Toxoplasma gondii 70 kDa Heat Shock Protein: Systemic Detection Is Associated with the Death of the Parasites by the Immune Response and Its Increased Expression in the Brain Is Associated with Parasite Replication

The heat shock protein of Toxoplasma gondii (TgHSP70) is a parasite virulence factor that is expressed during T. gondii stage conversion. To verify the effect of dexamethasone (DXM)-induced infection reactivation in the TgHSP70-specific humoral immune response and the presence of the protein in the mouse brain, we produced recombinant TgHSP70 and anti-TgHSP70 IgY antibodies to detect the protein, the specific antibody and levels of immune complexes (ICs) systemically, as well as the protein in the brain of resistant (BALB/c) and susceptible (C57BL/6) mice. It was observed higher TgHSP70-specific antibody titers in serum samples of BALB/c compared with C57BL/6 mice. However, the susceptible mice presented the highest levels of TgHSP70 systemically and no detection of specific ICs. The DXM treatment induced increased parasitism and lower inflammatory changes in the brain of C57BL/6, but did not interfere with the cerebral parasitism in BALB/c mice. Additionally, DXM treatment decreased the serological TgHSP70 concentration in both mouse lineages. C57BL/6 mice presented high expression of TgHSP70 in the brain with the progression of infection and under DXM treatment. Taken together, these data indicate that the TgHSP70 release into the bloodstream depends on the death of the parasites mediated by the host immune response, whereas the increased TgHSP70 expression in the brain depends on the multiplication rate of the parasite.     

Toxoplasma gondii: protection against colonization of the brain and muscles in a rat model

The objective was to test immune protection against the formation of Toxoplasma gondii tissue cysts in rats. It has been previously shown that 50 T. gondii tissue cysts of strain Me49 are not pathogenic for CF-1 mice, whereas 1 T. gondii tissue cyst of strain M-7741, can be lethal for mice 11-13 days after subcutaneous or oral administration. In the present study, ten rats were fed T. gondii oocysts of strain Me49 and after a further 30 days they were each orally challenged with T. gondii oocysts of strain M-7741. Thirty days after this, they were euthanased and brain and muscle samples inoculated subcutaneously or orally dosed, respectively, to mice for bioassay. None of the mice died, whereas all the mice that were inoculated with brain homogenates or were fed muscle samples from four non-immunized rats that had been inoculated with T. gondii oocysts of strain M-7741, died. These results encourage further research towards achieving vaccinal protection against the formation of T. gondii tissue cysts in meat animals and people.     

Genotypes and Mouse Virulence of Toxoplasma gondii Isolates from Animals and Humans in China

Background

Recent population structure studies of T. gondii revealed that a few major clonal lineages predominated in different geographical regions. T. gondii in South America is genetically and biologically divergent, whereas this parasite is remarkably clonal in North America and Europe with a few major lineages including Types I, II and III. Information on genotypes and mouse virulence of T. gondii isolates from China is scarce and insufficient to investigate its population structure, evolution, and transmission.

Methodology/Principal Findings

Genotyping of 23 T. gondii isolates from different hosts using 10 markers for PCR-restriction fragment length polymorphism analyses (SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico) revealed five genotypes; among them three genotypes were atypical and two were archetypal. Fifteen strains belong to the Chinese 1 lineage, which has been previously reported as a widespread lineage from swine, cats, and humans in China. Two human isolates fall into the type I and II lineages and the remaining isolates belong to two new atypical genotypes (ToxoDB#204 and #205) which has never been reported in China. Our results show that these genotypes of T. gondii isolates are intermediately or highly virulent in mice except for the strain TgCtwh6, which maintained parasitemia in mice for 35 days post infection although it possesses the uniform genotype of Chinese 1. Additionally, phylogenetic network analyses of all isolates of genotype Chinese 1 are identical, and there is no variation based on the sequence data generated for four introns (EF1, HP2, UPRT1 and UPRT7) and two dense granule proteins (GRA6 and GRA7).

Conclusion/Significance

A limited genetic diversity was found and genotype Chinese 1 (ToxoDB#9) is dominantly circulating in mainland China. The results will provide a useful profile for deep insight to the population structure, epidemiology and biological characteristics of T. gondii in China.     

Coordinated action of multiple transporters in the acquisition of essential cationic amino acids by the intracellular parasite Toxoplasma gondii

Intracellular parasites of the phylum Apicomplexa are dependent on the scavenging of essential amino acids from their hosts. We previously identified a large family of apicomplexan-specific plasma membrane-localized amino acid transporters, the ApiATs, and showed that the Toxoplasma gondii transporter TgApiAT1 functions in the selective uptake of arginine. TgApiAT1 is essential for parasite virulence, but dispensable for parasite growth in medium containing high concentrations of arginine, indicating the presence of at least one other arginine transporter. Here we identify TgApiAT6-1 as the second arginine transporter. Using a combination of parasite assays and heterologous characterisation of TgApiAT6-1 in Xenopus laevis oocytes, we demonstrate that TgApiAT6-1 is a general cationic amino acid transporter that mediates both the high-affinity uptake of lysine and the low-affinity uptake of arginine. TgApiAT6-1 is the primary lysine transporter in the disease-causing tachyzoite stage of T. gondii and is essential for parasite proliferation. We demonstrate that the uptake of cationic amino acids by TgApiAT6-1 is ‘trans-stimulated’ by cationic and neutral amino acids and is likely promoted by an inwardly negative membrane potential. These findings demonstrate that T. gondii has evolved overlapping transport mechanisms for the uptake of essential cationic amino acids, and we draw together our findings into a comprehensive model that highlights the finely-tuned, regulated processes that mediate cationic amino acid scavenging by these intracellular parasites.     

Isolation and Genotyping of Toxoplasma gondii Strains in Ovine Aborted Fetuses in Khorasan Razavi Province,Iran

Toxoplasmosis is an important zoonotic disease that can cause abortion in humans and animals. The aim of this study was isolation and subsequent genotyping of Toxoplasma gondii isolates in ovine aborted fetuses. During 2012-2013, 39 ovine aborted fetuses were collected from sheep flocks in Khorasan Razavi Province, Iran. The brain samples were screened for detection of the parasite DNA by nested PCR. The positive brain samples were bioassayed in Webster Swiss mice. The serum samples of mice were examined for T. gondii antibodies by IFAT at 6 weeks post inoculation, and T. gondii cysts were searched in brain tissue samples of seropositive mice. The positive samples were genotyped by using a PCR-RLFP method. Subsequently, GRA6 sequences of isolates were analyzed using a phylogenetic method. The results revealed that T. gondii DNA was detected in 54% (20/37, 95% CI 38.4-69.0%) brain samples of ovine aborted fetuses. In bioassay of mice, only 2 samples were virulent and the mice were killed at 30 days post inoculation, while the others were non-virulent to mice. The size of cysts ranged 7-22 µm. Complete genotyping data for GRA6 locus were observed in 5 of the 20 samples. PCR-RLFP results and phylogenetic analysis revealed that all of the isolated samples were closely related to type I. For the first time, we could genotype and report T. gondii isolates from ovine aborted fetuses in Khorasan Razavi Province, Iran. The results indicate that the T. gondii isolates are genetically related to type I, although most of them were non-virulent for mice.     

Inducible nitric oxide synthase in innate immune cells is important for restricting cyst formation of Toxoplasma gondii in the brain but not required for the protective immune process to remove the cysts

Significantly larger numbers of Toxoplasma gondii cysts were detected in the brains of RAG1^?/?NOS2^?/? than RAG1^?/? mice following infection. In contrast, the cyst numbers markedly decreased in a same manner in both strains of mice after receiving CD8⁺ immune T cells. Thus, NOS2-mediated innate immunity is important for inhibiting formation of cysts in the brain but not required for the T cell-initiated cyst removal, which is associated with phagocyte accumulation. Treatment with chloroquine, an inhibitor of endolysosomal acidification, partially but significantly inhibited the T cell-mediated cyst removal, suggesting that phagosome–lysosome fusion could be involved in the T. gondii cyst elimination.     

The distribution of Toxoplasma gondii cysts in the brain of a mouse with latent toxoplasmosis: implications for the behavioral manipulation hypothesis

Background

The highly prevalent parasite Toxoplasma gondii reportedly manipulates rodent behavior to enhance the likelihood of transmission to its definitive cat host. The proximate mechanisms underlying this adaptive manipulation remain largely unclear, though a growing body of evidence suggests that the parasite-entrained dysregulation of dopamine metabolism plays a central role. Paradoxically, the distribution of the parasite in the brain has received only scant attention.

Methodology/Principal Findings

The distributions of T. gondii cysts and histopathological lesions in the brains of CD1 mice with latent toxoplasmosis were analyzed using standard histological techniques. Mice were infected per orally with 10 tissue cysts of the avirulent HIF strain of T. gondii at six months of age and examined 18 weeks later. The cysts were distributed throughout the brain and selective tropism of the parasite toward a particular functional system was not observed. Importantly, the cysts were not preferentially associated with the dopaminergic system and absent from the hypothalamic defensive system. The striking interindividual differences in the total parasite load and cyst distribution indicate a probabilistic nature of brain infestation. Still, some brain regions were consistently more infected than others. These included the olfactory bulb, the entorhinal, somatosensory, motor and orbital, frontal association and visual cortices, and, importantly, the hippocampus and the amygdala. By contrast, a consistently low incidence of tissue cysts was recorded in the cerebellum, the pontine nuclei, the caudate putamen and virtually all compact masses of myelinated axons. Numerous perivascular and leptomeningeal infiltrations of inflammatory cells were observed, but they were not associated with intracellular cysts.

Conclusion/Significance

The observed pattern of T. gondii distribution stems from uneven brain colonization during acute infection and explains numerous behavioral abnormalities observed in the chronically infected rodents. Thus, the parasite can effectively change behavioral phenotype of infected hosts despite the absence of well targeted tropism.     

Phosphatidylethanolamine Synthesis in the Parasite Mitochondrion Is Required for Efficient Growth but Dispensable for Survival of Toxoplasma gondii

Toxoplasma gondii is a highly prevalent obligate intracellular parasite of the phylum Apicomplexa, which also includes other parasites of clinical and/or veterinary importance, such as Plasmodium, Cryptosporidium, and Eimeria. Acute infection by Toxoplasma is hallmarked by rapid proliferation in its host cells and requires a significant synthesis of parasite membranes. Phosphatidylethanolamine (PtdEtn) is the second major phospholipid class in T. gondii. Here, we reveal that PtdEtn is produced in the parasite mitochondrion and parasitophorous vacuole by decarboxylation of phosphatidylserine (PtdSer) and in the endoplasmic reticulum by fusion of CDP-ethanolamine and diacylglycerol. PtdEtn in the mitochondrion is synthesized by a phosphatidylserine decarboxylase (TgPSD1mt) of the type I class. TgPSD1mt harbors a targeting peptide at its N terminus that is required for the mitochondrial localization but not for the catalytic activity. Ablation of TgPSD1mt expression caused up to 45% growth impairment in the parasite mutant. The PtdEtn content of the mutant was unaffected, however, suggesting the presence of compensatory mechanisms. Indeed, metabolic labeling revealed an increased usage of ethanolamine for PtdEtn synthesis by the mutant. Likewise, depletion of nutrients exacerbated the growth defect (∼56%), which was partially restored by ethanolamine. Besides, the survival and residual growth of the TgPSD1mt mutant in the nutrient-depleted medium also indicated additional routes of PtdEtn biogenesis, such as acquisition of host-derived lipid. Collectively, the work demonstrates a metabolic cooperativity between the parasite organelles, which ensures a sustained lipid synthesis, survival and growth of T. gondii in varying nutritional milieus.     

Imidazo[1,2-b]pyridazines targeting Toxoplasma gondii calcium-dependent protein kinase 1 decrease the parasite burden in mice with acute toxoplasmosis

The current therapeutic arsenal for toxoplasmosis is restricted to drugs non-specific to the parasite which cause important side effects. Development of more efficient and specific anti-Toxoplasma compounds is urgently needed. Imidazo[1,2-b]pyridazines designed to inhibit the calcium-dependent protein kinase 1 of Toxoplasma gondii (TgCDPK1) and effective against tachyzoite growth in vitro at submicromolar ranges were modified into hydrochloride salts to be administered in vivo in a mouse model of acute toxoplasmosis. All protonated imidazo[1,2-b]pyridazine salts (SP230, SP231 and SP232) maintained their activity on TgCDPK1 and T. gondii tachyzoites. Rat and mouse liver microsomes were used to predict half-life and intrinsic clearance, and the pharmacokinetic profile of the most rapidly degraded imidazo[1,2b]pyridazine salt (SP230) was determined in serum, brain and lungs of mice after a single administration of 50?mg/kg. Compounds were then tested in vivo in a murine model of acute toxoplasmosis. Mice infected with tachyzoites of the ME49 strain of T. gondii were treated for 4, 7 or 8?days with 25 or 50?mg/kg/day of SP230, SP231 or SP232. The parasite burdens were strongly diminished (>90% reduction under some conditions) in the spleen and the lungs of mice treated with imidazo[1,2-b]pyridazine salts compared with untreated mice, without the need for pre-treatment. Moreover, no increases in the levels of hepatic and renal toxicity markers were observed, demonstrating no significant signs of short-term toxicity. To conclude, imidazo[1,2-b]pyridazine salts have strong efficacy in vivo on acute toxoplasmosis and should be further tested in a model of mouse congenital toxoplasmosis.     

Two viable Toxoplasma gondii isolates from red-necked wallaby (Macropus rufogriseus) and red kangaroo (M. rufus)

Wallabies and kangaroos are susceptible to Toxoplasma gondii. However, little information concerning T. gondii infection in captive macropods is available. Three dead macropods collected from a zoo exhibited no clinical symptoms associated with toxoplasmosis. Heart fluids were tested for T. gondii antibodies using a modified agglutination test. T. gondii DNA samples derived from macropod tissues were tested by Polymerase Chain Reaction. Viable T. gondii were isolated from myocardium of macropods via mouse bioassay. Tissues (brain, lungs, or mesenteric lymph nodes) from T. gondii-positive mice were seeded into Vero cell culture flasks. The virulence of the isolated T. gondii strains was evaluated in Swiss mice. The DNA from T. gondii tachyzoites obtained from cell cultures was characterized by 10 PCR-RFLP markers and the virulence genes, ROP18 and ROP5. T. gondii antibodies were identified in two of the three macropods (Macropod^#5 and ^#7). T. gondii DNA was obtained from the heart and lungs of Macropod^#7. Two viable T. gondii strains were isolated from the myocardium of Macropus rufogriseus (Macropod^#5) and M. rufus (Macropod^#7) via mouse bioassay and designated as TgRooCHn2 and TgRooCHn3, respectively. TgRooCHn2 was ToxoDB genotype^#3, and TgRooCHn3 was ToxoDB genotyp^#2. Both 10⁴ TgRooCHn2 and TgRooCHn3 tachyzoites had intermediate virulence in mice. M. rufogriseus (Macropod^#5) and M. rufus (Macropod^#7) may have been in the initial stages of toxoplasmosis, due to a recent T. gondii infection with oocysts. This study is the first to document the T. gondii ToxoDB^#3 isolate in macropods. T. gondii infection in captive macropods indicates the urgent need to control the transmission of this parasite in the environment, food and water of zoo animals.     

Isolation and Genetic Characterization of Toxoplasma gondii from Black Bears (Ursus americanus), Bobcats (Lynx rufus), and Feral Cats (Felis catus) from Pennsylvania

Toxoplasma gondii infects virtually all warm‐blooded hosts worldwide. Recently, attention has been focused on the genetic diversity of the parasite to explain its pathogenicity in different hosts. It has been hypothesized that interaction between feral and domestic cycles of T. gondii may increase unusual genotypes in domestic cats and facilitate transmission of potentially more pathogenic genotypes to humans, domestic animals, and wildlife. In the present study, we tested black bear (Ursus americanus), bobcat (Lynx rufus), and feral cat (Felis catus) from the state of Pennsylvania for T. gondii infection. Antibodies to T. gondii were found in 32 (84.2%) of 38 bears, both bobcats, and 2 of 3 feral cats tested by the modified agglutination test (cut off titer 1:25). Hearts from seropositive animals were bioassayed in mice, and viable T. gondii was isolated from 3 of 32 bears, 2 of 2 bobcats, and 2 of 3 feral cats. DNA isolated from culture‐derived tachyzoites of these isolates was characterized using multilocus PCR‐RFLP markers. Three genotypes were revealed, including ToxoDB PCR‐RFLP genotype #1 or #3 (Type II, 1 isolate), #5 (Type 12, 3 isolates), and #216 (3 isolates), adding to the evidence of genetic diversity of T. gondii in wildlife in Pennsylvania. Pathogenicity of 3 T. gondii isolates (all #216, 1 from bear, and 2 from feral cat) was determined in outbred Swiss Webster mice; all three were virulent causing 100% mortality. Results indicated that highly mouse pathogenic strains of T. gondii are circulating in wildlife, and these strains may pose risk to infect human through consuming of game meat.     

Phosphatidylthreonine and Lipid-Mediated Control of Parasite Virulence

The major membrane phospholipid classes, described thus far, include phosphatidylcholine (PtdCho), phosphatidylethanolamine (PtdEtn), phosphatidylserine (PtdSer), and phosphatidylinositol (PtdIns). Here, we demonstrate the natural occurrence and genetic origin of an exclusive and rather abundant lipid, phosphatidylthreonine (PtdThr), in a common eukaryotic model parasite, Toxoplasma gondii. The parasite expresses a novel enzyme PtdThr synthase (TgPTS) to produce this lipid in its endoplasmic reticulum. Genetic disruption of TgPTS abrogates de novo synthesis of PtdThr and impairs the lytic cycle and virulence of T. gondii. The observed phenotype is caused by a reduced gliding motility, which blights the parasite egress and ensuing host cell invasion. Notably, the PTS mutant can prevent acute as well as yet-incurable chronic toxoplasmosis in a mouse model, which endorses its potential clinical utility as a metabolically attenuated vaccine. Together, the work also illustrates the functional speciation of two evolutionarily related membrane phospholipids, i.e., PtdThr and PtdSer.     

In vivo characterization of a Toxoplasma gondii strain TgCatJpTy1/k-3 isolated from a stray cat in Japan

The Toxoplasma gondii strain TgCatJpTy1/k-3 (K-3), isolated from a stray cat in Tokyo, Japan, is categorized as a type II genotype. Since the K-3 strain is empirically known to form relatively larger cysts and exhibit weak pathogenesis in a mouse, it could serve as a useful model organism to study chronic T. gondii infection in the host. However, a detailed biological characterization of this strain had not been performed. In this study, we thoroughly assessed the K-3 strain in vivo using a mouse model. Tests indicated that pathogenicity of the K-3 strain was lower than that of the PLK strain, a clonal laboratory strain with a moderately pathogenic type II genotype. Further, cyst sizes of the K-3 strain were significantly larger than those of the PLK strain. Interestingly, K-3 cyst sizes in T. gondii-resistant ICR mice were larger than those in T. gondii-susceptible C57BL/6N mice.Our study suggests that the K-3 strain is suitable to study T. gondii cystogenesis and chronic infection, which are currently difficult to analyze using cell-adopted T. gondii strains.     

Virulence and molecular characterization of Toxoplasma gondii isolated from goats in Ceará, Brazil

One hundred and sixty-nine fragments of heart muscle collected from goats in the State of Ceará, Brazil, were analyzed by mouse bioassay with the aim of isolating Toxoplasma gondii. Two T. gondii isolates, named G1 and G2, were obtained and were characterized by PCR-RFLP. In addition, their virulence was evaluated by experimental inoculation into BALB/c mice. The G1 isolate presented high virulence leading to 100% mortality of mice after inoculations with 10¹, 10², and 10³ tachyzoites. The G2 isolate presented low virulence and none of the doses tested lead to mortality of mice. The PCR-RFLP analysis showed that the two isolates are recombinants of the types I/III. However, they differ in the haplotype combination for the genotypes analyzed.     

ROP18 and ROP5 alleles combinations are related with virulence of T. gondii isolates from Argentina

The allelic combination of ROP18/ROP5 genes of Toxoplasma gondii has been shown to be highly predictive of mouse virulence in canonical isolates and strains. The aims of this study were to analyze the alleles present in the ROP18/ROP5 genes from T. gondii isolates obtained in Argentina, to associate the results with the virulence registered in mouse model, and to compare with other isolates and reference strains using a phylogenetic network. Fourteen T. gondii isolates from Argentina were analyzed by nPCR-RFLP for ROP18/ROP5. Phylogenetic network analysis was inferred using the ToxoDB genotypes and the ROPs molecular markers. All isolates and reference strains were categorized as lethal or non-lethal. As results, combinations 2/2, 3/3 and 4/3 for ROP18/ROP5 were detected in 12 isolates, whereas only alleles 1 and 2 of ROP5 were detected in 2 isolates. The majority of the isolates had a mouse virulence matching to that predicted by the ROP18/ROP5 allele combination. The 3 isolates that differed from the expected virulence presented non-clonal genotypes. ROPs incorporation increased the accuracy of the phylogenetic network relations among the T. gondii samples, prevailing the clustering according to regions. Our results indicate a predominance of type 3 allele in both ROP18 and ROP5 markers and an association of allelic profiles 3/3 and 4/3 of non-clonal genotypes from Argentina, both with virulent and avirulent profiles in mice.     

Toxoplasma gondii: The immunogenic and protective efficacy of recombinant ROP2 and ROP4 rhoptry proteins in murine experimental toxoplasmosis

Toxoplasmosis is a one of the most world-wide spread zoonosis representing a very serious clinical and veterinary problem. In the presented study, we evaluated the protective efficacy of a combined recombinant ROP2 and ROP4 subunit vaccine in a chronic Toxoplasma gondii infection in mice. The recombinant ROP2 (rROP2) and ROP4 (rROP4) proteins were cloned and expressed in Escherichia coli and then used for the immunization of C3H/HeJ mice. Both antigens generated a strong systemic mixed Th1/Th2 response polarized towards IgG1 antibody isotype. In contrast to rROP2 stimulating only the specific IL-2 release, rROP4 and crude toxoplasma lysate antigen (TLA) used as a source of native forms of the parasite proteins induced significant proliferation of splenocytes and specific production of IFN-γ as well as IL-2, the Th1-type cytokines. Challenge of rROP2 and rROP4-vaccinated mice with cysts of low virulent T. gondii DX strain resulted in a partial protection effect with a significantly lower brain parasites load when compared with control animals. In the immunized group of mice the brain cysts number was reduced by nearly 46% as was determined in two independent experiments. These results suggest that, similar to ROP2, rhoptry protein ROP4 could be a very good candidate for future anti-T. gondii multicomponent vaccine based on the recombinant forms of different parasite proteins.