

Toxoplasma gondii (T. gondii) is widely spread around the world, which can cause serious harm to immunosuppressed patients. Currently, the commercial test kits are poor at assessing T. gondii infection and vaccine effectiveness, making an urgent need to exploit effective enzyme-linked immunosorbent assay with great performance to compensate for this deficiency. Here, the TgIMP1 recombinant protein was expressed in E. coli BL(21) cells. The TgIMP1 was purified with affinity chromatography and the reactivity was retained with anti-TgIMP1 antibodies. The TgIMP1 was then used to develop an indirect ELISA (IMP1-iELISA) and the reaction conditions of IMP1-iELISA were optimized. As a result, the cut-off value was determined to be 0.2833 by analyzing the OD450nm values of forty T. gondii-negative sera. The coefficient of variation of 6 T. gondii-positive sera within and between runs were both less than 10%. The IMP1-iELISA was non-cross-reactive with the sera of cytomegalovirus, herpes virus, rubella virus, Cryptosporidium spp., Theileria spp., Neospora spp. and Plasmodium spp.. Furthermore, the sensitivity and specificity of IMP1-iELISA were 98.9% and 96.7%, respectively, based on testing 150 serum samples. The results suggest that this IMP1-iELISA is specific, sensitive, repeatable and can be applied to the detection of T. gondii infections in the medical and health industries.

TgMIC6 inhibition of autophagy is partially responsible for the phenotypic differences between Chinese 1 Toxoplasma gondii strains.

Chinese1 is the predominant Toxoplasma gondii lineage in China, and significant phenotypic differences are observed within the lineage. WH3 and WH6 are two representative strains of Chinese 1, which exhibit divergent virulence and pathogenicity in mice. However, virulence determinants and their modulating mechanisms remain elusive. A global genome expression analysis of the WH3 and WH6 transcriptional profiles identified microneme secretory protein 6 (MIC6), which may be associated with the phenotypic difference observed in WH3. In the present study, the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome-editing technique was used to generate a T. gondii microneme secretory protein (TgMIC6) knockout in WH3. Wild-type mice and different mouse and human cell lines were infected with the WH3, WH3-Δmic6, and WH6 strains. The survival rate of mice, related cytokine levels in serum, and the proliferation of parasites were observed. These results suggested that TgMIC6 is an important effector molecule that determines the differential virulence of WH3 in vivo and in vitro. Furthermore, MIC6 may enhance WH3 virulence via inhibition of host cell autophagy and activation of key molecules in the epidermal growth factor receptor (EGFR)-Akt-mammalian target of rapamycin (mTOR) classical autophagy pathway. CD40L was cleared in vivo by i.p injection of CD40L monoclonal antibody, and it was found that the virulence of WH3-Δmic6 to mice was restored to a certain extent in the absence of CD40L. This study elucidates the virulence determinants and immune escape strategies of Toxoplasma gondii in China. Moreover, these data will aid the development of effective strategies for the prevention and control of toxoplasmosis.

Constitutive upregulation of transcription factors underlies permissive bradyzoite differentiation in a natural isolate of Toxoplasma gondii.

Toxoplasma gondii bradyzoites play a critical role in pathology due to their long-term persistence in intermediate hosts and their potential to reactivate, resulting in severe diseases in immunocompromised individuals. Currently, there is no effective treatment for eliminating bradyzoites. Hence, better in vitro models of T. gondii bradyzoite development would facilitate identification of therapeutic targets for bradyzoites. Herein, we characterized a natural isolate of T. gondii, called Tg68, which showed slower in vitro replication of tachyzoites, and permissive bradyzoite development under stress conditions in vitro. Transcriptional analysis revealed constitutive expression in Tg68 tachyzoites of the key regulators of bradyzoite development including BFD1, BFD2, and several AP2 factors. Consistent with this finding, Tg68 tachyzoites expressed high levels of bradyzoite-specific genes including BAG1, ENO1, and LDH2. Moreover, after stress-induced differentiation, Tg68 bradyzoites exhibited gene expression profiles of mature bradyzoites, even at early time points. These data suggest that Tg68 tachyzoites exist in a pre-bradyzoite stage primed to readily develop into mature bradyzoites under stress conditions in vitro. Tg68 presents a novel model for differentiation in vitro that will serve as a useful tool for the investigation of bradyzoite biology and the development of therapeutics. IMPORTANCE: Toxoplasma gondii is a widespread protozoan that chronically infects ~30% of the world's population. T. gondii can differentiate between the fast-growing life stage that causes acute infection and the slow-growing stage that persists in the host for extended periods of time. The slow-growing stage cannot be eliminated by the host immune response or currently known antiparasitic drugs. Studies on the slow-growing stage have been limited due to the limitations of in vivo experiments and the challenges of in vitro manipulation. Here, we characterize a natural isolate of T. gondii, which constitutively expresses factors that drive development and that is permissive to convert to the slow-growing stage under stress conditions in vitro. The strain presents a novel in vitro model for studying the chronic phase of toxoplasmosis and identifying new therapeutic treatments for chronic infections.

Evaluation of the efficacy of Chitosan nanoparticles based on Rosuvastatin in the treatment of acute toxoplasmosis: An In vitro and In vivo study.

Toxoplasma gondii (T.gondii) is an obligate intracellular protozoan that infects warm-blooded animals and has a global distribution. Acute toxoplasmosis is commonly reported in patients with acquired/congenital toxoplasmosis and immune deficiency. New methods are needed to prevent the sideffects of classical treatment. In this study, Rosuvastatin loaded chitosan nanoparticle (CH-NP-ROS) were synthesized and zeta potential and size were determined, and an MTT assay was performed to evaluate the cell toxicity on Macrophage cells (MQ) and anti-Toxoplasma activity using Trypan-blue staining by different concentrations of Rosuvastatin (ROS), and Rosuvastatin loaded chitosan nanoparticle (CH-NP-ROS). The cell viability assay demonstrated that CH-NP-ROS had lower cell toxicity (<15 %) compared to ROS (<30 %). Statistical analysis showed that CH-NP-ROS significantly killed 98.950 ± 1.344; P < 0.05) of Toxoplasma gondii tachyzoites. In vivo results of perituneal fluid showed that CH-NP significantly reduced the parasite load in the CH-NP-ROS group, compared to that in negative control group (P < 0.001). Growth inhibition rates of tachyzoites in mice receiving free ROS and CH-NP-ROS (injection and oral form) were found to be 166.125 + 4.066, 118.750 + 4.596 and 124.875 + 2.652, respectively, compared to mice in Sulfadiazine/Pyrimethamine treated group (positive control). In the infected untreated mice (control +), the mean tachyzoite counts per oil immersion field in the spleen was 8.25 respectively. The mean survival time in all the groups treated with ROS and CH-NP-ROS was longer than that in the negative control group Therefore, nanoformulation is a promising approach for the delivery and is safe for using therapeutic effects in acute toxoplasmosis.

Calcium-binding protein TgpCaBP regulates calcium storage of the zoonotic parasite Toxoplasma gondii.

Toxoplasma gondii, the causative parasite of toxoplasmosis, is an apicomplexan parasite that infects warm-blooded mammals. The ability of the calcium-binding proteins (CBPs) to transport large amounts of Ca2+ appears to be critical for the biological activity of T. gondii. However, the functions of some members of the CBP family have not yet been deciphered. Here, we characterized a putative CBP of T. gondii, TgpCaBP (TGME49_229480), which is composed of four EF-hand motifs with Ca2+-binding capability. TgpCaBP was localized in the cytosol and ER of T. gondii, and parasites lacking the TgpCaBP gene exhibited diminished abilities in cell invasion, intracellular growth, egress, and motility. These phenomena were due to the abnormalities in intracellular Ca2+ efflux and ER Ca2+ storage, and the reduction in motility was associated with a decrease in the discharge of secretory proteins. Therefore, we propose that TgpCaBP is a Ca2+ transporter and signaling molecule involved in Ca2+ regulation and parasitization in the hosts.IMPORTANCECa2+ signaling is essential in the development of T. gondii. In this study, we identified a calcium-binding protein in T. gondii, named TgpCaBP, which actively regulates intracellular Ca2+ levels in the parasite. Deletion of the gene coding for TgpCaBP caused serious deficits in the parasite's ability to maintain a stable intracellular calcium environment, which also impaired the secretory protein discharged from the parasite, and its capacity of gliding motility, cell invasion, intracellular growth, and egress from host cells. In summary, we have identified a novel calcium-binding protein, TgpCaBP, in the zoonotic parasite T. gondii, which is a potential therapeutic target for toxoplasmosis.

Toxoplasma gondii rhoptry discharge factor 3 is essential for invasion and microtubule-associated vesicle biogenesis.

Rhoptries are specialized secretory organelles conserved across the Apicomplexa phylum, essential for host cell invasion and critical for subverting of host cellular and immune functions. They contain proteins and membranous materials injected directly into the host cells, participating in parasitophorous vacuole formation. Toxoplasma gondii tachyzoites harbor 8 to 12 rhoptries, 2 of which are docked to an apical vesicle (AV), a central element associated with a rhoptry secretory apparatus prior to injection into the host cell. This parasite is also equipped with 5 to 6 microtubule-associated vesicles, presumably serving as AV replenishment for iterative rhoptry discharge. Here, we characterized a rhoptry protein, rhoptry discharge factor 3 (RDF3), crucial for rhoptry discharge and invasion. RDF3 enters the secretory pathway, localizing near the AV and associated with the rhoptry bulb. Upon invasion, RDF3 dynamically delocalizes, suggesting a critical role at the time of rhoptry discharge. Cryo-electron tomography analysis of RDF3-depleted parasites reveals irregularity in microtubule-associated vesicles morphology, presumably impacting on their preparedness to function as an AV. Our findings suggest that RDF3 is priming the microtubule-associated vesicles for rhoptry discharge by a mechanism distinct from the rhoptry secretory apparatus contribution.

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

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.

Toxoplasma gondii and Toxocara spp. contamination in university area.

Toxoplasma gondii and Toxocara spp. zoonotic infections may cause severe systemic and ocular illness in infected individuals. Cats play a significant role in environmental contamination and the transmission of parasites. The goal of the present study was to investigate the prevalence of Toxoplasma gondii (T. gondii) and Toxocara spp. infection among stray cats at Ahvaz Jundishapur University of Medical Sciences campus. The current descriptive study began with the collection of 170 fresh cat faecal samples from various sites in the Ahvaz Jundishapur University of Medical Sciences area. Sheather's sugar flotation method was applied to all specimens, and parasites were identified and examined microscopically. Next, a nested-PCR assay, sequencing, and real-time PCR with high-resolution melting curve (HRM) analysis were performed. In this study, out of 170 cat faecal samples microscopically evaluated, 8 (4.70%) and 37 (21.76%) were infected with T. gondii oocysts and Toxocara eggs, respectively. Using nested PCR, 8 out of 170 samples (4.70%) were found to be infected with T. gondii. HRM analysis showed that all isolates could be classified into three genetic lineages. Considerable prevalence, exceeding 50% for Toxocara and surpassing 25% for Toxoplasma in certain instances, along with genetic diversity, was observed in the present study. Hence, it is suggested that all individuals, including kindergarten children, students, employees, workers, and pregnant women who are in contact with their surroundings, take the necessary precautions.

Decoding Toxoplasma gondii virulence: the mechanisms of IRG protein inactivation.

Toxoplasmosis is a common parasitic zoonosis that can be life-threatening in immunocompromised patients. About one-third of the human population is infected with Toxoplasma gondii. Primary infection triggers an innate immune response wherein IFN-γ-induced host cell GTPases, namely IRG and GBP proteins, serve as a vital component for host cell resistance. In the past decades, interest in elucidating the function of these GTPase families in controlling various intracellular pathogens has emerged. Numerous T. gondii effectors were identified to inactivate particular IRG proteins. T. gondii is re-optimizing its effectors to combat IRG function and in this way secures transmission. We discuss the IRG-specific effectors employed by the parasite in murine infections, contributing to a better understanding of T. gondii virulence.

miRNA, New Perspective to World of Intestinal Protozoa and Toxoplasma.

BACKGROUND: miRNAs are known as non-coding RNAs that can regulate gene expression. They are reported in many microorganisms and their host cells. Parasite infection can change or shift host miRNAs expression, which can aim at both parasite eradication and infection. PURPOSE: This study dealt with examination of miRNA expressed in intestinal protozoan, coccidia , as well as profile changes in host cell miRNA after parasitic infection and their role in protozoan clearance/ survival. METHODS: The authors searched ISI Web of Sciences, Pubmed, Scholar, Scopus, another databases and articles published up to 2024 were included. The keywords of miRNA, intestinal protozoa, toxoplasma and some words associated with topics were used in this search. RESULTS: Transfection of miRNA mimics or inhibitors can control parasitic diseases, and be introduced as a new therapeutic option in parasitology. CONCLUSION: This review can be used to provide up-to date knowledge for future research on these issues.