PLK Strain Research Articles

Unique asymmetric distribution of phosphatidylserine and phosphatidylethanolamine in Toxoplasma gondii revealed by nanoscale analysis.

Toxoplasma gondii is a highly prevalent obligate apicomplexan parasite that is important in clinical and veterinary medicine. It is known that glycerophospholipids phosphatidylserine (PtdSer) and phosphatidylethanolamine (PtdEtn), especially their expression levels and flip-flops between cytoplasmic and exoplasmic leaflets, in the membrane of T. gondii play important roles in efficient growth in host mammalian cells, but their distributions have still not been determined because of technical difficulties in studying intracellular lipid distribution at the nanometer level. In this study, we developed an electron microscopy method that enabled us to determine the distributions of PtdSer and PtdEtn in individual leaflets of cellular membranes by using quick-freeze freeze-fracture replica labeling. Our findings show that PtdSer and PtdEtn are asymmetrically distributed, with substantial amounts localized at the luminal leaflet of the inner membrane complex (IMC), which comprises flattened vesicles located just underneath the plasma membrane (see Figs. 2B and 7). We also found that PtdSer was absent in the cytoplasmic leaflet of the inner IMC membrane, but was present in considerable amounts in the cytoplasmic leaflet of the middle IMC membrane, suggesting a barrier-like mechanism preventing the diffusion of PtdSer in the cytoplasmic leaflets of the two membranes. In addition, the expression levels of both PtdSer and PtdEtn in the luminal leaflet of the IMC membrane in the highly virulent RH strain were higher than those in the less virulent PLK strain. We also found that the amount of glycolipid GM3, a lipid raft component, was higher in the RH strain than in the PLK strain. These results suggest a correlation between lipid raft maintenance, virulence, and the expression levels of PtdSer and PtdEtn in T. gondii.

Toxoplasma gondii GRA15 DNA Vaccine with a Liposomal Nanocarrier Composed of an SS-Cleavable and pH-Activated Lipid-like Material Induces Protective Immunity against Toxoplasmosis in Mice.

Toxoplasma gondii affects the health of humans and livestock and causes severe illness in the fetus and immunocompromised individuals. Because of the high incidence and severe consequences of T. gondii infection, a safe and suitable vaccine is needed. We found that lipid nanoparticles (LNPs) consisting of a series of functional materials prepared with vitamin E, such as SS-cleavable and pH-activated lipid-like materials (ssPalmE), were a safe and efficient way to develop next-generation DNA vaccines. In this study, we prepared ssPalmE-LNP to encapsulate pCpG-free-T. gondii dense granule protein 15 DNA (ssPalmE-LNPTgGRA15). Following a challenge infection with avirulent PLK strain of T. gondii, the mice immunized with ssPalmE-LNPTgGRA15 had a significantly higher survival rate and lower clinical scores compared with unimmunized and ssPalmE-LNPnon-coding-immunized mice. Immunization of mice with the ssPalmE-LNPTgGRA15 led to a significantly higher production of specific IgG1 and IG2c antibodies compared with unimmunized and ssPalmE-LNPnon-coding-immunized mice, while there was no statistically significant difference in the concentration of serum interferon-gamma at the acute stage of the infection. These findings indicate that ssPalmE-LNP is an effective cargo for the transportation of DNA vaccines for protozoan infections. To explore the mechanism of protective immunity induced by ssPalmE-LNPTgGRA15, further immunological study is needed in the future.

Expression of immunoproteasome subunits in the brains of Toxoplasma gondii-infected mice

The immunoproteasomes are specific proteasomes that clear oxidant-damaged proteins under inflammatory conditions in various diseases. Toxoplasma gondii (T. gondii) infects the central nervous system and causeencephalitis. However, the relationship between the immunoproteasomes and brain inflammation during T. gondii infection is not well characterized. In this study, we established an in vivo mouse model of T. gondii PLK strain infection via intraperitoneal injection and evaluated the expression of immunoproteasome subunits in the brains of infected mice. The results demonstrated that first, pathological changes in the brains of infected mice increase in severity over time. Second, following T. gondii infection, activated microglia and astrocytes undergo a series of functional alterations and morphological transformations, including proliferation and migration. Third, T. gondii infection induces expression of inflammatory cytokines, including IFN-γ, IL-1β, TNF-α, and IL-6. Fourth, the immunoproteasome subunits low-molecular-weight polypeptide 2 (LMP2), LMP7, and LMP10 mRNA and protein levels are significantly upregulated in T. gondii-infected mouse brains, as shown by RT-qPCR and western blot analysis, compared with that in vehicle-treated brains, and their expression is localized in the microglia, astrocytes, and neurons of T. gondii-infected brains, as determined via immunofluorescence staining. Furthermore, the western blot mean gray value for the immunoproteasome subunits and the positive microglia and astrocyte immunohistochemical signals in the brains of T. gondii-infected mice were positively correlated, indicating that the observed relationships were highly significant. Therefore, it was concluded that the induction of the immunoproteasomes is a pathogenic mechanism underlying T. gondii infection-induced inflammation.

PLK:Δgra9 Live Attenuated Strain Induces Protective Immunity Against Acute and Chronic Toxoplasmosis.

Toxoplasmosis is a zoonotic parasitic disease caused by the obligate intracellular protozoa Toxoplasma gondii, which threatens a range of warm-blooded mammals including humans. To date, it remains a challenge to find safe and effective drug treatment or vaccine against toxoplasmosis. In this study, our results found that the development of a mutant strain based on gene disruption of dense granule protein 9 (gra9) in type II PLK strain decreased parasite replication in vivo, severely attenuated virulence in mice, and significantly reduced the formation of cysts in animals. Hence, we developed an immunization scheme to evaluate the protective immunity of the attenuated strain of Δgra9 in type II PLK parasite as a live attenuated vaccine against toxoplasmosis in the mouse model. Δgra9 vaccination-induced full immune responses characterized by significantly high levels of pro-inflammatory cytokine interferon gamma (IFN-γ) and interleukin-12 (IL-12), maintained the high T. gondii-specific immunoglobulin G (IgG) level, and mixed high IgG1/IgG2a levels. Their levels provided the complete protective immunity which is a combination of cellular and humoral immunity in mouse models against further infections of lethal doses of type I RH, type II PLK wild-type tachyzoites, or type II PLK cysts. Results showed that Δgra9 vaccination proved its immunogenicity and potency conferring 100% protection against acute and chronic T. gondii challenges. Together, Δgra9 vaccination provided safe and efficient immune protection against challenging parasites, suggesting that PLK:Δgra9 is a potentially promising live attenuated vaccine candidate.

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.

Establishment of BV2 microglia polarization model and its effect on Toxoplasma gondii proliferation

Toxoplasma gondii is an intracellular opportunistic, parasitic protozoan. Microglia have been classified into two main types: M1 (classically activated macrophages) and M2 (alternatively activated macrophages). BV2 cells were used in this study, together with lipopolysaccharide (LPS) and interferon (IFN)-γ or interleukin (IL)-4, which were used to induce resting microglia. Expression levels of M1/M2 markers were determined at both mRNA and protein levels, using PCR, western blot, and flow cytometry. Furthermore, cells were infected with T. gondii PLK strain, and the dynamic changes in M1/M2 marker expression levels were determined. An in vitro polarization model was successfully established. Expression of Nos2 and M1-associated markers was significantly upregulated at 12 h post-infection in BV2 cells. Further, the JAK/STAT1 and NF-κB signaling pathways were also activated following T. gondii infection. This demonstrated that T. gondii infection induces M1-type microglial polarization in vitro. The present study demonstrated that T. gondii infection affects microglial activation in vitro and elucidated the effects of activated microglia on T. gondii proliferation. This data may serve as a useful reference for more detailed elucidation of interactions between T. gondii and the innate immune system.

Characterization of strain-specific phenotypes associated with knockout of dense granule protein 9 in Toxoplasma gondii

Toxoplasma gondii is an obligate intracellular parasite that can invade any nucleated cell of mammals and cause toxoplasmosis. Dense granule proteins play major structural functions within the parasitophorous vacuole (PV) and the cyst wall of T. gondii. Moreover, their particular location within the PV allows them to be involved in various interactions between parasites and the host cells. Dense granule protein 9 (GRA9) gene has been identified in T. gondii, although its role in the lytic cycle remains unclear. In the current study, the function of GRA9 in type I and type II Toxoplasma parasites was characterized. T. gondii GRA9 sequence and its expression were analyzed and derivatives of T. gondii RH and PLK strains with a null mutation in GRA9 were generated using CRISPR/Cas9 system. The phenotypes of GRA9 in wild types, knockout and complemented strains were analyzed in vitro and in vivo using Vero cells and BALB/c mice, respectively. Alignment of the amino acid sequence indicated that RH strain GRA9 contained one amino acid substitution when compared with PLK strain. Western blot analysis revealed that PLK strain had a higher expression level of GRA9 than RH strain. The phenotype analysis revealed that knockout of GRA9 in PLK parasites inhibited the plaque formation and egress from PV. Both the plaque formation and egress ability of PLKΔGRA9 strain were restored by complementation with a synonymous allele of PLK strain GRA9. Mouse experiments demonstrated that loss of GRA9 in PLK strain significantly reduced the pathogenicity of T. gondii. However, there was no phenotypic diferences between RH and RHΔGRA9 strains except the defect in host cell invasion. Overall, T. gondii GRA9 knockout only influenced the growth and virulence of PLK strain. These results indicate that GRA9 may be involved in parasite egress and virulence in mice in a strain-specific manner.

Translationally controlled tumor protein is required for the fast growth of Toxoplasma gondii and maintenance of its intracellular development.

Translationally controlled tumor protein (TCTP) is a highly conserved, multifunctional protein that has been implicated in a range of cell physiologic processes, especially cell growth and development. A TCTP-like gene has been identified in the Toxoplasma genome [ Toxoplasma gondii TCTP ( TgTCTP)], although its function remains unknown. The sequence analysis of TgTCTP indicated that it is a highly conserved protein in eukaryotes. We found that the expression level of TgTCTP in the virulent RH strain was significantly higher than that in the avirulent PLK strain. Indirect immunofluorescence showed that TgTCTP was expressed in the parasite cytoplasm. The localization of TgTCTP was unchanged during the replication of the parasite. We expressed a functional recombinant TgTCTP (r TgTCTP) protein in Escherichia coli and found that the recombinant protein could form a multimer. We then evaluated the function of TgTCTP using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 knockout (KO) system. Phenotypic analysis of the KO strain (Δ TgTCTP) revealed that TgTCTP is required for the robust growth of the parasites. TgTCTP deficiency also led to early egress of the parasites and subsequent impairment in their invasion and attachment abilities. We subsequently found that the multimer form of TgTCTP might not be necessary for the growth and replication of the parasite. Then the expression profiling of genes in the Δ TgTCTP and complement strains were analyzed. The results revealed that 988 genes were regulated in Δ TgTCTP compared with the complement strain. Overall, although not essential, TgTCTP is required for the fast growth of Tg and maintenance of its intracellular development.-Zheng, J., Chen, Y., Li, Z., Cao, S., Zhang, Z., Jia, H. Translationally controlled tumor protein is required for the fast growth of Toxoplasma gondii and maintenance of its intracellular development.

Effects of Extracts from Thai Piperaceae Plants against Infection with Toxoplasma gondii.

Herbal medicines and natural herb extracts are widely used as alternative treatments for various parasitic diseases, and such extracts may also have potential to decrease the side effects of the standard regimen drugs used to treat toxoplasmosis (sulfadiazine-pyrimethamine combination). We evaluated how effective the Thai piperaceae plants Piper betle, P. nigrum and P. sarmentosum are against Toxoplasma gondii infection in vitro and in vivo. Individually, we extracted the piperaceae plants with ethanol, passed them through a rotary evaporator and then lyophilized them to obtain crude extracts for each one. The in vitro study indicated that the P. betle extract was the most effective extract at inhibiting parasite growth in HFF cells (IC50 on RH-GFP: 23.2 μg/mL, IC50 on PLK-GFP: 21.4 μg/mL). Furthermore, treatment of experimental mice with the P. betle extract for 7 days after infection with 1,000 tachyzoites of the T. gondii PLK strain increased their survival (survival rates: 100% in 400 mg/kg-treated, 83.3% in 100 mg/kg-treated, 33.3% in 25 mg/kg-treated, 33.3% in untreated mice). Furthermore, treatment with 400 mg/kg of the P. betle extract resulted in 100% mouse survival following infection with 100,000 tachyzoites. The present study shows that P. betle extract has the potential to act as a medical plant for the treatment of toxoplasmosis.

Mechanisms of interferon-beta-induced inhibition ofToxoplasma gondiigrowth in murine macrophages and embryonic fibroblasts: role of immunity-related GTPase M1

The apical complex of Toxoplasma gondii enables it to invade virtually all nucleated cells in warm-blooded animals, including humans, making it a parasite of global importance. Anti-T. gondii cellular defence mechanisms depend largely on interferon (IFN)-γ production by immune cells. However, the molecular mechanism of IFN-β-mediated defence remains largely unclear. Here, mouse peritoneal macrophages and murine embryonic fibroblasts (MEFs) primed with recombinant IFN-β and IFN-γ showed different pathways of activation. Treatment of these cells with IFN-β or IFN-γ inhibited T. gondii (type II PLK strain) growth. Priming macrophages with IFN-β had no effect on inflammatory cytokine expression, inducible nitric oxide synthase or indoleamine 2,3-dioxygenase, nor did it have an effect on their metabolites, nitric oxide and kynurenine respectively. In contrast, IFN-γ stimulation was characterized by classical macrophage activation and T. gondii elimination. IFN-β activation recruited the immunity-related GTPase M1 (IRGM1) to the parasitophorous vacuole in the macrophages and MEFs. Anti-toxoplasma activities induced by IFN-β were significantly reduced after IRGM1 knockdown in murine macrophages and in IRGM1-deficient MEFs. Thus, this study unravels an alternative pathway of macrophage activation by IFN-β and provides a mechanistic explanation for the contribution of IRGM1 induced by IFN-β to the elimination of T. gondii.

Transcriptome Analysis of Mouse Brain Infected with Toxoplasma gondii

Toxoplasma gondii is an obligate intracellular parasite that invades a wide range of vertebrate host cells. Chronic infections with T. gondii become established in the tissues of the central nervous system, where the parasites may directly or indirectly modulate neuronal function. However, the mechanisms underlying parasite-induced neuronal disorder in the brain remain unclear. This study evaluated host gene expression in mouse brain following infection with T. gondii. BALB/c mice were infected with the PLK strain, and after 32 days of infection, histopathological lesions in the frontal lobe were found to be more severe than in other areas of the brain. Total RNA extracted from infected and uninfected mouse brain samples was subjected to transcriptome analysis using RNA sequencing (RNA-seq). In the T. gondii-infected mice, 935 mouse brain genes were upregulated, whereas 12 genes were downregulated. GOstat analysis predicted that the upregulated genes were primarily involved in host immune responses and cell activation. Positive correlations were found between the numbers of parasites in the infected mouse brains and the expression levels of genes involved in host immune responses. In contrast, genes that had a negative correlation with parasite numbers were predicted to be involved in neurological functions, such as small-GTPase-mediated signal transduction and vesicle-mediated transport. Furthermore, differential gene expression was observed between mice exhibiting the clinical signs of toxoplasmosis and those that did not. Our findings may provide insights into the mechanisms underlying neurological changes during T. gondii infection.

Toxoplasma gondii tachyzoite-infected peripheral blood mononuclear cells are enriched in mouse lungs and liver

The intracellular parasite Toxoplasma gondii is thought to disseminate throughout the host by circulation of tachyzoite-infected leukocytes in the blood, and adherence and migration of such leukocytes into solid tissues. However, it is unclear whether T. gondii-infected leukocytes can migrate to solid organs via the general circulation. In this study, we developed a real-time quantitative PCR (qRT-PCR) method to determine the rate of infection of peripheral blood mononuclear cells (PBMCs) flowing into and remaining within solid organs in mice. A transgenic T. gondii parasite line derived from the PLK strain that expresses DsRed Express, and transgenic green fluorescent protein-positive PBMCs, were used for these experiments. Tachyzoite-infected PBMCs were injected into mouse tail veins and qRT-PCR was used to measure the infection rates of the PBMCs remaining in the lungs, liver, spleen and brain. We found that the PBMCs in the lungs and liver had statistically higher infection rates than that of the original inoculum; this difference was statistically significant. However, the PBMC infection rate in the spleen showed no such enhancement. These results show that tachyzoite-infected PBMCs in the general circulation remain in the lungs and liver more effectively than non-infected PBMCs.

Detection of the initial site of Toxoplasma gondii reactivation in brain tissue

Detection of the initial site of Toxoplasma gondii reactivation in brain tissue is difficult because the number of latent cysts is small and reactivation is a transient event. To detect the early stage of reactivation in mouse brain tissue, we constructed a cyst-forming strain of T. gondii in the tachyzoite stage, specifically expressing red fluorescence. The PLK strain of T. gondii was stably transfected with a red fluorescent protein gene, DsRed Express, under the control of a tachyzoite-specific SAG-1 promoter and the resulting parasite was designated as PLK/RED. Tachyzoites of PLK/RED growing in Vero cells showed red fluorescence. When C57BL/6J mice were i.p. infected with tachyzoites of PLK/RED, red fluorescent tachyzoites were detected in their brains at the fourth day p.i. However, red fluorescent tachyzoites were not detected in BALB/c mice latently infected with PLK/RED, although non-fluorescent cysts were detected in their brains. After treatment of latently infected mice with dexamethasone for 1 month, the mice showed neurological symptoms. In mice with symptoms, red fluorescent tachyzoites were again detected in their brains and in other organs. To detect the initial site of reactivation, BALB/c mice latently infected with the strain were treated with dexamethasone for 3 weeks, and brains were excised before any symptoms appeared. Excised brains were examined for red fluorescence-positive sites. By a histological study of red fluorescent-positive sites, we detected a cyst containing red fluorescent zoites, which still had a PAS stain-positive cyst wall. A few red fluorescent zoites breaking away from the cyst were also observed. The stage-specific expression of fluorescent protein facilitates detection of a rare transient event and makes it possible to detect the initial site of reactivation.

Toxoplasma gondii: Detection of MIC10 antigen in sera of experimentally infected mice

We developed a sandwich ELISA for the detection of circulating Toxoplasma gondii MIC10 antigens. In T. gondii culture supernatant, MIC10 was detected in a growth dependent manner. Mice were infected with a lethal dose of either a virulent RH strain, an avirulent Beverley strain or a sub-lethal dose of a PLK strain of T. gondii. MIC10 appeared 2 days after infection and increased gradually in the sera of RH-infected mice. A detectable but significantly lower amount of MIC10 was observed in the sera of mice infected intraperitoneally with Beverley tachyzoites. In contrast, the MIC10 antigen in mice sera following oral infection with Beverley cysts was below detectable levels during the course of the experiment. In sera of PLK-infected mice, MIC10 was predominantly observed between late acute and early chronic phase. Our data show that the kinetics of circulating MIC10 differs depending on the strain and route of infection.

Experimental Ocular Toxoplasmosis in Genetically Susceptible and Resistant Mice

Genetic factors determining the pathogenesis and course of ocular toxoplasmosis are poorly understood. In this study, we explored the development of experimental ocular pathogenesis in genetically dissimilar mice infected with either the RH strain, the PLK strain, or the immunodominant surface antigen 1 (SAG1 [P30])-deficient mutant of the RH strain of Toxoplasma gondii. At 11 days postinfection, ocular infection of C57BL/6 mice with all of the strains of parasites resulted in severe inflammatory lesions and high numbers of parasites in eye tissue; less severe ocular lesions at earlier histopathology and prolonged survival were observed in this mouse strain infected with either the major surface antigen 1-deficient SAG1(-/-) strain or the less virulent PLK strain compared with RH infection. In contrast, both BALB/c and CBA/J mice had less severe lesions and low numbers of parasites in their eye tissue, and infection developed into the chronic stage in these mice. There were significantly higher serum levels of gamma interferon and tumor necrosis factor alpha in C57BL/6 mice than in BALB/c and CBA/J mice following ocular infection. These observations confirm earlier reports on systemic immunity to these parasites that the route of Toxoplasma infection markedly influences survival of mice. Our data indicate that genetic factors of the host as well as the parasite strain are critical in determining susceptibility to experimental ocular toxoplasmosis in murine models.

Infection of mice by a Toxoplasma gondii isolate from an AIDS patient: virulence and activation of hosts' immune responses are independent of parasite genotype.

Virulence of a Toxoplasma gondii isolate from an AIDS patient (designated as PTN) was compared with that of PLK, a variant of P-strain. Virulence was assessed in term of host survival upon inoculation in different strains of mice. All C57BL/6 mice died of acute toxoplasmosis by 7-10 days following intraperitoneal infection with 1 x 105 tachyzoites of PTN and 40% of BALB/c died on day 23 of infection, whereas 100% CBA/J infected with the same dose of PTN survived, as did outbred Swiss Webster mice. All C57BL/6, BALB/c, CBA/J, or Swiss Webster died of acute toxoplasmosis by 3-9 days postinfection upon inoculation with same dose of tachyzoites of the PLK strain. Further studies in CBA/J mice demonstrated that mice infected with PTN elicited a significantly higher lymphoproliferative response to crosslinked anti-CD3 mAb or Con A than PLK infected mice, and augmented production of TNFalpha, lower levels of nitrite and a higher number of NK cells. Genetical analysis indicated that both PLK and PTN strains of T. gondii are from type ll. Interestingly, being of the same genotype, the later showed less virulence upon inoculation in mice and had greater capacity to activate host immune system than the PLK strain.

Protection against Lethal Toxoplasmosis in Mice by an Avirulent Strain of Toxoplasma gondii: Stimulation of IFN-γ and TNF-α Response

Haque, S., Franck, J., Dumon, H., Kasper, L. H., and Haque, A. 1999. Protection against lethal toxoplasmosis in mice by an avirulent strain of Toxoplasma gondii: Stimulation of IFN-γ and TNF-α response. Experimental Parasitology93, 231–240. In this study, we examined whether the PTN strain (isolated from an AIDS patient) of Toxoplasma gondii could induce cross-protection in mice against infection with a lethal dose of the PLK strain. Mice were first infected with tachyzoites (5 × 105) of PTN and 5 days later challenged with PLK (1 × 105, LD90) parasites. None of these mice succumbed to infection until day 21 after infection, whereas 100% of the mice given the same dose of PLK infection alone died between 5 and 11 days after infection. The protection was accompanied by an increased expansion of NK cells and CD4 + T cells. This condition was associated by increased production of IFN-γ and an augmented number of IFN-γ-producing cells in the spleen. Further, PTN + PLK-infected mice showed higher production of TNF-α and nitrite compared to PLK-infected mice. Mice infected with the PTN strain had an enhanced capacity to activate the immune system early in infection since they produced higher levels of IFN-γ, TNF-α, and NO than PLK-infected mice. Administration of anti-IFN-γ mAb or anti-asialo GM1 antibody resulted in 100 and 20% mortality, respectively, in PTN-infected mice but no death in PTN + PLK-infected mice. Together, these results suggest that early production of IFN-γ and NK-cell activity is important in protection against PTN infection, whereas in PTN + PLK infection components of adaptive immunity rapidly developed following elaboration of an effective early innate immune response.

Disruption of the Toxoplasma gondii bradyzoite-specific gene BAG1 decreases in vivo cyst formation.

The bradyzoite stage of the Apicomplexan protozoan parasite Toxoplasma gondii plays a critical role in maintenance of latent infection. We reported previously the cloning of a bradyzoite-specific gene BAG1/hsp30 (previously referred to as BAG5) encoding a cytoplasmic antigen related to small heat shock proteins. We have now disrupted BAG1 in the T. gondii PLK strain by homologous recombination. H7, a cloned null mutant, and Y8, a control positive for both cat and BAG1, were chosen for further characterization. Immunofluorescence and Western blot analysis of bradyzoites with BAG1 antisera demonstrated expression of BAG1 in the Y8 and the PLK strain but no expression in H7. All three strains expressed a 116 kDa bradyzoite cyst wall antigen, a 29 kDa matrix antigen and the 65 kDa matrix reactive antigen MAG1. Mice inoculated with H7 parasites formed significantly fewer cysts than those inoculated with the Y8 and the PLK strains. H7 parasites were complemented with BAG1 using phleomycin selection. Cyst formation in vivo for the BAG1-complemented H7 parasites was similar to wild-type parasites. We therefore conclude that BAG1 is not essential for cyst formation, but facilitates formation of cysts in vivo.

Experimental ocular toxoplasmosis induced in naive and preinfected mice by intracameral inoculation

We have developed a murine model to investigate the pathogenesis of acquired ocular toxoplasmosis. Tachyzoites of PLK strain of Toxoplasma gondii were intracamerally inoculated under anesthesia into the right eyes of naive or perorally preinfected C57BL/6 and MRL-MpJ mice. Clinical and histopathological observations of responses to intraocular infection were analyzed. Ocular inflammation from Toxoplasma gondii is dose-dependent in both strains of mice. After inoculation of fifty parasites, no evidence of inflammation was observed in the eyes of naive mice. The eyes of naive mice that received 500 or 5,000 parasites developed inflammatory changes by day 6 post challenge. By day 8, the changes progressed to moderate to severe intraocular inflammation. Histologic analysis of the ocular lesions demonstrated mononuclear cell infiltration and necrosis predominantly in the anterior segment of the eyes of the naive mice. Inoculation of 50,000 tachyzoites induced a destructive ocular inflammatory response and was uniformly lethal to the mice by approximately one week after challenge. In contrast, eyes from mice previously orally infected with Toxoplasma gondii and that received a 50 or 500 parasite intracameral challenge revealed no inflammation, but the eyes receiving 5,000 parasites demonstrated necrotic focal retinochoroiditis with vitreitis by day 8 after challenge. The murine model reproduces some features of ocular toxoplasmosis in humans and may be suitable for large-scale controlled studies of the pathogenesis and therapeutics of acquired ocular toxoplasmosis as well as for study of the mechanisms of immune privilege in the eye.

Basis for substrate specificity of the Toxoplasma gondii nucleoside triphosphate hydrolase

The Toxoplasma gondii nucleoside triphosphate hydrolase is the most active E-type ATPase yet identified, and was the first member of this new gene family to be cloned (Bermudes D, Peck KR, Afifi-Afifi M, Beckers CJM, Joiner KA. J Biol Chem 1994;269:29252–29260. Previous work also identified two isoforms of the enzyme in the virulent RH strain, and demonstrated that internal fragments of the genes encoding these isoforms were found differentially in virulent versus avirulent organisms (Asai T, Miura S, Sibley D, Okabayashi H, Tsutomu T, J Biol Chem 1995;270:11391–11397). We now show that the NTPase 1 isoform is expressed in avirulent strains, whereas virulent strains express both the NTPase 1 and NTPase 3 isoforms. The avirulent PLK strain lacks the gene for NTPase 3, explaining the absence of expression. Despite the fact that NTPase 1 and NTPase 3 are 97% identical at the amino acid level, recombinant NTPase 1 is a true apyrase, whereas recombinant NTPase 3 cleaves predominantly nucleotide triphosphates. Furthermore, native and recombinant NTPase 3 but neither native nor recombinant NTPase 1 bind to ATP-agarose, further distinguishing the two isoforms. Using chimeras between the NTP1 and NTP3 genes, we show that a block of twelve residues at the C-terminus dictates substrate specificity. These residues lie outside the regions conserved among other E-ATPases, and therefore provide new insight into substrate recognition by this class of enzymes.