Toxoplasma gondii chitinase-like protein TgCLP1 regulates the parasite cyst burden.

Toxoplasma, an important intracellular parasite of humans and animals, causes life-threatening toxoplasmosis in immunocompromised individuals. Although Toxoplasma secretory proteins during acute infection (tachyzoite, which divides rapidly and causes inflammation) have been extensively characterized, those involved in chronic infection (bradyzoite, which divides slowly and is surrounded by a cyst wall) remain uncertain. Regulation of the cyst wall is essential to the parasite life cycle, and polysaccharides, such as chitin, in the cyst wall are necessary to sustain latent infection. Toxoplasma secretory proteins during the bradyzoite stage may have important roles in regulating the cyst wall via polysaccharides. Here, we focused on characterizing the hypothetical T. gondii chitinase, chitinase-like protein 1 (TgCLP1). We found that the chitinase-like domain containing TgCLP1 is partially present in the bradyzoite microneme and confirmed, albeit partially, its previous identification in the tachyzoite microneme. Furthermore, although parasites lacking TgCLP1 could convert from tachyzoites to bradyzoites and make an intact cyst wall, they failed to convert from bradyzoites to tachyzoites, indicating that TgCLP1 is necessary for bradyzoite reactivation. Taken together, our findings deepen our understanding of the molecular basis of recrudescence and could contribute to the development of novel strategies for the control of toxoplasmosis.

Translation initiation factor eIF1.2 promotes Toxoplasma stage conversion by regulating levels of key differentiation factors.

The parasite Toxoplasma gondii persists in its hosts by converting from replicating tachyzoites to latent bradyzoites housed in tissue cysts. The molecular mechanisms that mediate T. gondii differentiation remain poorly understood. Through a mutagenesis screen, we identified translation initiation factor eIF1.2 as a critical factor for T. gondii differentiation. A F97L mutation in eIF1.2 or the genetic ablation of eIF1.2 (∆eif1.2) markedly impeded bradyzoite cyst formation in vitro and in vivo. We demonstrated, at single-molecule level, that the eIF1.2 F97L mutation impacts the scanning process of the ribosome preinitiation complex on a model mRNA. RNA sequencing and ribosome profiling experiments unveiled that ∆eif1.2 parasites are defective in upregulating bradyzoite induction factors BFD1 and BFD2 during stress-induced differentiation. Forced expression of BFD1 or BFD2 significantly restored differentiation in ∆eif1.2 parasites. Together, our findings suggest that eIF1.2 functions by regulating the translation of key differentiation factors necessary to establish chronic toxoplasmosis.

iNOS is necessary for GBP-mediated T. gondii clearance in murine macrophages via vacuole nitration and intravacuolar network collapse.

Toxoplasma gondii is an obligate intracellular parasite of rodents and humans. Interferon-inducible guanylate binding proteins (GBPs) are mediators of T. gondii clearance, however, this mechanism is incomplete. Here, using automated spatially targeted optical micro proteomics we demonstrate that inducible nitric oxide synthetase (iNOS) is highly enriched at GBP2+ parasitophorous vacuoles (PV) in murine macrophages. iNOS expression in macrophages is necessary to limit T. gondii load in vivo and in vitro. Although iNOS activity is dispensable for GBP2 recruitment and PV membrane ruffling; parasites can replicate, egress and shed GBP2 when iNOS is inhibited. T. gondii clearance by iNOS requires nitric oxide, leading to nitration of the PV and collapse of the intravacuolar network of membranes in a chromosome 3 GBP-dependent manner. We conclude that reactive nitrogen species generated by iNOS cooperate with GBPs to target distinct structures in the PV that are necessary for optimal parasite clearance in macrophages.

Iron Stress Affects the Growth and Differentiation of Toxoplasma gondii.

Iron is an indispensable nutrient for the survival of Toxoplasma gondii; however, excessive amounts can lead to toxicity. The parasite must overcome the host's "nutritional immunity" barrier and compete with the host for iron. Since T. gondii can infect most nucleated cells, it encounters increased iron stress during parasitism. This study assessed the impact of iron stress, encompassing both iron depletion and iron accumulation, on the growth of T. gondii. Iron accumulation disrupted the redox balance of T. gondii while enhancing the parasite's ability to adhere in high-iron environments. Conversely, iron depletion promoted the differentiation of tachyzoites into bradyzoites. Proteomic analysis further revealed proteins affected by iron depletion and identified the involvement of phosphotyrosyl phosphatase activator proteins in bradyzoite formation.

Myosin F controls actin organization and dynamics in Toxoplasma gondii.

Intracellular cargo transport is a ubiquitous cellular process in all eukaryotes. In many cell types, membrane bound cargo is associated with molecular motors which transport cargo along microtubule and actin tracks. In Toxoplasma gondii (T. gondii), an obligate intracellular parasite in the phylum Apicomplexa, organization of the endomembrane pathway depends on actin and an unconventional myosin motor, myosin F (MyoF). Loss of MyoF and actin disrupts vesicle transport, organelle positioning, and division of the apicoplast, a nonphotosynthetic plastid organelle. How this actomyosin system contributes to these cellular functions is still unclear. Using live-cell imaging, we observed that MyoF-EmeraldFP (MyoF-EmFP) displayed a dynamic and filamentous-like organization in the parasite cytosol, reminiscent of cytosolic actin filament dynamics. MyoF was not associated with the Golgi, apicoplast or dense granule surfaces, suggesting that it does not function using the canonical cargo transport mechanism. Instead, we found that loss of MyoF resulted in a dramatic rearrangement of the actin cytoskeleton in interphase parasites accompanied by significantly reduced actin dynamics. However, actin organization during parasite replication and motility was unaffected by the loss of MyoF. These findings revealed that MyoF is an actin organizing protein in Toxoplasma and facilitates cargo movement using an unconventional transport mechanism.

Mechanisms of adaptation and evolution in Toxoplasma gondii.

Toxoplasma has high host flexibility, infecting all nucleated cells of mammals and birds. This implies that during its infective process the parasite must constantly adapt to different environmental situations, which in turn leads to modifications in its metabolism, regulation of gene transcription, translation of mRNAs and stage specific factors. There are conserved pathways that support these adaptations, which we aim to elucidate in this review. We begin by exploring the widespread epigenetic mechanisms and transcription regulators, continue with the supportive role of Heat Shock Proteins (Hsp), the translation regulation, stress granules, and finish with the emergence of contingency genes in highly variable genomic domains, such as subtelomeres. Within epigenetics, the discovery of a new histone variant of the H2B family (H2B.Z), contributing to T. gondii virulence and differentiation, but also gene expression regulation and its association with the metabolic state of the parasite, is highlighted. Associated with the regulation of gene expression are transcription factors (TFs). An overview of the main findings on TF and development is presented. We also emphasize the role of Hsp90 and Tgj1 in T. gondii metabolic fitness and the regulation of protein translation. Translation regulation is also highlighted as a mechanism for adaptation to conditions encountered by the parasite as well as stress granules containing mRNA and proteins generated in the extracellular tachyzoite. Another important aspect in evolution and adaptability are the subtelomeres because of their high variability and gene duplication rate. Toxoplasma possess multigene families of membrane proteins and contingency genes that are associated with different metabolic stresses. Among them parasite differentiation and environmental stresses stand out, including those that lead tachyzoite to bradyzoite conversion. Finally, we are interested in positioning protozoa as valuable evolution models, focusing on research related to the Extended Evolutionary Synthesis, based on models recently generated, such as extracellular adaptation and ex vivo cyst recrudescence.

Host E3 ubiquitin ligase ITCH mediates Toxoplasma gondii effector GRA35-triggered NLRP1 inflammasome activation and cell-autonomous immunity.

Toxoplasma gondii is an intracellular parasite that can activate the NLRP1 inflammasome leading to macrophage pyroptosis in Lewis rats, but the underlying mechanism is not well understood. In this study, we performed a genome-wide CRISPR screen and identified the dense granule proteins GRA35, GRA42, and GRA43 as the Toxoplasma effectors mediating cell death in Lewis rat macrophages. GRA35 localizes on the parasitophorous vacuole membrane, where it interacts with the host E3 ubiquitin ligase ITCH. Inhibition of proteasome activity or ITCH knockout prevented pyroptosis in Toxoplasma-infected Lewis rat macrophages, consistent with the "NLRP1 functional degradation model." However, there was no evidence that ITCH directly ubiquitinates or interacts with rat NLRP1. We also found that GRA35-ITCH interaction affected Toxoplasma fitness in IFNγ-activated human fibroblasts, likely due to ITCH's role in recruiting ubiquitin and the parasite-restriction factor RNF213 to the parasitophorous vacuole membrane. These findings identify a new role of host E3 ubiquitin ligase ITCH in mediating effector-triggered immunity, a critical concept that involves recognizing intracellular pathogens and initiating host innate immune responses.IMPORTANCEEffector-triggered immunity represents an innate immune defense mechanism that plays a crucial role in sensing and controlling intracellular pathogen infection. The NLRP1 inflammasome in the Lewis rats can detect Toxoplasma infection, which triggers proptosis in infected macrophages and eliminates the parasite's replication niche. The work reported here revealed that host E3 ubiquitin ligase ITCH is able to recognize and interact with Toxoplasma effector protein GRA35 localized on the parasite-host interface, leading to NLRP1 inflammasome activation in Lewis rat macrophages. Furthermore, ITCH-GRA35 interaction contributes to the restriction of Toxoplasma in human fibroblasts stimulated by IFNγ. Thus, this research provides valuable insights into understanding pathogen recognition and restriction mediated by host E3 ubiquitin ligase.

Protozoan-Derived Cytokine-Transgenic Macrophages Reverse Hepatic Fibrosis.

Macrophage therapy for liver fibrosis is on the cusp of meaningful clinical utility. Due to the heterogeneities of macrophages, it is urgent to develop safer macrophages with a more stable and defined phenotype for the treatment of liver fibrosis. Herein, a new macrophage-based immunotherapy using macrophages stably expressing a pivotal cytokine from Toxoplasma gondii, a parasite that infects ≈ 2 billion people is developed. It is found that Toxoplasma gondii macrophage migration inhibitory factor-transgenic macrophage (Mφtgmif) shows stable fibrinolysis and strong chemotactic capacity. Mφtgmif effectively ameliorates liver fibrosis and deactivates aHSCs by recruiting Ly6Chi macrophages via paracrine CCL2 and polarizing them into the restorative Ly6Clo macrophage through the secretion of CX3CL1. Remarkably, Mφtgmif exhibits even higher chemotactic potential, lower grade of inflammation, and better therapeutic effects than LPS/IFN-γ-treated macrophages, making macrophage-based immune therapy more efficient and safer. Mechanistically, TgMIF promotes CCL2 expression by activating the ERK/HMGB1/NF-κB pathway, and this event is associated with recruiting endogenous macrophages into the fibrosis liver. The findings do not merely identify viable immunotherapy for liver fibrosis but also suggest a therapeutic strategy based on the evolutionarily designed immunomodulator to treat human diseases by modifying the immune microenvironment.

Exploring the potential of Toxoplasma gondii in drug development and as a delivery system.

Immune-mediated inflammatory diseases are various groups of conditions that result in immune system disorders and increased cancer risk. Despite the identification of causative cytokines and pathways, current clinical treatment for immune-mediated inflammatory diseases is limited. In addition, immune-mediated inflammatory disease treatment can increase the risk of cancer. Several previous studies have demonstrated that Toxoplasma gondii manipulates the immune response by inhibiting or stimulating cytokines, suggesting the potential for controlling and maintaining a balanced immune system. Additionally, T. gondii also has the unique characteristic of being a so-called "Trojan horse" bacterium that can be used as a drug delivery system to treat regions that have been resistant to previous drug delivery therapies. In this study, we reviewed the potential of T. gondii in drug development and as a delivery system through current research on inflammation-regulating mechanisms in immune-mediated inflammatory diseases.

Genome-wide and targeted CRISPR screens identify RNF213 as a mediator of interferon gamma-dependent pathogen restriction in human cells.

To define cellular immunity to the intracellular pathogen Toxoplasma gondii, we performed a genome-wide CRISPR loss-of-function screen to identify genes important for (interferon gamma) IFN-γ-dependent growth restriction. We revealed a role for the tumor suppressor NF2/Merlin for maximum induction of Interferon Stimulated Genes (ISG), which are positively regulated by the transcription factor IRF-1. We then performed an ISG-targeted CRISPR screen that identified the host E3 ubiquitin ligase RNF213 as necessary for IFN-γ-mediated control of T. gondii in multiple human cell types. RNF213 was also important for control of bacterial (Mycobacterium tuberculosis) and viral (Vesicular Stomatitis Virus) pathogens in human cells. RNF213-mediated ubiquitination of the parasitophorous vacuole membrane (PVM) led to growth restriction of T. gondii in response to IFN-γ. Moreover, overexpression of RNF213 in naive cells also impaired growth of T. gondii. Surprisingly, growth inhibition did not require the autophagy protein ATG5, indicating that RNF213 initiates restriction independent of a previously described noncanonical autophagy pathway. Mutational analysis revealed that the ATPase domain of RNF213 was required for its recruitment to the PVM, while loss of a critical histidine in the RZ finger domain resulted in partial reduction of recruitment to the PVM and complete loss of ubiquitination. Both RNF213 mutants lost the ability to restrict growth of T. gondii, indicating that both recruitment and ubiquitination are required. Collectively, our findings establish RNF213 as a critical component of cell-autonomous immunity that is both necessary and sufficient for control of intracellular pathogens in human cells.

The Toxoplasma protein phosphatase 6 catalytic subunit (TgPP6C) is essential for cell cycle progression and virulence.

Protein phosphatases are post-translational regulators of Toxoplasma gondii proliferation, tachyzoite-bradyzoite differentiation and pathogenesis. Here, we identify the putative protein phosphatase 6 (TgPP6) subunits of T. gondii and elucidate their role in the parasite lytic cycle. The putative catalytic subunit TgPP6C and regulatory subunit TgPP6R likely form a complex whereas the predicted structural subunit TgPP6S, with low homology to the human PP6 structural subunit, does not coassemble with TgPP6C and TgPP6R. Functional studies showed that TgPP6C and TgPP6R are essential for parasite growth and replication. The ablation of TgPP6C significantly reduced the synchronous division of the parasite's daughter cells during endodyogeny, resulting in disordered rosettes. Moreover, the six conserved motifs of TgPP6C were required for efficient endodyogeny. Phosphoproteomic analysis revealed that ablation of TgPP6C predominately altered the phosphorylation status of proteins involved in the regulation of the parasite cell cycle. Deletion of TgPP6C significantly attenuated the parasite virulence in mice. Immunization of mice with TgPP6C-deficient type I RH strain induced protective immunity against challenge with a lethal dose of RH or PYS tachyzoites and Pru cysts. Taken together, the results show that TgPP6C contributes to the cell division, replication and pathogenicity in T. gondii.

Global Proteome-Wide Analysis of Cysteine S-Nitrosylation in Toxoplasma gondii.

Toxoplasma gondii transmits through various routes, rapidly proliferates during acute infection and causes toxoplasmosis, which is an important zoonotic disease in human and veterinary medicine. T. gondii can produce nitric oxide and derivatives, and S-nitrosylation contributes to their signaling transduction and post-translation regulation. To date, the S-nitrosylation proteome of T. gondii remains mystery. In this study, we reported the first S-nitrosylated proteome of T. gondii using mass spectrometry in combination with resin-assisted enrichment. We found that 637 proteins were S-nitrosylated, more than half of which were localized in the nucleus or cytoplasm. Motif analysis identified seven motifs. Of these motifs, five and two contained lysine and isoleucine, respectively. Gene Ontology enrichment revealed that S-nitrosylated proteins were primarily located in the inner membrane of mitochondria and other organelles. These S-nitrosylated proteins participated in diverse biological and metabolic processes, including organic acid binding, carboxylic acid binding ribose and phosphate biosynthesis. T. gondii S-nitrosylated proteins significantly contributed to glycolysis/gluconeogenesis and aminoacyl-tRNA biosynthesis. Moreover, 27 ribosomal proteins and 11 microneme proteins were identified as S-nitrosylated proteins, suggesting that proteins in the ribosome and microneme were predominantly S-nitrosylated. Protein-protein interaction analysis identified three subnetworks with high-relevancy ribosome, RNA transport and chaperonin complex components. These results imply that S-nitrosylated proteins of T. gondii are associated with protein translation in the ribosome, gene transcription, invasion and proliferation of T. gondii. Our research is the first to identify the S-nitrosylated proteomic profile of T. gondii and will provide direction to the ongoing investigation of the functions of S-nitrosylated proteins in T. gondii.

ATP synthase-associated coiled-coil-helix-coiled-coil-helix (CHCH) domain-containing proteins are critical for mitochondrial function in Toxoplasma gondii.

IMPORTANCE: Members of the coiled-coil-helix-coiled-coil-helix (CHCH) domain protein family are transported into the mitochondrial intermembrane space, where they play important roles in the biogenesis and function of the organelle. Unexpectedly, the ATP synthase of the apicomplexan Toxoplasma gondii harbors CHCH domain-containing subunits of unknown function. As no other ATP synthase studied to date contains this class of proteins, characterizing their function will be of broad interest to the fields of molecular parasitology and mitochondrial evolution. Here, we demonstrate that that two T. gondii ATP synthase subunits containing CHCH domains are required for parasite survival and for stability and function of the ATP synthase. We also show that knockdown disrupts multiple aspects of the mitochondrial morphology of T. gondii and that mutation of key residues in the CHCH domains caused mis-localization of the proteins. This work provides insight into the unique features of the apicomplexan ATP synthase, which could help to develop therapeutic interventions against this parasite and other apicomplexans, such as the malaria-causing parasite Plasmodium falciparum.

An ex vivo model of Toxoplasma recrudescence reveals developmental plasticity of the bradyzoite stage.

IMPORTANCE: The classical depiction of the Toxoplasma lifecycle is bradyzoite excystation conversion to tachyzoites, cell lysis, and immune control, followed by the reestablishment of bradyzoites and cysts. In contrast, we show that tachyzoite growth slows independent of the host immune response at a predictable time point following excystation. Furthermore, we demonstrate a host cell-dependent pathway of continuous amplification of the cyst-forming bradyzoite population. The developmental plasticity of the excysted bradyzoites further underlines the critical role the cyst plays in the flexibility of the lifecycle of this ubiquitous parasite. This revised model of Toxoplasma recrudescence uncovers previously unknown complexity in the clinically important bradyzoite stage of the parasite, which opens the door to further study these novel developmental features of the Toxoplasma intermediate life cycle.

F-actin and myosin F control apicoplast elongation dynamics which drive apicoplast-centrosome association in Toxoplasma gondii.

IMPORTANCE: Toxoplasma gondii and most other parasites in the phylum Apicomplexa contain an apicoplast, a non-photosynthetic plastid organelle required for fatty acid, isoprenoid, iron-sulfur cluster, and heme synthesis. Perturbation of apicoplast function results in parasite death. Thus, parasite survival critically depends on two cellular processes: apicoplast division to ensure every daughter parasite inherits a single apicoplast, and trafficking of nuclear encoded proteins to the apicoplast. Despite the importance of these processes, there are significant knowledge gaps in regards to the molecular mechanisms which control these processes; this is particularly true for trafficking of nuclear-encoded apicoplast proteins. This study provides crucial new insight into the timing of apicoplast protein synthesis and trafficking to the apicoplast. In addition, this study demonstrates how apicoplast-centrosome association, a key step in the apicoplast division cycle, is controlled by the actomyosin cytoskeleton.

Assessment of tissue levels of miR-146a and proinflammatory cytokines in experimental cerebral toxoplasmosis following atovaquone and clindamycin treatment: An in vivo study.

BACKGROUND: Despite recent advances for treating cerebral toxoplasmosis (CT), monitoring the parasite burden and treatment response is still challenging. miRNAs are small non-coding RNAs with regulatory functions that can be used in diagnosis and treatment monitoring. We investigated the changes in miR-146a, BAG-1 gene, IL-6, and IL-10 tissue levels in the brain of BALB/c mice with chronic CT caused by the PRU strain of T. gondii following anti-parasitic and antibiotic treatment. METHOD: Fifty-three 6-to 8-week-old BALB/c mice were infected using intraperitoneal inoculation of cerebral cysts of T. gondii PRU strain and then divided into five groups as follows: group 1 included mice treated with 100 mg/kg/d Atovaquone (AT), group 2 included mice treated with 400 mg/kg/d clindamycin (CL), group 3 included mice treated with combination therapy (AT + CL), group 4 included infected untreated mice as a positive control (PC), and; group 5 included uninfected untreated mice as negative control (NC). After the completion of the treatment course, tissue level of mir-146a, miR-155, BAG-1 gene, IL-6, and IL-10 was investigated with real-time polymerase chain reaction. The IL-6/IL-10 ratio was calculated as an indicator of immune response. Moreover, brain cyst numbers were counted on autopsy samples. RESULTS: miR-146a, IL-6, IL-10, and BAG-1 genes were expressed in PC, but not in the NC group; miR-146a, IL-6, IL-10, and BAG-1 gene expression were significantly lower in AT, CL, and AT + CL compared with PC. MiR-146a and BAG-1 levels in AT and CL were not different statistically, however, they both had lower levels compared to AT + CL (P < 0.01). There was no difference in the expression of IL-6 and IL-10 between treatment groups. BAG-1 expression was significantly lower in AT, than in CL and AT + CL (P < 0.0089 and < 0.002, respectively). The PC group showed a higher ratio of IL-6/IL-10, although this increase was not statistically significant. It is noteworthy that the treatment with AT reduced this ratio; in the inter-group comparison, this ratio showed a decrease in the AT and AT + CL compared to the PC. The number of brain tissue cysts was significantly lower in AT, CL, and AT + CL, than in PC (p < 0.0001). AT had significantly lower brain cysts than CL and AT + CL (P < 0.0001). CONCLUSION: It seems that the factors studied in the current research (microRNA and cytokines) are a suitable index for evaluating the response to antiparasitic and antibiotic treatment. However, more studies should be conducted in the future to confirm our findings.

The Toxoplasma gondii effector GRA83 modulates the host's innate immune response to regulate parasite infection.

Toxoplasma gondii's propensity to infect its host and cause disease is highly dependent on its ability to modulate host cell functions. One of the strategies the parasite uses to accomplish this is via the export of effector proteins from the secretory dense granules. Dense granule (GRA) proteins are known to play roles in nutrient acquisition, host cell cycle manipulation, and immune regulation. Here, we characterize a novel dense granule protein named GRA83, which localizes to the parasitophorous vacuole (PV) in tachyzoites and bradyzoites. Disruption of GRA83 results in increased virulence, weight loss, and parasitemia during the acute infection, as well as a marked increase in the cyst burden during the chronic infection. This increased parasitemia was associated with an accumulation of inflammatory infiltrates in tissues in both acute and chronic infections. Murine macrophages infected with ∆gra83 tachyzoites produced less interleukin-12 (IL-12) in vitro, which was confirmed with reduced IL-12 and interferon-gamma in vivo. This dysregulation of cytokines correlates with reduced nuclear translocation of the p65 subunit of the nuclear factor-κB (NF-κB) complex. While GRA15 similarly regulates NF-κB, infection with ∆gra83/∆gra15 parasites did not further reduce p65 translocation to the host cell nucleus, suggesting these GRAs function in converging pathways. We also used proximity labeling experiments to reveal candidate GRA83 interacting T. gondii-derived partners. Taken together, this work reveals a novel effector that stimulates the innate immune response, enabling the host to limit the parasite burden. Importance Toxoplasma gondii poses a significant public health concern as it is recognized as one of the leading foodborne pathogens in the United States. Infection with the parasite can cause congenital defects in neonates, life-threatening complications in immunosuppressed patients, and ocular disease. Specialized secretory organelles, including the dense granules, play an important role in the parasite's ability to efficiently invade and regulate components of the host's infection response machinery to limit parasite clearance and establish an acute infection. Toxoplasma's ability to avoid early clearance, while also successfully infecting the host long enough to establish a persistent chronic infection, is crucial in allowing for its transmission to a new host. While multiple GRAs directly modulate host signaling pathways, they do so in various ways highlighting the parasite's diverse arsenal of effectors that govern infection. Understanding how parasite-derived effectors harness host functions to evade defenses yet ensure a robust infection is important for understanding the complexity of the pathogen's tightly regulated infection. In this study, we characterize a novel secreted protein named GRA83 that stimulates the host cell's response to limit infection.

A transcriptional network required for bradyzoite development in Toxoplasma gondii is dispensable for recrudescent disease.

Identification of regulators of Toxoplasma gondii bradyzoite development and cyst formation is the most direct way to address the importance of parasite development in long-term persistence and reactivation of this parasite. Here we show that a T. gondii gene (named Regulator of Cystogenesis 1; ROCY1) is sufficient for T. gondii bradyzoite formation in vitro and in vivo. ROCY1 encodes an RNA binding protein that has a preference for 3' regulatory regions of hundreds of T. gondii transcripts, and its RNA-binding domains are required to mediate bradyzoite development. Female mice infected with ΔROCY1 parasites have reduced (>90%) cyst burden. While viable parasites can be cultivated from brain tissue for up to 6 months post-infection, chronic brain-resident ΔROCY1 parasites have reduced oral infectivity compared to wild type. Despite clear defects in bradyzoite formation and oral infectivity, ΔROCY1 parasites were able to reactivate with similar timing and magnitude as wild type parasites for up to 5 months post-infection. Therefore while ROCY1 is a critical regulator of the bradyzoite developmental pathway, it is not required for parasite reactivation, raising new questions about the persisting life stage responsible for causing recrudescent disease.

Quercetin inhibits Toxoplasma gondii tachyzoite proliferation and acts synergically with azithromycin.

Quercetin (QUE) is a natural polyphenol known to have numerous pharmacological properties against infectious and non-infectious diseases. Azithromycin (AZ) is an antibiotic that belongs to the azalide class of antimicrobials and an antiparasitic that is known to be effective in combination with clindamycin against pyrimethamine/sulfadiazine-resistant Toxoplasma gondii tachyzoites in clinical settings. Both compounds are known to target protein synthesis and have anti-inflammatory properties. However, little is known about QUE and AZ synergistic interaction against T. gondii growth. Here, we report for the first time the effects of the combination of QUE and AZ on T. gondii growth. The 50% inhibitory concentration (IC50) for QUE at 72 h of interaction was determined to be 0.50 µM, whereas AZ gave an IC50 value of 0.66 µM at 72 h of interaction with parasites. Combination testing of QUE and AZ in a ratio of 2:1 (QUE:AZ) showed an IC50 value of 0.081 µM. Interestingly, a fractional inhibitory index value of 0.28 was observed, indicating a strong synergy. QUE was also found to upregulate the generation of reactive oxygen species and cause dysfunction of the mitochondria membrane of both intracellular and extracellular T. gondii tachyzoites. Overall, the results indicate that QUE is a novel lead capable of synergizing with AZ for inhibiting T. gondii growth and may merit future investigation in vivo for possible combination drug development.

The vacuolar iron transporter mediates iron detoxification in Toxoplasma gondii.

Iron is essential to cells as a cofactor in enzymes of respiration and replication, however without correct storage, iron leads to the formation of dangerous oxygen radicals. In yeast and plants, iron is transported into a membrane-bound vacuole by the vacuolar iron transporter (VIT). This transporter is conserved in the apicomplexan family of obligate intracellular parasites, including in Toxoplasma gondii. Here, we assess the role of VIT and iron storage in T. gondii. By deleting VIT, we find a slight growth defect in vitro, and iron hypersensitivity, confirming its essential role in parasite iron detoxification, which can be rescued by scavenging of oxygen radicals. We show VIT expression is regulated by iron at transcript and protein levels, and by altering VIT localization. In the absence of VIT, T. gondii responds by altering expression of iron metabolism genes and by increasing antioxidant protein catalase activity. We also show that iron detoxification has an important role both in parasite survival within macrophages and in virulence in a mouse model. Together, by demonstrating a critical role for VIT during iron detoxification in T. gondii, we reveal the importance of iron storage in the parasite and provide the first insight into the machinery involved.