[Preliminary observation on the development and dynamic changes of chronic toxoplasmosis in mice].

OBJECTIVE: To investigate the development and dynamic changes of cysts in the brain of mice following infection with different forms of Toxoplasma gondii, so as to provide insights into for toxoplasmosis prevention and control. METHODS: ICR mice at ages of 6 to 8 weeks, each weighing 20 to 25 g, were intraperitoneally injected with tachyzoites of the T. gondii PRU strain at a dose of 1 × 105 tachyzoites per mouse, orally administered with cysts at a dose of 20 oocysts per mouse or oocysts at a dose of 200 oocysts per mouse for modeling chronic T. gondii infection in mice, and the clinical symptoms and survival of mice were observed post-infection. Mice were orally infected with T. gondii cysts at doses of 10 (low-dose group), 20 (medium-dose group), 40 cysts per mouse (high-dose group), and the effect of different doses of T. gondii infections on the number of cysts was examined in the mouse brain. Mice were orally administered with T. gondii cysts at a dose of 20 cysts per mouse, and grouped according to gender (female and male) and time points of infections (20, 30, 60, 90, 120, 150, 180 days post-infection), and the effects of gender and time points of infections on the number of cysts was examined in the mouse brain. In addition, mice were divided into the tachyzoite group (Group T), the first-generation cyst group (Group C1), the second-generation cyst group (Group C2), the third-generation cyst (Group C3) and the fourth-generation cyst group (Group C4). Mice in the Group T were intraperitoneally injected with T. gondii tachyzoites at a dose of 1 × 105 tachyzoites per mouse, and the cysts were collected from the mouse brain tissues 30 days post-infection, while mice in the Group C1 were orally infected with the collected cysts at a dose of 30 cysts per mouse. Continuous passage was performed by oral administration with cysts produced by the previous generation in mice, and the effect of continuous passage on the number of cysts was examined in the mouse brain. RESULTS: Following infection with T. gondii tachyzoites, cysts and oocysts in mice, obvious clinical symptoms were observed on days 6 to 13 and mice frequently died on days 7 to 12. The survival rates of mice were 67.0%, 87.0% and 53.0%, and the mean numbers of cysts were (516.0 ± 257.2), (1 203.0 ± 502.0) and (581.0 ± 183.1) in the mouse brain (F = 11.94, P < 0.01) on day 30 post-infection with T. gondii tachyzoites, cysts and oocysts, respectively, and the numbers of cysts in the brain tissues were significantly lower in mice infected with T. gondii tachyzoites and oocysts than in those infected with cysts (all P values < 0.01). The survival rates of mice were 87.0%, 87.0% and 60.0%, and the mean numbers of cysts were (953.0 ± 355.5), (1 084.0 ± 474.3) and (1 113.0 ± 546.0) in the mouse brain in the low-, medium- and high-dose groups on day 30 post-infection, respectively (F = 0.42, P > 0.05). The survival rates of male and female mice were 73.0% and 80.0%, and the mean numbers of cysts were (946.4 ± 411.4) and (932.1 ± 322.4) in the brain tissues of male and female mice, respectively (F = 1.63, P > 0.05). Following continuous passage, the mean numbers of cysts were (516.0 ± 257.2), (1 203.0 ± 502.0), (896.8 ± 332.3), (782.5 ± 423.9) and (829.2 ± 306.0) in the brain tissues of mice in the T, C1, C2, C3 and C4 groups, respectively (F = 4.82, P < 0.01), and the number of cysts was higher in the mouse brain in Group 1 than in Group T (P < 0.01). Following oral administration of 20 T. gondii cysts in mice, cysts were found in the moues brain for the first time on day 20 post-infection, and the number of cysts gradually increased over time, peaked on days 30 and 90 post-infection and then gradually decreased; however, the cysts were still found in the mouse brain on day 180 post-infection. CONCLUSIONS: There is a higher possibility of developing chronic T. gondii infection in mice following infection with cysts than with oocysts or tachyzoites and the most severe chronic infection is seen following infection with cysts. The number of cysts does not correlate with the severity of chronic T. gondii infection, and the number of cysts peaks in the mouse brain on days 30 and 90 post-infection.

Evaluation of In Vitro Cytotoxic and Apoptotic Effects of Miltefosine on the Toxoplasma gondii RH Strain.

Background: We aimed to investigate the cytotoxic and apoptotic effects of miltefosine on Toxoplasma gondii RH strain by various techniques. Methods: The study was conducted at the Department of Parasitology and Mycology, Urmia University of Medical Sciences, Iran in 2020. Four groups of five BALB/c mice were selected. The cytotoxicity test was conducted by adding miltefosine to T. gondii tachyzoites; control tachyzoites received PBS and MTT assay was done on each suspension. For evaluating the Th1-type immune responses, the serum levels of IFN-γ and nitric oxide (NO) were assessed in mice after injecting tachyzoites and miltefosine, respectively. The flow cytometry technique was performed on T. gondii tachyzoites challenged with IC50 and IC90 doses of miltefosine and unchallenged cells. DNA fragments in T. gondii tachyzoites were detected by Terminal dUTPnick-end labeling (TUNEL) method. Results: Overall, 256, 64, 32, and 16 µg concentrations of miltefosine, respectively could kill more than 50% of viable T. gondii tachyzoites. The infected mice group, treated with miltefosine, significantly produced more IFN-γ relative to other groups (P< 0.001). Moreover, a significant difference was found in inducible NO synthase between the experimental and control groups (P<0.05). The flow cytometry results demonstrated a concentration-dependent apoptosis rate in tachyzoites incubated with miltefosine, though the necrosis rate was non-significant. DNA fragmentation analysis indicated oligonucleotides (18-200 bp) in tachyzoites treated with 11µg of miltefosine for 24, 48 and 72 h. However, this pattern was not observed in untreated control microorganisms. Conclusion: Miltefosine could be a favorable candidate for use as a new treatment for toxoplasmosis.

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.

Nanoemulsion of Spiramycin against Tachyzoites of Toxoplasma gondii, RH Strain: Preparation, Toxicology, and Efficacy Studies.

Background: Toxoplasma infection is caused by Toxoplasma gondii, which is an intracellular protozoan parasite. This infection consequently lead various congenital disabilities during pregnancy in patients. Spiramycin (Spi), a macrolide antibiotic, is typically recommended for T. gondii infection in pregnant women. We aimed to prepare the nanoemulsion of spiramycin (NE-Spi) and to evaluate the activity of this formulation in tachyzoites of T. gondii, RH strain. Methods: This study was conducted in 2019-2021 at the School of Public Health, Tehran University of Medical Sciences, Tehran, Iran. NE-Spi was prepared by spontaneous emulsification. The effects of this nanoemulsion on the viability of cultured cells were measured using MTT assay. To estimate the effects of NE-Spi on tachyzoites of T. gondii, RH strain, different concentrations of NE-Spi, S-Spi (suspension of spiramycin), and NE (nanoemulsion without any spiramycin) were added to tachyzoites and then stored for 30, 60, 90, 120 min and 24 h in 250 µg/ml concentration at room temperature. Finally, Tachyzoites mortality rates were evaluated by trypan blue staining. Of note, flow cytometry was conducted to confirm the obtained results. Results: The final particle size of NE-Spi was calculated to be 11.3 nm by DLS and TEM. Thereafter, using MTT assay, in 62.5 µg/ml concentration of NE-Spi, the Vero cells viability was obtained as 82%. The highest mortality rates of tachyzoites of T.gondii, RH strain were observed at 250 µg/ml concentration and after 120 min of exposure, but it was not significantly different from 24 h of exposure. Conclusion: NE-Spi has lethal efficacy on T. gondii RH strain in-vitro.

In silico design of a novel peptide-based vaccine against the ubiquitous apicomplexan Toxoplasma gondii using surface antigens.

Human toxoplasmosis is a global public health concern and a commercial vaccine is still lacking. The present in silico study was done to design a novel vaccine candidate using tachyzoite-specific SAG1-realted sequence (SRS) proteins. Overlapping B-cell and strictly-chosen human MHC-I binding epitopes were predicted and connected together using appropriate spacers. Moreover, a TLR4 agonist, human high mobility group box protein 1 (HMGB1), and His-tag were added to the N- and C-terminus of the vaccine sequence. The final vaccine had 442 residues and a molecular weight of 47.71 kDa. Physico-chemical evaluation showed a soluble, highly antigenic and non-allergen protein, with coils and helices as secondary structures. The vaccine 3D model was predicted by ITASSER server, subsequently refined and was shown to possess significant interactions with human TLR4. As well, potent stimulation of cellular and humoral immunity was demonstrated upon chimeric vaccine injection. Finally, the outputs showed that this vaccine model possesses top antigenicity, which could provoke significant cell-mediated immune profile including IFN-γ, and can be utilized towards prophylactic purposes. Supplementary Information: The online version contains supplementary material available at 10.1007/s40203-023-00140-w.

The Therapeutic Efficacy of Zinc Oxide Nanoparticles on Acute Toxoplasmosis in BALB/c Mice.

Background: Toxoplasma gondii infects nearly one-third of the world's population. Due to the significant side effects of current treatment options, identifying safe and effective therapies seems crucial. Nanoparticles (NPs) are new promising compounds in treating pathogenic organisms. Currently, no research has investigated the effects of zinc oxide NPs (ZnO-NPs) on Toxoplasma parasite. We aimed to investigate the therapeutic efficacy of ZnO-NPs against tachyzoite forms of T. gondii, RH strain in BALB/c mice. Methods: In an experiment with 35 female BALB/c mice infected with T. gondii tachyzoites, colloidal ZnO-NPs at concentrations of 10, 20, and 50 ppm, as well as a 50 ppm ZnO solution and a control group, were orally administered four hours after inoculation and continued daily until the mices' death. Survival rates were calculated and tachyzoite counts were evaluated in the peritoneal fluids of infected mice. Results: The administration of ZnO-NPs resulted in the reduction of tachyzoite counts in infected mice compared to both the ZnO-treated and control group (P<0.001). Intervention with ZnO-NPs significantly increased the survival time compared to the control group (6.2±0.28 days, P-value <0.05), additionally, the highest dose of ZnO-NPs (50 ppm) showed the highest mice survival time (8.7±0.42 days). Conclusion: ZnO-NPs were effective in decreasing the number of tachyzoites and increasing mice survival time in vivo. Moreover, there were no significant differences in survival time between the untreated control group and the group treated with zinc oxide, suggesting that, bulk ZnO is not significantly effective in comparison with ZnONPs.

Plasticity and therapeutic potential of cAMP and cGMP-specific phosphodiesterases in Toxoplasma gondii.

Toxoplasma gondii is a common zoonotic protozoan pathogen adapted to intracellular parasitism in many host cells of diverse organisms. Our previous work has identified 18 cyclic nucleotide phosphodiesterase (PDE) proteins encoded by the parasite genome, of which 11 are expressed during the lytic cycle of its acutely-infectious tachyzoite stage in human cells. Here, we show that ten of these enzymes are promiscuous dual-specific phosphodiesterases, hydrolyzing cAMP and cGMP. TgPDE1 and TgPDE9, with a Km of 18 µM and 31 µM, respectively, are primed to hydrolyze cGMP, whereas TgPDE2 is highly specific to cAMP (Km, 14 µM). Immuno-electron microscopy revealed various subcellular distributions of TgPDE1, 2, and 9, including in the inner membrane complex, apical pole, plasma membrane, cytosol, dense granule, and rhoptry, indicating spatial control of signaling within tachyzoites. Notably, despite shared apical location and dual-catalysis, TgPDE8 and TgPDE9 are fully dispensable for the lytic cycle and show no functional redundancy. In contrast, TgPDE1 and TgPDE2 are individually required for optimal growth, and their collective loss is lethal to the parasite. In vitro phenotyping of these mutants revealed the roles of TgPDE1 and TgPDE2 in proliferation, gliding motility, invasion and egress of tachyzoites. Moreover, our enzyme inhibition assays in conjunction with chemogenetic phenotyping underpin TgPDE1 as a target of commonly-used PDE inhibitors, BIPPO and zaprinast. Finally, we identified a retinue of TgPDE1 and TgPDE2-interacting kinases and phosphatases, possibly regulating the enzymatic activity. In conclusion, our datasets on the catalytic function, physiological relevance, subcellular localization and drug inhibition of key phosphodiesterases highlight the previously-unanticipated plasticity and therapeutic potential of cyclic nucleotide signaling in T. gondii.

The determinants regulating Toxoplasma gondii bradyzoite development.

Toxoplasma gondii is an obligate intracellular zoonotic pathogen capable of infecting almost all cells of warm-blooded vertebrates. In intermediate hosts, this parasite reproduces asexually in two forms, the tachyzoite form during acute infection that proliferates rapidly and the bradyzoite form during chronic infection that grows slowly. Depending on the growth condition, the two forms can interconvert. The conversion of tachyzoites to bradyzoites is critical for T. gondii transmission, and the reactivation of persistent bradyzoites in intermediate hosts may lead to symptomatic toxoplasmosis. However, the mechanisms that control bradyzoite differentiation have not been well studied. Here, we review recent advances in the study of bradyzoite biology and stage conversion, aiming to highlight the determinants associated with bradyzoite development and provide insights to design better strategies for controlling toxoplasmosis.

Systematic identification of the lysine lactylation in the protozoan parasite Toxoplasma gondii.

BACKGROUND: Lysine lactylation (Kla) is a novelposttranslational modification (PTM) identified in histone and nonhistone proteins of several eukaryotic cells that directly activates gene expression and DNA replication. However, very little is known about the scope and cellular distribution of Kla in apicomplexan parasites despite its significance in public and animal health care. METHODS: Toxoplasma gondii, the causative agent of toxoplasmosis, is an obligate intracellular apicomplexan parasite that can infect different nucleated cell types of animals and humans. We used this parasite as a model organism and extracted the total protein of tachyzoites to produce the first global lysine lactylome profile of T. gondii through liquid chromatography-tandem mass spectrometry. We also investigated the level and localization of the Kla protein in T. gondii using western blotting and the indirect fluorescent antibody test (IFA), respectively. RESULTS: A total of 983 Kla sites occurring on 523 lactylated proteins were identified in the total protein extracted from Toxoplasma tachyzoites, the acute toxoplasmosis-causing stage. Bioinformatics analysis revealed that the lactylated proteins were evolutionarily conserved and involved in a wide variety of cellular functions, such as energy metabolism, gene regulation and protein biosynthesis. Subcellular localization analysis and IFA results further revealed that most of the lactylated T. gondii proteins were localized in the nucleus, indicating the potential impact of Kla on gene regulation in the T. gondii model. Notably, an extensive batch of parasite-specific proteins unique to phylum Apicomplexa is lactylated in T. gondii. CONCLUSIONS: This study revealed that Kla is widespread in early dividing eukaryotic cells. Lactylated proteins, including a batch of unique parasite proteins, are involved in a remarkably diverse array of cellular functions. These valuable data will improve our understanding of the evolution of Kla and potentially provide the basis for developing novel therapeutic avenues.

A functionally divergent transcription elongation factor 1-like protein in Toxoplasma gondii.

Zinc ribbons, one of the largest fold groups among zinc fingers, often include proteins involved in the transcription machinery. Here, we identify and characterize one such zinc ribbon-bearing protein in the apicomplexan parasite Toxoplasma gondii, annotated as putative transcription elongation factor 1 (ELF1), with predicted functions in transcription and chromatin maintenance. We show that this ELF1 homolog, referred to as T. gondii ELF1-like divergent (TgELD), is expressed in both tachyzoite and bradyzoite developmental stages. TgELD associates with the cytoskeleton in the tachyzoites, while it transiently becomes a part of the cyst wall in the early bradyzoites, followed by a cytosolic and peripheral localization in late bradyzoites. TgELD is phosphorylated by a casein kinase 2-like protein, which has potential implications for its localization and function in the parasite.

Characterization of a MOB1 Homolog in the Apicomplexan Parasite Toxoplasma gondii.

Monopolar spindle One Binder1 (MOB1) proteins are conserved components of the tumor-suppressing Hippo pathway, regulating cellular processes such as cytokinesis. Apicomplexan parasites present a life cycle that relies on the parasites' ability to differentiate between stages and regulate their proliferation; thus, Hippo signaling pathways could play an important role in the regulation of the apicomplexan life cycle. Here, we report the identification of one MOB1 protein in the apicomplexan Toxoplasma gondii. To characterize the function of MOB1, we generated gain-of-function transgenic lines with a ligand-controlled destabilization domain, and loss-of-function clonal lines obtained through CRISPR/Cas9 technology. Contrary to what has been characterized in other eukaryotes, MOB1 is not essential for cytokinesis in T. gondii. However, this picture is complex since we found MOB1 localized between the newly individualized daughter nuclei at the end of mitosis. Moreover, we detected a significant delay in the replication of overexpressing tachyzoites, contrasting with increased replication rates in knockout tachyzoites. Finally, using the proximity-biotinylation method, BioID, we identified novel members of the MOB1 interactome, a probable consequence of the observed lack of conservation of some key amino acid residues. Altogether, the results point to a complex evolutionary history of MOB1 roles in apicomplexans, sharing properties with other eukaryotes but also with divergent features, possibly associated with their complex life cycle.

In vitro activity of N-phenyl-1,10-phenanthroline-2-amines against tachyzoites and bradyzoites of Toxoplasma gondii.

Toxoplasma gondiiis an apicomplexan parasite, the causative agent of toxoplasmosis, a common disease in the world. Toxoplasmosis could be severe, especially in immunocompromised patients. The current therapy is limited, where pyrimethamine and sulfadiazine are the best choices despite being associated with side effects and ineffective against the bradyzoites, the parasitic form present during the chronic phase of the infection. Thus, new therapies against both tachyzoites and bradyzoites from T. gondii are urgent. Herein, we present the anti-T. gondii effect of 1,10-phenanthroline and its N-phenyl-1,10-phenanthroline-2-amine derivatives. The chemical modification of 1,10-phenanthroline tonew derivatives improved the anti-T. gondiiactivity 3.4 fold. The most active derivative presented ED50in the nanomolar range, the smallest value found was for Ph8, 0.1 µM for 96 h of treatment. The host cell viability was maintained after the treatment with the compounds, which were found to be highly selective presenting large selectivity indexes. Treatment with derivatives for 96 h was able to eliminate the T. gondii infection irreversibly. The ultrastructural alterations caused after the treatment with the most effective derivative (Ph8) included signs of cell death, specifically revealed by the Tunel assay for detection of DNA fragmentation. The Phen derivatives were also able to control the growth of the in vitro-derived bradyzoite forms of T. gondii EGS strain, causing its lysis and death. These findings promote the 1,10-phenanthroline derivatives as potential lead compounds for the development of a treatment for acute and chronic phases of toxoplasmosis.

In vitro anti-Toxoplasma gondii efficacy of synthesised benzyltriazole derivatives.

Toxoplasma gondii, an obligate intracellular parasite, is the aetiological agent of toxoplasmosis, a disease that affects approximately 25% - 30% of the world's population. At present, no safe and effective vaccine exists for the prevention of toxoplasmosis. Current treatment options for toxoplasmosis are active only against tachyzoites and may also cause bone marrow toxicity. To contribute to the global search for novel agents for the treatment of toxoplasmosis, we herein report the in vitro activities of previously synthesised benzyltriazole derivatives. The effects of these compounds against T. gondii in vitro were evaluated by using a expressing green fluorescent protein (GFP) type I strain parasite (RH-GFP) and a type II cyst-forming strain of parasite (PruΔku80Δhxgprt). The frontline antitubercular drug isoniazid, designated as Frans J. Smit -isoniazid (FJS-INH), was also included in the screening as a preliminary test in view of future repurposing of this agent. Of the compounds screened, FJS-302, FJS-303, FJS-403 and FJS-INH demonstrated 80% parasite growth inhibition with IC50 values of 5.6 µg/mL, 6.8 µg/µL, 7.0 µg/mL and 19.8 µg/mL, respectively. FJS-302, FJS-303 and FJS-403 inhibited parasite invasion and replication, whereas, sulphadiazine (SFZ), the positive control, was only effective against parasite replication. In addition, SFZ induced bradyzoite differentiation in vitro, whilst FJS-302, FJS-303 and FJS-403 did not increase the bradyzoite number. These results indicate that FJS-302, FJS-303 and FJS-403 have the potential to act as a viable source of antiparasitic therapeutic agents.

Dehydroepiandrosterone Effect on Toxoplasma gondii: Molecular Mechanisms Associated to Parasite Death.

Toxoplasmosis is a zoonotic disease caused by the apicomplexa protozoan parasite Toxoplasma gondii. This disease is a health burden, mainly in pregnant women and immunocompromised individuals. Dehydroepiandrosterone (DHEA) has proved to be an important molecule that could drive resistance against a variety of infections, including intracellular parasites such as Plasmodium falciparum and Trypanozoma cruzi, among others. However, to date, the role of DHEA on T. gondii has not been explored. Here, we demonstrated for the first time the toxoplasmicidal effect of DHEA on extracellular tachyzoites. Ultrastructural analysis of treated parasites showed that DHEA alters the cytoskeleton structures, leading to the loss of the organelle structure and organization as well as the loss of the cellular shape. In vitro treatment with DHEA reduces the viability of extracellular tachyzoites and the passive invasion process. Two-dimensional (2D) SDS-PAGE analysis revealed that in the presence of the hormone, a progesterone receptor membrane component (PGRMC) with a cytochrome b5 family heme/steroid binding domain-containing protein was expressed, while the expression of proteins that are essential for motility and virulence was highly reduced. Finally, in vivo DHEA treatment induced a reduction of parasitic load in male, but not in female mice.

Engineering and Functional Evaluation of Neutralizing Antibody Fragments Against Congenital Toxoplasmosis.

Maternal-fetal transmission of Toxoplasma gondii tachyzoites acquired during pregnancy has potentially dramatic consequences for the fetus. Current reference-standard treatments are not specific to the parasite and can induce severe side effects. In order to provide treatments with a higher specificity against toxoplasmosis, we developed antibody fragments-single-chain fragment variable (scFv) and scFv fused with mouse immunoglobulin G2a crystallizable fragment (scFv-Fc)-directed against the major surface protein SAG1. After validating their capacity to inhibit T. gondii proliferation in vitro, the antibody fragments' biological activity was assessed in vivo using a congenital toxoplasmosis mouse model. Dams were treated by systemic administration of antibody fragments and with prevention of maternal-fetal transmission being used as the parameter of efficacy. We observed that both antibody fragments prevented T. gondii dissemination and protected neonates, with the scFv-Fc format having better efficacy. These data provide a proof of concept for the use of antibody fragments as effective and specific treatment against congenital toxoplasmosis and provide promising leads.

Lysine crotonylation is widespread on proteins of diverse functions and localizations in Toxoplasma gondii.

Lysine crotonylation (Kcr) is an evolutionally conserved post-translational modification (PTM) on histone proteins. However, information about Kcr and its involvement in the biology and metabolism of Toxoplasma gondii is limited. In the present study, a global Kcr proteome analysis using LC-MS/MS in combination with immune-affinity method was performed. A total of 12,152 Kcr sites distributed over 2719 crotonylated proteins were identified. Consistent with lysine acetylation and succinylation in Apicomplexa, Kcr was associated with various metabolic pathways, including carbon metabolism, pyrimidine metabolism, glycolysis, gluconeogenesis, and proteasome. Markedly, many stage-specific proteins, histones, and histone-modifying enzymes related to the stage transition were found to have Kcr sites, suggesting a potential involvement of Kcr in the parasite stage transformation. Most components of the apical secretory organelles were identified as crotonylated proteins which were associated with the attachment, invasion, and replication of T. gondii. These results expanded our understanding of Kcr proteome and proposed new hypotheses for further research of the Kcr roles in the pathobiology of T. gondii infection.

Toxoplasma gondii induces robust humoral immune response against cyst wall antigens in chronically infected animals and humans.

Toxoplasma gondii differentiation from proliferating tachyzoites into latent bradyzoites is central to pathogenesis and transmission. Strong humoral immune response has been reported against tachyzoite antigens, however, antibody-mediated response towards bradyzoite antigens is poorly characterized. This work aimed to study the humoral immune response towards bradyzoite and associated cyst wall antigens particularly CST1. The immunoreactivity of 404 goats, 88 sheep and 92 human sera to recombinant (CST1 and SRS9) and native proteins of encysted bradyzoite along with well-established tachyzoite antigens (SAG1 and GRA7) was determined using ELISA, Western blot and immunofluorescence analysis (IFA). ELISA results revealed nearly 50% of sera contain T. gondii specific antibodies. Results were further validated using Western blot and IFA. T. gondii positive sera predominantly recognized the cyst wall besides the known tachyzoite surface antigens. The presence of CST1 antibodies in seropositive samples were in line with the staining patterns which were consistent with CST localization. Notably, T. gondii IgM- IgG+ sera recognize the cyst wall whereas IgM + IgG-sera recognize tachyzoite antigens indicating acute infection consistent with presence of parasite DNA. The study demonstrates a strong humoral response against bradyzoite associated cyst wall antigens across naturally infected animals and humans. CST1 emerged as a key immunomodulatory antigen which may have direct implications for clinical immunodiagnostics.

The protozoan parasite Toxoplasma gondii encodes a gamut of phosphodiesterases during its lytic cycle in human cells.

Cyclic nucleotide signaling is pivotal to the asexual reproduction of Toxoplasma gondii, however little do we know about the phosphodiesterase enzymes in this widespread obligate intracellular parasite. Here, we identified 18 phosphodiesterases (TgPDE1-18) in the parasite genome, most of which form apicomplexan-specific clades and lack archetypal regulatory motifs often found in mammalian PDEs. Genomic epitope-tagging in the tachyzoite stage showed the expression of 11 phosphodiesterases with diverse subcellular distributions. Notably, TgPDE8 and TgPDE9 are located in the apical plasma membrane to regulate cAMP and cGMP signaling, as suggested by their dual-substrate catalysis and structure modeling. TgPDE9 expression can be ablated with no apparent loss of growth fitness in tachyzoites. Likewise, the redundancy in protein expression, subcellular localization and predicted substrate specificity of several other PDEs indicate significant plasticity and spatial control of cyclic nucleotide signaling during the lytic cycle. Our findings shall enable a rational dissection of signaling in tachyzoites by combinatorial mutagenesis. Moreover, the phylogenetic divergence of selected Toxoplasma PDEs from human counterparts can be exploited to develop parasite-specific inhibitors and therapeutics.

Key Limitations and New Insights Into the Toxoplasma gondii Parasite Stage Switching for Future Vaccine Development in Human, Livestock, and Cats.

Toxoplasmosis is a parasitic disease affecting human, livestock and cat. Prophylactic strategies would be ideal to prevent infection. In a One Health vaccination approach, the objectives would be the prevention of congenital disease in both women and livestock, prevention/reduction of T. gondii tissue cysts in food-producing animals; and oocyst shedding in cats. Over the last few years, an explosion of strategies for vaccine development, especially due to the development of genetic-engineering technologies has emerged. The field of vaccinology has been exploring safer vaccines by the generation of recombinant immunogenic proteins, naked DNA vaccines, and viral/bacterial recombinants vectors. These strategies based on single- or few antigens, are less efficacious than recombinant live-attenuated, mostly tachyzoite T. gondii vaccine candidates. Reflections on the development of an anti-Toxoplasma vaccine must focus not only on the appropriate route of administration, capable of inducing efficient immune response, but also on the choice of the antigen (s) of interest and the associated delivery systems. To answer these questions, the choice of the animal model is essential. If mice helped in understanding the protection mechanisms, the data obtained cannot be directly transposed to humans, livestock and cats. Moreover, effectiveness vaccines should elicit strong and protective humoral and cellular immune responses at both local and systemic levels against the different stages of the parasite. Finally, challenge protocols should use the oral route, major natural route of infection, either by feeding tissue cysts or oocysts from different T. gondii strains. Effective Toxoplasma vaccines depend on our understanding of the (1) protective host immune response during T. gondii invasion and infection in the different hosts, (2) manipulation and modulation of host immune response to ensure survival of the parasites able to evade and subvert host immunity, (3) molecular mechanisms that define specific stage development. This review presents an overview of the key limitations for the development of an effective vaccine and highlights the contributions made by recent studies on the mechanisms behind stage switching to offer interesting perspectives for vaccine development.

Global profiling of lysine 2-hydroxyisobutyrylome in Toxoplasma gondii using affinity purification mass spectrometry.

Lysine 2-hydroxyisobutyrylation (Khib) is a recently discovered and evolutionarily conserved form of protein post-translational modification (PTM) found in mammalian and yeast cells. Previous studies have shown that Khib plays roles in the activity of gene transcription and Khib-containing proteins are closely related to the cellular metabolism. In this study, a global Khib-containing analysis using the latest databases (ToxoDB 46, 8322 sequences, downloaded on April 16, 2020) and sensitive immune-affinity enrichment coupled with liquid chromatography-tandem mass spectrometry was performed. A total of 1078 Khib modification sites across 400 Khib-containing proteins were identified in tachyzoites of Toxoplasma gondii RH strain. Bioinformatics and functional enrichment analysis showed that Khib-modified proteins were associated with various biological processes, such as ribosome, glycolysis/gluconeogenesis, and central carbon metabolism. Interestingly, many proteins of the secretory organelles (e.g., microneme, rhoptry, and dense granule) that play roles in the infection cycle of T. gondii were found to be Khib-modified, suggesting the involvement of Khib in key biological process during T. gondii infection. We also found that histone proteins, key enzymes related to cellular metabolism, and several glideosome components had Khib sites. These results expanded our understanding of the roles of Khib in T. gondii and should promote further investigations of how Khib regulates gene expression and key biological functions in T. gondii.