Cyst formation by Toxoplasma gondii in vivo and in brain-cell culture: a comparative morphology and immunocytochemistry study

Formation of Toxoplasma gondii cysts was examined in cultured murine brain cells and was compared with the development of cysts in mouse-brain tissue. Cultures of mixed glial cells from neonatal mouse brain were infected with bradyzoites of the avirulent T. gondii strain DX. The development and maturation of Toxoplasma cysts was monitored for up to 63 days after inoculation. Transmission electron microscopy indicated that in-vitro-derived cysts were morphologically similar to tissue cysts and were located intracellularly, even for up to 63 days postinfection. For immunohistological and immunocytochemical examination of both in-vivo- and in-vitro-infected material, monoclonal antibody (mAb) CC2 was used. MAb CC2 was shown to detect specifically the underlying granular material of the cyst wall without binding to the limiting membrane of the parasitophorous vacuole. This reactivity of mAb CC2 allows the distinction of bradyzoite-containing cysts from parasitophorous vacuoles harboring tachyzoites both in vitro and in vivo. Received: 15 February 1997 / Accepted: 18 March 1997     

In vitro generation of cloned populations of Toxoplasma gondii

Cloned populations were generated from Indian isolates of Toxoplasma gondii by transferring single tissue cysts from the brains of chronically infected mice to confluent murine macrophage (J774A.1) monolayers. The clones were then maintained continuously as tachyzoites in culture. Physical rupture of the tissue cysts and release of bradyzoites prior to seeding was found to be necessary for establishment of the parasite in culture. Although intact tissue cysts seeded over monolayers released bradyzoites spontaneously, they did not succeed in setting up an infection in the monolayers. Random amplified polymorphic DNA (RAPD)-PCR, which revealed distinct patterns for a clone and its progenitor, further confirmed the efficiency of the technique. The cloning technique was found to be simple and rapid compared to those involving limiting dilutions.     

Partially Protective Vaccination Permits the Development of Latency in a Normally Virulent Strain of Toxoplasma gondii

The virulent RH strain of Toxoplasma gondii is acutely lethal in mice and fails to establish chronic infection. Vaccination of BALB/c mice with a soluble tachyzoite antigen preparation, STAg, in combination with the immunostimulatory cytokine interleukin-12 results in partial protection against RH lethal challenge. Nevertheless, brain tissue obtained from surviving, vaccinated mice as late as 1 year after RH infection contained latent parasite forms as demonstrated by subinoculation into naive recipients. The tachyzoites arising in the subinoculated animals were genetically indistinguishable from the original RH inoculum. Microscopic examination revealed that the persistent parasite forms present in the brains of vaccinated and challenged mice have a tissue cyst-like morphology and express the bradyzoite antigen BAG-1 but not the tachyzoite-specific antigen SAG-2 but are different from the cysts formed by avirulent T. gondii strains in that the internal parasite stages display ultrastructural features intermediate between tachyzoites and bradyzoites. Moreover, the zoites within the RH tissue cysts are clearly distinct from conventional bradyzoites in their sensitivity to pepsin-HCl digestion. In contrast to the observations made with partially resistant STAg/interleukin-12-vaccinated animals, no latent forms could be detected in brain tissue after RH challenge of mice immunized with a live attenuated tachyzoite vaccine which confers total protection against this parasite isolate. The above findings demonstrate the potential of a virulent T. gondii strain to generate latent parasite stages, a process which may be promoted under conditions of incomplete vaccination.     

Toxoplasma gondii: determinants of tachyzoite to bradyzoite conversion

Apicomplexa are primarily obligate intracellular protozoa that have evolved complex developmental stages important for pathogenesis and transmission. Toxoplasma gondii, responsible for the disease toxoplasmosis, has the broadest host range of the Apicomplexa as it infects virtually any warm-blooded vertebrate host. Key to T. gondii’s pathogenesis is its ability to differentiate from a rapidly replicating tachyzoite stage during acute infection to a relatively non-immunogenic, dormant bradyzoite stage contained in tissue cysts. These bradyzoite cysts can reconvert back to tachyzoites years later causing serious pathology and death if a person becomes immune-compromised. Like the sexual stage sporozoites, bradyzoites are also orally infectious and a major contributor to transmission. Because of the critical role of stage conversion to pathogenesis and transmission, a major research focus is aimed at identifying molecular mediators and pathways that regulate differentiation. Tachyzoite to bradyzoite development can occur spontaneously in vitro and be induced in response to exogenous stress including but not limited to host immunity. The purpose of this review is to explore the potential contributors to stage differentiation in infection and how a determination is made by the parasite to differentiate from tachyzoites to bradyzoites.     

Toxoplasma gondii: localization of purine nucleoside phosphorylase activity in vitro and in vivo by electron microscopy

Purine nucleoside phosphorylase (PNP, EC 2.4.2.1) activity was revealed by enzyme histochemistry in Toxoplasma gondii ME49 strain isolated from murine cerebral cysts and from in vitro cultivation. The activity of the enzyme was revealed by an insoluble electron-opaque precipitate of lead phosphate at the site of the reaction. In bradyzoites and tachyzoites of T. gondii, the enzyme activity could be observed only in the cytoplasm. In bradyzoites, one or two foci of important PNP activity were detected near the nucleus. In tachyzoites, an important PNP activity underlined the plasma membrane. For both bradyzoites and tachyzoites, localization neither in the nucleus nor in cytoplasmic organelles could be detected.     

Removal of Toxoplasma gondii Cysts from the Brain by Perforin-Mediated Activity of CD8+ T Cells

Chronic infection with Toxoplasma gondii is one of the most common parasitic infections in humans. Formation of tissue cysts is the basis of persistence of the parasite in infected hosts, and this cyst stage has generally been regarded as untouchable. Here we provide the first evidence that the immune system can eliminate T. gondii cysts from the brains of infected hosts when immune T cells are transferred into infected immunodeficient animals that have already developed large numbers of cysts. This T cell-mediated immune process was associated with accumulation of microglia and macrophages around tissue cysts. CD8⁺ immune T cells possess a potent activity to remove the cysts. The initiation of this process by CD8⁺ T cells does not require their production of interferon-γ, the major mediator to prevent proliferation of tachyzoites during acute infection, but does require perforin. These results suggest that CD8⁺ T cells induce elimination of T. gondii cysts through their perforin-mediated cytotoxic activity. Our findings provide a new mechanism of the immune system to fight against chronic infection with T. gondii and suggest a possibility of developing a novel vaccine to eliminate cysts from patients with chronic infection and to prevent the establishment of chronic infection after a newly acquired infection.Toxoplasma gondii is an obligate intracellular protozoan parasite capable of infecting many warm-blooded mammals including humans. Acute infection is characterized by proliferation of tachyzoites and is known to cause various diseases including lymphadenitis and congenital infection of fetuses.¹ Interferon (IFN)-γ-mediated immune responses limit proliferation of tachyzoites, but the parasite establishes a chronic infection by forming cysts, which can contain hundreds to thousands of bradyzoites, primarily in the brain. Chronic infection with T. gondii is one of the most common parasitic infections in humans. It is estimated that 5 × 10⁸ people worldwide are chronically infected with this parasite.² The tissue cysts remain largely quiescent for the life of the host, but can reactivate and cause life-threatening toxoplasmic encephalitis in immunocompromised patients, such as those with AIDS, neoplastic diseases and organ transplants.^3,4 In immunocompetent individuals, recent studies suggested that T. gondii is an important cause of cryptogenic epilepsy,^5,6 and is likely involved in the etiology of schizophrenia.^7,8 The tissue cyst is not affected by any of the current drug treatments and it has been generally regarded as untouchable. However, the immune responses against T. gondii cysts remain largely unexplored.Resistance to T. gondii is under genetic control in humans^9,10 and mice.^11,12 BALB/c mice are genetically resistant and have only small numbers of cysts in their brains at 2 to 3 months after infection.^11,12 These mice may be able to prevent formation of cysts by efficiently controlling proliferation of tachyzoites during the acute stage of infection. However, it is also possible that the immune system of these animals has the capability to recognize T. gondii cysts and eliminate them from their brains. To examine whether immune cells have an activity to remove cysts that have already been formed in the brain, we adoptively transferred immune cells obtained from chronically infected BALB/c mice into infected, sulfadiazine-treated athymic nude or severe combined immunodeficient (SCID) mice, both of which lack T cells and developed large numbers of cysts in their brains. We present evidence for a potent capability of CD8⁺ immune T cells to eliminate T. gondii cysts from the brains through their perforin-mediated activity.     

Neurological and behavioral abnormalities, ventricular dilatation, altered cellular functions, inflammation, and neuronal injury in brains of mice due to common, persistent, parasitic infection

Background

Worldwide, approximately two billion people are chronically infected with Toxoplasma gondii with largely unknown consequences.

Methods

To better understand long-term effects and pathogenesis of this common, persistent brain infection, mice were infected at a time in human years equivalent to early to mid adulthood and studied 5–12 months later. Appearance, behavior, neurologic function and brain MRIs were studied. Additional analyses of pathogenesis included: correlation of brain weight and neurologic findings; histopathology focusing on brain regions; full genome microarrays; immunohistochemistry characterizing inflammatory cells; determination of presence of tachyzoites and bradyzoites; electron microscopy; and study of markers of inflammation in serum. Histopathology in genetically resistant mice and cytokine and NRAMP knockout mice, effects of inoculation of isolated parasites, and treatment with sulfadiazine or αPD1 ligand were studied.

Results

Twelve months after infection, a time equivalent to middle to early elderly ages, mice had behavioral and neurological deficits, and brain MRIs showed mild to moderate ventricular dilatation. Lower brain weight correlated with greater magnitude of neurologic abnormalities and inflammation. Full genome microarrays of brains reflected inflammation causing neuronal damage (Gfap), effects on host cell protein processing (ubiquitin ligase), synapse remodeling (Complement 1q), and also increased expression of PD-1L (a ligand that allows persistent LCMV brain infection) and CD 36 (a fatty acid translocase and oxidized LDL receptor that mediates innate immune response to beta amyloid which is associated with pro-inflammation in Alzheimer's disease). Immunostaining detected no inflammation around intra-neuronal cysts, practically no free tachyzoites, and only rare bradyzoites. Nonetheless, there were perivascular, leptomeningeal inflammatory cells, particularly contiguous to the aqueduct of Sylvius and hippocampus, CD4+ and CD8+ T cells, and activated microglia in perivascular areas and brain parenchyma. Genetically resistant, chronically infected mice had substantially less inflammation.

Conclusion

In outbred mice, chronic, adult acquired T. gondii infection causes neurologic and behavioral abnormalities secondary to inflammation and loss of brain parenchyma. Perivascular inflammation is prominent particularly contiguous to the aqueduct of Sylvius and hippocampus. Even resistant mice have perivascular inflammation. This mouse model of chronic T. gondii infection raises questions of whether persistence of this parasite in brain can cause inflammation or neurodegeneration in genetically susceptible hosts.     

Functional and biophysical analyses of the class XIV Toxoplasma gondii Myosin D

Summary The obligate intracellular parasite Toxoplasma gondii uses gliding motility to migrate across the biological barriers of the host and to invade cells. This unique form of locomotion requires an intact actin cytoskeleton and involves at least one motor protein (TgMyoA) that belongs to the class XIV of the myosin superfamily. TgMyoA is anchored in the inner membrane complex and is essential for the gliding motion, host cell invasion and egress of T. gondii tachyzoites. TgMyoD is the smallest T. gondii myosin and is structurally very closely related to TgMyoA. We show here that TgMyoD exhibits similar transient kinetic properties as the fast single-headed TgMyoA. To determine if TgMyoD also contributes to parasite gliding motility, the TgMyoD gene was disrupted by double homologous recombination. In contrast to TgMyoA, TgMyoD gene is dispensable for tachyzoite propagation and motility. Parasites lacking TgMyoD glide normally and their virulence is not compromised in mice. The fact that TgMyoD is predominantly expressed in bradyzoites explains the absence of a phenotype observed with myodko in tachyzoites and does not exclude a role of this motor in gliding that would be restricted to the cyst forming but nevertheless motile stage of the parasite.Both authors contributed equally to the work.     

Structures of Toxoplasma gondii Tachyzoites, Bradyzoites, and Sporozoites and Biology and Development of Tissue Cysts

Infections by the protozoan parasite Toxoplasma gondii are widely prevalent worldwide in animals and humans. This paper reviews the life cycle; the structure of tachyzoites, bradyzoites, oocysts, sporocysts, sporozoites and enteroepithelial stages of T. gondii; and the mode of penetration of T. gondii. The review provides a detailed account of the biology of tissue cysts and bradyzoites including in vivo and in vitro development, methods of separation from host tissue, tissue cyst rupture, and relapse. The mechanism of in vivo and in vitro stage conversion from sporozoites to tachyzoites to bradyzoites and from bradyzoites to tachyzoites to bradyzoites is also discussed.     

Reassessment of the Role of Aromatic Amino Acid Hydroxylases and the Effect of Infection by Toxoplasma gondii on Host Dopamine

Toxoplasma gondii infection has been described previously to cause infected mice to lose their fear of cat urine. This behavioral manipulation has been proposed to involve alterations of host dopamine pathways due to parasite-encoded aromatic amino acid hydroxylases. Here, we report successful knockout and complementation of the aromatic amino acid hydroxylase AAH2 gene, with no observable phenotype in parasite growth or differentiation in vitro and in vivo. Additionally, expression levels of the two aromatic amino acid hydroxylases were negligible both in tachyzoites and in bradyzoites. Finally, we were unable to confirm previously described effects of parasite infection on host dopamine either in vitro or in vivo, even when AAH2 was overexpressed using the BAG1 promoter. Together, these data indicate that AAH enzymes in the parasite do not cause global or regional alterations of dopamine in the host brain, although they may affect this pathway locally. Additionally, our findings suggest alternative roles for the AHH enzymes in T. gondii, since AAH1 is essential for growth in nondopaminergic cells.     

Expressed Sequence Tag Analysis of the Bradyzoite Stage of Toxoplasma gondii: Identification of Developmentally Regulated Genes

Toxoplasma gondii is a protozoan parasite responsible for widespread infections in humans and animals. Two major asexual forms are produced during the life cycle of this parasite: the rapidly dividing tachyzoite and the more slowly dividing, encysted bradyzoite. To further study the differentiation between these two forms, we have generated a large number of expressed sequence tags (ESTs) from both asexual stages. Previously, we obtained data on ∼7,400 ESTs from tachyzoites (J. Ajioka et al., Genome Res. 8:18–28, 1998). Here, we report the results from analysis of ∼2,500 ESTs from bradyzoites purified from the cysts of infected mice. We also report the results from analysis of 760 ESTs from parasites induced to differentiate from tachyzoites to bradyzoites in vitro. Comparison of the data sets from bradyzoites and tachyzoites reveals many previously uncharacterized sequence clusters which are largely or completely specific to one or other developmental stage. This class includes a bradyzoite-specific form of enolase. Combined with the previously identified bradyzoite-specific form of lactate dehydrogenase, this finding suggests significant differences in flux through the lower end of the glycolytic pathway in this stage. Thus, the generation of this data set provides valuable insights into the metabolism and growth of the parasite in the encysted form and represents a substantial body of information for further study of development in Toxoplasma.Toxoplasma gondii is a member of the protozoan phylum Apicomplexa, which also includes the causative agents of malaria (Plasmodium spp.), coccidiosis (Eimeria spp.), and cryptosporidiosis (Cryptosporidium spp.). T. gondii is a major pathogen of a broad range of warm-blooded animals, including humans, livestock, and domestic pets (11). The parasite is of clinical importance both for the devastating disease it causes in the developing fetus and as an opportunistic infection in patients immunocompromised through disease or transplantation (19).The parasite has a complex life cycle that includes sexual and asexual stages. The sexual cycle occurs exclusively in the guts of felines, while asexual growth can occur in almost any tissue of its broad range of hosts. The asexual cycle has two major forms: the rapidly dividing tachyzoite and the more slowly dividing, encysted bradyzoite. Tachyzoites are not normally responsible for host-to-host transmission and instead serve to disseminate infection within a given animal by invading and rapidly multiplying in a wide range of nucleated cells. In apparent response to immune pressure from the host, T. gondii tachyzoites differentiate into bradyzoites, which grow within cyst-like structures in the host tissue. When ingested, bradyzoites are infectious both to cats (resulting in entry into the sexual cycle) and other intermediate hosts, where further asexual growth can occur. Spontaneous reactivation of the disease through rupture of the cysts and dissemination of T. gondii tachyzoites can result in fatal encephalitis in patients with AIDS.Tachyzoites and bradyzoites differ in a number of surface antigens (6) as well as important metabolic enzymes (10, 33, 34). Because of the difficulty of obtaining large amounts of tissue cysts from infected animals, however, it has been difficult to characterize bradyzoites in detail. Tachyzoites can be induced to differentiate in vitro through a variety of stresses (reviewed in reference 4) into forms which resemble bradyzoites by both morphological and antigenic criteria. Such in vitro bradyzoites have proved invaluable both in enabling the identification of bradyzoite-specific genes (16) and in providing initial insights into the mechanisms that control gene expression in this stage (5). However, the extent to which in vitro bradyzoites resemble parasites found in vivo is unclear.To understand more about this important stage in the asexual cycle of the parasite, we have generated a large number of bradyzoite expressed sequence tags (ESTs) that complement the data already obtained from the tachyzoite stage (1). We also report here the results of an EST analysis from tachyzoites induced to switch to bradyzoites in vitro following 6 days of growth at high pH (29). As with a similar study of mammalian cells (17) in various states of differentiation, this analysis reveals many genes that appear to be developmentally regulated in addition to a large number of putative housekeeping genes that are expressed constitutively.     

Psychosis may be associated with toxoplasmosis

Many parasites induce characteristic changes in their host. The effect of Toxoplasma gondii infection on the cerebrum and neuropsychiatric patients has been increasingly emphasized in recent years. T. gondii has a high affinity for brain tissue where tachyzoites may form tissue cysts and persist for a life long time. Some psychiatric symptoms such as schizophrenia and mental retardation may be induced by the infection of T. gondii. Furthermore, experiments demonstrated that some antipsychotics and mood stabilizers used to treat psychosis displayed the function of inhibiting T. gondii replication. Investigations from various regions in China in psychotic patients support the hypotheses that psychosis may be linked to T. gondii infection.     

The Surface of Toxoplasma Tachyzoites Is Dominated by a Family of Glycosylphosphatidylinositol-Anchored Antigens Related to SAG1

Toxoplasma gondii is an Apicomplexan parasite with a complex life cycle that includes a rapidly dividing asexual stage known as the tachyzoite. The tachyzoite surface has been reported to comprise five major antigens, the most abundant of which is designated SAG1 (for surface antigen 1). At least one of the other four (SAG3) and another recently described minor antigen (SRS1 [for SAG1-related sequence 1]) have previously been shown to be structurally related to SAG1. To determine if further SAG1 homologs exist, we searched a Toxoplasma expressed sequence tag (EST) database and found numerous ESTs corresponding to at least three new genes related to SAG1. Like SAG1, these new SRS genes encode apparently glycosylphosphatidylinositol-anchored proteins that share several motifs and a set of conserved cysteine residues. This family appears to have arisen by divergence from a common ancestor under selection for the conservation of overall topology. The products of two of these new genes (SRS2 and SRS3) are shown to be expressed on the surface of Toxoplasma tachyzoites by immunofluorescence. We also identified strain-specific differences in relative expression levels. A total of 10 members of the SAG1 gene family have now been identified, which apparently include three of the five major surface antigens previously described and one antigen expressed only in bradyzoites. The function of this family may be to provide a redundant system of receptors for interaction with host cells and/or to direct the immune responses that limit acute T. gondii infections.     

A Toxoplasma Patatin-Like Protein Changes Localization and Alters the Cytokine Response during Toxoplasmic Encephalitis

Toxoplasma gondii is an obligate intracellular parasite that forms a lifelong infection within the central nervous system of its host. The T. gondii genome encodes six members of the patatin-like phospholipase family; related proteins are associated with host-microbe interactions in bacteria. T. gondii patatin-like protein 1 (TgPL1) was previously determined to be necessary for parasites to suppress nitric oxide and prevent degradation in activated macrophages. Here, we show that in the rapidly replicating tachyzoite stage, TgPL1 is localized within vesicles inside the parasite that are distinct from the dense granules; however, in the encysted bradyzoite stage, TgPL1 localizes to the parasitophorous vacuole (PV) and cyst wall. While we had not previously seen a defect of the TgPL1 deletion mutant (ΔTgPL1) during acute and early chronic infection, the localization change of TgPL1 in bradyzoites caused us to reevaluate the ΔTgPL1 mutant during late chronic infection and in a toxoplasmic encephalitis (TE) mouse model. Mice infected with ΔTgPL1 are more resistant to TE and have fewer inflammatory lesions than mice infected with the wild type and ΔTgPL1 genetically complemented with TgPL1. This increased resistance to TE could result from several contributing factors. First, we found that ΔTgPL1 bradyzoites did not convert back to tachyzoites readily in tissue culture. Second, a subset of cytokine levels were higher in ΔTgPL1-infected mice, including gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), and monocyte chemotactic protein 1 (MCP-1). These studies suggest that TgPL1 plays a role in the maintenance of chronic T. gondii infection.     

A cluster of four surface antigen genes specifically expressed in bradyzoites, SAG2CDXY, plays an important role in Toxoplasma gondii persistence

Toxoplasma gondii is one of the most successful protozoan parasites of warm-blooded animals. Stage-specific expression of its surface molecules is thought to be key to its ability to establish chronic infection in immunocompetent animals. The rapidly dividing tachyzoite stage displays a different subset of family of surface antigen 1 (SAG1)-related sequences (SRSs) from that displayed by the encysted bradyzoite stage. It is possible that this switch is necessary to protect the bradyzoites against an immune response raised against the tachyzoite stage. Alternatively, it might be that bradyzoite SRSs evolved to facilitate invasion of different cell types, such as those found in the brain, where cysts develop, or the small intestine, where bradyzoites must enter after oral infection. Here we studied the function of a cluster of four tandem genes, encoding bradyzoite SRSs called SAG2C, -D, -X, and -Y. Using bioluminescence imaging of mice infected with parasites expressing firefly luciferase (FLUC) driven by the SAG2D promoter, we show stage conversion for the first time in living animals. A truncated version of the SAG2D promoter (SAG2Dmin) gave efficient expression of FLUC in both tachyzoites and bradyzoites, indicating that the bradyzoite specificity of the complete SAG2D promoter is likely due to an element(s) that normally suppresses expression in tachyzoites. Comparing mice infected with the wild type or a mutant where the SAG2CDXY cluster of genes has been deleted (DeltaSAG2CDXY), we demonstrate that whereas DeltaSAG2CDXY parasites are less capable of maintaining a chronic infection in the brain, they do not show a defect in oral infectivity.     

T cell receptor specificities of Toxoplasma gondii-reactive mouse CD4+ T lymphocytes and Th1 clones

The Toxoplasma gondii-directed CD4⁺ T cell response in chronically infected mice was studied with respect to both T cell receptor diversity and antigen specificities. T cell receptor chains Vβ4, 6, 8, 10, and 14 were predominantly found on toxoplasma-reactive CD4⁺ splenocytes. This repertoire was also detected among T. gondii-specific CD4⁺ T cell clones. Analysis of clonotypic cytokine profiles revealed typical Th1 clones secreting interleukin-2, interferon-γ and tumour necrosis factor activity and Th2 clones producing interleukin-4 and interleukin-10. Five distinct toxoplasma antigens (p26, p40, p55, p58 and p60) were detected in electrophoretically separated toxoplasma lysate by five individual Th1 clones. Parallel testing of CD4⁺ T lymphocytes from infected mice confirmed that these specificities constitute the peak immunogenic fractions of toxoplasma lysate. The expression patterns of two clonotypic, T cell-stimulatory parasite antigens were studied in detail. While p55 was expressed by mouse-virulent and avirulent T. gondii isolates and in both the tachyzoite and bradyzoite stages, p58 was detected only in virulent strains from intraspecies subgroup I. Thus, we describe the heterogeneity of toxoplasmic immunodominant T cell antigens including a 58-kDa group I-restricted molecule which may provide a marker for virulent isolates. Received: 3 February 1997     

Improved methods for examination of Toxoplasma gondii cytoskeleton at ultrastructural level

To evaluate an improved method for identifying the presence of the structural elements of the cytoskeleton of Toxoplasma gondii and their influence on invasion of the parasite in host cells, copper grids coated with plastic film were used for adhesion of whole parasites. Tachyzoites were incubated with 0.5% Triton X-100 in PHEM buffer containing protease inhibitors, post-fixed in 1% glutaraldehyde, stained with uranyl acetate and submitted to critical point drying. In order to analyze the presence of the structural elements of the cytoskeleton, immunolocalization was carried out using colloidal gold. Invasion of the parasite was examined on cell culture after treatment of tachyzoites with cytochalasin B (CB). In order to observe this effect, an immunocytochemical assay using alkaline phosphatase was carried out. A very well conserved extraction of the cytoskeleton elements of T. gondii, such as conoid and microtubules, as well as the rhoptries, was observed. By immunolocalization with colloidal gold, it was possible to detect the actin in its globular form. In the assay of invasion of the parasite on the host cell, after treatment of the T. gondii tachyzoites with CB, the invasion process was totally inhibited. Received: 18 April 2000 / Accepted: 19 August 2000     

MHC I presentation of Toxoplasma gondii immunodominant antigen does not require Sec22b and is regulated by antigen orientation at the vacuole membrane

The intracellular Toxoplasma gondii parasite replicates within a parasitophorous vacuole (PV). T. gondii secretes proteins that remain soluble in the PV space, are inserted into PV membranes or are exported beyond the PV boundary. In addition to supporting T. gondii growth, these proteins can be processed and presented by MHC I for CD8⁺ T‐cell recognition. Yet it is unclear whether membrane binding influences the processing pathways employed and if topology of membrane antigens impacts their MHC I presentation. Here we report that the MHC I pathways of soluble and membrane‐bound antigens differ in their requirement for host ER recruitment. In contrast to the soluble SAG1‐OVA model antigen, we find that presentation of the membrane‐bound GRA6 is independent from the SNARE Sec22b, a key molecule for transfer of host endoplasmic reticulum components onto the PV. Using parasites modified to secrete a transmembrane antigen with opposite orientations, we further show that MHC I presentation is highly favored when the C‐terminal epitope is exposed to the host cell cytosol, which corresponds to GRA6 natural orientation. Our data suggest that the biochemical properties of antigens released by intracellular pathogens critically guide their processing pathway and are valuable parameters to consider for vaccination strategies.