Toxoplasma gondii: Identification and characterization of bradyzoite-specific deoxyribose phosphate aldolase-like gene (TgDPA)

Toxoplasma gondii undergoes stage conversion from tachyzoites to bradyzoites in intermediate hosts. There have been many reports on bradyzoite-specific genes which are thought to be involved in stage conversion. Here, we described a novel T. gondii deoxyribose phosphate aldolase-like gene (TgDPA) expressing predominantly in bradyzoites. The TgDPA gene encodes 286 amino acids having a predicted molecular weight of 31 kDa. Sequence analysis revealed that TgDPA had a deoxyribose phosphate aldolase (DeoC) domain with about 30% homology with its Escherichia coli counterpart. RT- and quantitative PCR analyses showed that the TgDPA gene was more expressed in bradyzoites and that its expression gradually increased during in vitro tachyzoite-to-bradyzoite stage conversion. A polyclonal antibody against recombinant TgDPA protein was raised in rabbits, and immunofluorescent analysis demonstrated that TgDPA was expressed in bradyzoites in vivo and in vitro. These findings indicate that the TgDPA gene is a new bradyzoite-specific marker and might play a role in bradyzoites.     

Toxoplasma gondii: Simple duplex RT-PCR assay for detecting SAG1 and BAG1 genes during stage conversion in immunosuppressed mice

Toxoplasmic encephalitis (TE) is caused by reactivation of dormant bradyzoites into rapidly dividing tachyzoites of the apicomplexan parasite Toxoplasma gondii in immune-compromised hosts. Diagnosis of this life-threatening disease is complicated, since it is difficult to distinguish between these two stages. It is, therefore, mainly based on a test positive for T. gondii antibodies, and specific clinical symptoms. We developed a duplex RT-PCR to detect the expression of bradyzoite (BAG1) and tachyzoite (SAG1) specific genes simultaneously during tachyzoite/bradyzoite stage conversion. The conversion reaction was observed in many organs of experimental mice, indicated by tachyzoites in the cerebrum, cerebellum, heart and lung, beginning in week 1 after the suppression period, and continuing until the end. Bradyzoites were also detected in nearly all organs throughout the study, suggesting that during the reactivation period, bradyzoites not only escape from cysts and reinvade neighboring cells as tachyzoites, but are also driven into developing new bradyzoites. The results of our study show that duplex RT-PCR is an easy, rapid, sensitive, and reproducible method, which is particularly valuable when numerous samples must be analyzed. This technique may usefully serve as an alternate tool for diagnosing TE in severely immunocompromised patients.     

Microplate assay for screening Toxoplasma gondii bradyzoite differentiation with DUAL luciferase assay

Toxoplasma gondii can differentiate into tachyzoites or bradyzoites. To accelerate the investigation of bradyzoite differentiation mechanisms, we constructed a reporter parasite, PLK/DLUC_1C9, for a high-throughput assay. PLK/DLUC_1C9 expressed firefly luciferase under the bradyzoite-specific BAG1 promoter. Firefly luciferase activity was detected with a minimum of 10² parasites induced by pH 8.1. To normalize bradyzoite differentiation, PLK/DLUC_1C9 expressed Renilla luciferase under the parasite’s α-tubulin promoter. Renilla luciferase activity was detected with at least 10² parasites. By using PLK/DLUC_1C9 with this 96-well format screening system, we found that the protein kinase inhibitor analogs, bumped kinase inhibitors 1NM-PP1, 3MB-PP1, and 3BrB-PP1, had bradyzoite-inducing effects.     

Role of amylopectin synthesis in Toxoplasma gondii and its implication in vaccine development against toxoplasmosis

Toxoplasma gondii is a ubiquitous pathogen infecting one-third of the global population. A significant fraction of toxoplasmosis cases is caused by reactivation of existing chronic infections. The encysted bradyzoites during chronic infection accumulate high levels of amylopectin that is barely present in fast-replicating tachyzoites. However, the physiological significance of amylopectin is not fully understood. Here, we identified a starch synthase (SS) that is required for amylopectin synthesis in T. gondii. Genetic ablation of SS abolished amylopectin production, reduced tachyzoite proliferation, and impaired the recrudescence of bradyzoites to tachyzoites. Disruption of the parasite Ca²⁺-dependent protein kinase 2 (CDPK2) was previously shown to cause massive amylopectin accumulation and bradyzoite death. Therefore, the Δcdpk2 mutant is thought to be a vaccine candidate. Notably, deleting SS in a Δcdpk2 mutant completely abolished starch accrual and restored cyst formation as well as virulence in mice. Together these results suggest that regulated amylopectin production is critical for the optimal growth, development and virulence of Toxoplasma. Not least, our data underscore a potential drawback of the Δcdpk2 mutant as a vaccine candidate as it may regain full virulence by mutating amylopectin synthesis genes like SS.     

Toxoplasma gondii within skeletal muscle cells: a critical interplay for food-borne parasite transmission

Toxoplasma gondii infects virtually any nucleated cell type of warm-blooded animals and humans including skeletal muscle cells (SkMCs). Infection of SkMCs by T. gondii, differentiation from the highly replicative tachyzoites to dormant bradyzoites and tissue cyst formation are crucial for parasite persistence in muscle tissue. These processes are also prerequisites for one of the major routes of transmission to humans via undercooked or cured meat products. Evidence obtained in vitro and in vivo indicates that SkMCs are indeed a preferred cell type for tissue cyst formation and long-term persistence of T. gondii. This raises intriguing questions about what makes SkMCs a suitable environment for parasite persistence and how the SkMC–T. gondii interaction is regulated. Recent data from our laboratory show that differentiation of SkMCs from myoblasts to syncytial myotubes, rather than the cell type itself, is critical for parasite growth, bradyzoite formation and tissue cyst maturation. Myotube formation is accompanied by a permanent withdrawal from the cell cycle, and the negative cell cycle regulator cell division autoantigen (CDA)-1 directly or indirectly promotes T. gondii stage conversion in SkMCs. Moreover, host cell cycle regulators are specifically modulated in mature myotubes, but not myoblasts, following infection. Myotubes also up-regulate the expression of various pro-inflammatory cytokines and chemokines after T. gondii infection and they respond to IFN-γ by exerting potent anti-parasitic activity. This highlights that mature myotubes are active participants rather than passive targets of the local immune response to T. gondii which may also govern the interaction between SkMCs and the parasite.     

Functional analysis of Toxoplasma lactate dehydrogenases suggests critical roles of lactate fermentation for parasite growth in vivo

Glycolysis was thought to be the major pathway of energy supply in both fast‐replicating tachyzoites and slowly growing bradyzoites of Toxoplasma gondii. However, its biological significance has not been clearly verified. The genome of T. gondii encodes two lactate dehydrogenases (LDHs), which are differentially expressed in tachyzoites and bradyzoites. In this study, we knocked out the two LDH genes individually and in combination and found that neither gene was required for tachyzoite growth in vitro under standard growth conditions. However, during infection in mice, Δldh1 and Δldh1 Δldh2 mutants were unable to propagate and displayed significant virulence attenuation and cyst formation defects. LDH2 only played minor roles in these processes. To further elucidate the mechanisms underlying the critical requirement of LDH in vivo, we found that Δldh1 Δldh2 mutants replicated significantly more slowly than wild‐type parasites when cultured under conditions with physiological levels of oxygen (3%). In addition, Δldh1 Δldh2 mutants were more susceptible to the oxidative phosphorylation inhibitor oligomycin A. Together these results suggest that lactate fermentation is critical for parasite growth under physiological conditions, likely because energy production from oxidative phosphorylation is insufficient when oxygen is limited and lactate fermentation becomes a key supplementation.     

Glycolysis is important for optimal asexual growth and formation of mature tissue cysts by Toxoplasma gondii

Toxoplasma gondii can grow and replicate using either glucose or glutamine as the major carbon source. Here, we have studied the essentiality of glycolysis in the tachyzoite and bradyzoite stages of T. gondii, using transgenic parasites that lack a functional hexokinase gene (Δhk) in RH (Type-1) and Prugniaud (Type-II) strain parasites. Tachyzoite stage Δhk parasites exhibit a fitness defect similar to that reported previously for the major glucose transporter mutant, and remain virulent in mice. However, although Prugniaud strain Δhk tachyzoites were capable of transforming into bradyzoites in vitro, they were severely compromised in their ability to make mature bradyzoite cysts in the brain tissue of mice. Isotopic labelling studies reveal that glucose-deprived tacyzoites utilise glutamine to replenish glycolytic and pentose phosphate pathway intermediates via gluconeogenesis. Interestingly, while glutamine-deprived intracellular Δhk tachyzoites continued to replicate, extracellular parasites were unable to efficiently invade host cells. Further, studies on mutant tachyzoites lacking a functional phosphoenolpyruvate carboxykinase (Δpepck1) revealed that glutaminolysis is the sole source of gluconeogenic flux in glucose-deprived parasites. In addition, glutaminolysis is essential for sustaining oxidative phosphorylation in Δhk parasites, while wild type (wt) and Δpepck1 parasites can obtain ATP from either glycolysis or oxidative phosphorylation. This study provides insights into the role of nutrient metabolism during asexual propagation and development of T. gondii, and validates the versatile nature of central carbon and energy metabolism in this parasite.     

Monoclonal antibodies against nucleoside triphosphate hydrolase-II can reduce the replication of Toxoplasma gondii

Nucleoside triphosphate hydrolase (NTPase) is an abundant protein secreted by the obligate protozoan parasite Toxoplasma gondii, which has a wide specificity toward NTP. In the present study, two monoclonal antibodies (mAbs, MNT1 and MNT2) against recombinant T. gondii NTPase-II (rTgNTPase-II) were developed. Western blot analysis displayed that these two mAbs can recognize specifically rTgNTPase-II as well as a 63 kDa molecule in tachyzoites soluble antigens that corresponded to native NTPase-II. T. gondii tachyzoites pretreated with two mAbs were observed under Confocal Laser Microscope and a specific reaction was displayed on tachyzoites after indirect fluorescence antibody test (IFAT). When COS-7 cells were co-cultured with tachyzoites pretreated with two mAbs, the number of intracellular parasites per infected cell was significantly decreased compared with the control. Furthermore, incubation of T. gondii tachyzoites with two mAbs can inhibit NTPase activity in the presence of dithiothreitol, which hinted that the reduction of tachyzoite replication might be owing to the inhibition of NTPase-II by the mAbs. The passive immunization test indicated that the transferred mAbs can significantly prolong the survival time of challenge infected mice. Taken together, we concluded that the mAbs against NTPase-II can reduce the replication of T. gondii and have a crucial effect on the protection of host from T. gondii infection.     

Identification of Toxoplasma gondii cAMP dependent protein kinase and its role in the tachyzoite growth

Background

cAMP-dependent protein kinase (PKA) has been implicated in the asexual stage of the Toxoplasma gondii life cycle through assaying the effect of a PKA-specific inhibitor on its growth rate. Since inhibition of the host cell PKA cannot be ruled out, a more precise evaluation of the role of PKA, as well as characterization of the kinase itself, is necessary.

Methodology/Principal Finding

The inhibitory effects of two PKA inhibitors, H89, an ATP-competitive chemical inhibitor, and PKI, a substrate-competitive mammalian natural peptide inhibitor, were estimated. In the in vitro kinase assay, the inhibitory effect of PKI on a recombinant T. gondii PKA catalytic subunit (TgPKA-C) was weaker compared to that on mammalian PKA-C. In a tachyzoite growth assay, PKI had little effect on the growth of tachyzoites, whereas H89 strongly inhibited it. Moreover, T. gondii PKA regulatory subunit (TgPKA-R)-overexpressing tachyzoites showed a significant growth defect.

Conclusions/Significance

Our data suggest that PKA plays an important role in the growth of tachyzoites, and the inhibitory effect of substrate-competitive inhibitor PKI on T. gondii PKA was low compared to that of the ATP competitive inhibitor H89.     

Toxoplasma gondii-positive human sera recognise intracellular tachyzoites and bradyzoites with diverse patterns of immunoreactivity

Antibody detection assays have long been the first line test to confirm infection with the zoonotic parasite Toxoplasma gondii. However, challenges exist with serological diagnosis, especially distinguishing between acute, latent and reactivation disease states. The sensitivity and specificity of serological tests might be improved by testing for antibodies against parasite antigens other than those typically found on the parasite surface during the acute stage. To this end, we analysed the reactivity profile of human sera, identified as positive for anti-Toxoplasma gondii IgG in traditional assays, by indirect immunofluorescence reactivity to acute stage intracellular tachyzoites and in vitro-induced latent stage bradyzoites. The majority of anti-Toxoplasma gondii IgG positive sera recognised both intracellularly replicating tachyzoites and in vitro-induced bradyzoites with varying patterns of immune-reactivity. Furthermore, anti-bradyzoite antibodies were not detected in sera that were IgM-positive/IgG-negative. These results demonstrate that anti-Toxoplasma gondii-positive sera may contain antibodies to a variety of antigens in addition to those traditionally used in serological tests, and suggest the need for further investigations into the utility of anti-bradyzoite-specific antibodies to aid in diagnosis of Toxoplasma gondii infection.     

Suppressed CD31 Expression in Sarcoma-180 Tumors after Injection with Toxoplasma gondii Lysate Antigen in BALB/c Mice

The anti-tumorigenic effects of Toxoplasma gondii (RH) antigens were studied in a murine sarcoma-180 tumor model. To determine the anti-tumor effects, the reduction in tumor size and expression of CD31 (an angiogenesis marker in the tumor tissue) were examined after injection of BALB/c mice with T. gondii lysate antigen (TLA) or formalin-fixed, proliferation-inhibited, T. gondii tachyzoites. Tumors were successfully produced by an intradermal injection of sarcoma-180 cells with plain Matrigel in the mid-backs of mice. After injection with TLA or formalin-fixed T. gondii tachyzoites, the increase in tumor size and weight nearly stopped while tumor growth continued in control mice that were injected with PBS. CD31 expression in TLA-treated or formalin-fixed T. gondii-injected mice was lower than the control mice. Accordingly, the present study shows that the treatment of mice with formalin-fixed T. gondii or TLA in the murine sarcoma-180 tumor model results in a decrease of both tumor size and CD31 expression.     

Toxoplasma gondii: an improved rat model of congenital infection

The objective of this study was to refine the rat model of congenital toxoplasmosis. In Fischer rats we found that visualization of spermatozoa in vaginal exudates and the detection of at least 6 g body weight increase between days 9 and 12 of pregnancy, allowed the diagnosis and timing of pregnancy with 60% specificity and 84% sensitivity. A dose of 10⁴Toxoplasma gondii bradyzoites or 10²T. gondii oocysts of the Prugniaud strain resulted in more than 50% of congenital infection of the rat litters. Transmission of T. gondii via lactation was not detected in rats inoculated with either bradyzoites or oocysts. Bioassays of 51 neonates born from mothers inoculated with bradyzoites (in tissue cysts) and 29 neonates from mothers inoculated with oocysts demonstrated that both liver and lungs can be used for the diagnosis of congenital transmission in this model.     

Pathogenicity of Five Strains of Toxoplasma gondii from Different Animals to Chickens

Toxoplasma gondii is a protozoan parasite with a broad range of intermediate hosts. Chickens as important food-producing animals can also serve as intermediate hosts. To date, experimental studies on the pathogenicity of T. gondii in broiler chickens were rarely reported. The objective of the present study was to compare the pathogenicity of 5 different T. gondii strains (RH, CN, JS, CAT2, and CAT3) from various host species origin in 10-day-old chickens. Each group of chickens was infected intraperitoneally with 5×10⁸, 1×10⁸, 1×10⁷, and 1×10⁶ tachyzoites of the 5 strains, respectively. The negative control group was mockly inoculated with PBS alone. After infection, clinical symptoms and rectal temperatures of all the chickens were checked daily. Dead chickens during acute phage of the infection were checked for T. gondii tachyzoites by microscope, while living cases were checked for T. gondii infection at day 53 post-inoculation (PI) by PCR method. Histopathological sections were used to observe the pathological changes in the dead chickens and the living animals at day 53 PI. No significant differences were found in survival periods, histopathological findings, and clinical symptoms among the chickens infected with the RH, CN, CAT2, and CAT3 strains. Histopathological findings and clinical symptoms of the JS (chicken origin) group were similar to the others. However, average survival times of infected chickens of the JS group inoculated with 5×10⁸ and 1×10⁸ tachyzoites were 30.0 and 188.4 hr, respectively, significantly shorter than those of the other 4 mammalian isolates. Chickens exposed to 10⁸ of T. gondii tachyzoites and higher showed acute signs of toxoplasmosis, and the lesions were relatively more severe than those exposed to lower doses. The results indicated that the pathogenicity of JS strain was comparatively stronger to the chicken, and the pathogenicity was dose-dependent.     

Sterculic Acid and Its Analogues Are Potent Inhibitors of Toxoplasma gondii

Toxoplasmosis is a serious disease caused by Toxoplasma gondii, one of the most widespread parasites in the world. Lipid metabolism is important in the intracellular stage of T. gondii. Stearoyl-CoA desaturase (SCD), a key enzyme for the synthesis of unsaturated fatty acid is predicted to exist in T. gondii. Sterculic acid has been shown to specifically inhibit SCD activity. Here, we examined whether sterculic acid and its methyl ester analogues exhibit anti-T. gondii effects in vitro. T. gondii-infected Vero cells were disintegrated at 36 hr because of the propagation and egress of intracellular tachyzoites. All test compounds inhibited tachyzoite propagation and egress, reducing the number of ruptured Vero cells by the parasites. Sterculic acid and the methyl esters also inhibited replication of intracellular tachyzoites in HFF cells. Among the test compounds, sterculic acid showed the most potent activity against T. gondii, with an EC₅₀ value of 36.2 μM, compared with EC₅₀ values of 248-428 μM for the methyl esters. Our study demonstrated that sterculic acid and its analogues are effective in inhibition of T. gondii growth in vitro, suggesting that these compounds or analogues targeting SCD could be effective agents for the treatment of toxoplasmosis.