Sandra K. Halonen

Gamma interferon-induced inhibition of Toxoplasma gondii in astrocytes is mediated by IGTP.

ABSTRACT Toxoplasma gondii is an important pathogen in the central nervous system, causing a severe and often fatal encephalitis in patients with AIDS. Gamma interferon (IFN-γ) is the main cytokine preventing reactivation of Toxoplasma encephalitis in the brain. Microglia are important IFN-γ-activated effector cells controlling the growth of T. gondii in the brain via a nitric oxide (NO)-mediated mechanism. IFN-γ can also activate astrocytes to inhibit the growth of T. gondii. Previous studies found that the mechanism in murine astrocytes is independent of NO and all other known anti-Toxoplasma mechanisms. In this study we investigated the role of IGTP, a recently identified IFN-γ-regulated gene, in IFN-γ inhibition of T. gondii in murine astrocytes. Primary astrocytes were cultivated from IGTP-deficient mice, treated with IFN-γ, and then tested for anti-Toxoplasma activity. In wild-type astrocytesT. gondii growth was significantly inhibited by IFN-γ, whereas in astrocytes from IGTP-deficient mice IFN-γ did not cause a significant inhibition of growth. Immunoblot analysis confirmed that IFN-γ induced significant levels of IGTP in wild-type murine astrocytes within 24 h. These results indicate that IGTP plays a central role in the IFN-γ-induced inhibition of T. gondii in murine astrocytes.

Initial Characterization of CST1, a Toxoplasma gondii Cyst Wall Glycoprotein

ABSTRACT Toxoplasma gondii is an important protozoan pathogen of humans that can cause encephalitis in immunocompromised individuals such as those with AIDS. This encephalitis is due to reactivation of latent infection in T. gondii-seropositive patients. Latent organisms survive within tissue cysts, which are specialized parasitophorous vacuoles containing bradyzoites. The cyst wall of this structure is produced by modification of the parasitophorous vacuole by the parasite and is important in cyst survival. The components of the cyst wall have been poorly characterized. By using immunofluorescence and immunoelectron microscopy, we have identified a monoclonal antibody (MAb 93.18) that reacts with the cyst wall. This antibody recognizes a 116-kDa glycoprotein, which we have termed CST1, containing sugar residues that bind Dolichos biflorans lectin (DBA). CST1 is distinct from T. gondii antigen labeled with succinylTriticum vulgare lectin (S-WGA) and represents the major DBA-binding component in T. gondii. The carbohydrate components of the tissue cyst, such as CST1, are probably important in both providing stability and facilitating persistence in its host. As is seen in the carbohydrate capsules of fungi, glycoproteins in theT. gondii cyst wall may protect cysts from the immune response of the host. Further characterization of the formation of the cyst wall and its components should lead to insights into the mechanism of tissue cyst persistence and may suggest novel therapeutic approaches to eliminate tissue cysts of this organism.

The Gamma Interferon (IFN-γ)-Inducible GTP-Binding Protein IGTP Is Necessary for Toxoplasma Vacuolar Disruption and Induces Parasite Egression in IFN-γ-Stimulated Astrocytes

ABSTRACT Toxoplasma gondii is a common central nervous system infection in individuals with immunocompromised immune systems, such as AIDS patients. Gamma interferon (IFN-γ) is the main cytokine mediating protection against T. gondii. Our previous studies found that IFN-γ significantly inhibits T. gondii in astrocytes via an IFN-γ-inducible GTP-binding protein (IGTP)-dependent mechanism. The IGTP-dependent-, IFN-γ-stimulated inhibition is not understood, but recent studies found that IGTP induces disruption of the parasitophorous vacuole (PV) in macrophages. In the current study, we have further investigated the mechanism of IFN-γ inhibition and the role of IGTP in the vacuolar disruption in murine astrocytes. Vacuolar disruption was found to be dependent upon IGTP, as PV disruption was not observed in IGTP-deficient (IGTP−/−) astrocytes and PV disruption could be induced in IGTP−/− astrocytes transfected with IGTP. Live-cell imaging studies using green fluorescent protein-IGTP found that IGTP is delivered to the PV via the host cell endoplasmic reticulum (ER) early after invasion and that IGTP condenses into vesicle-like structures on the vacuole just prior to PV disruption, suggesting that IGTP is involved in PV disruption. Intravacuolar movement of the parasite occurred just prior to PV disruption. In some instances, IFN-γ induced parasite egression. Electron microscopy and immunofluorescence studies indicate that the host cell ER fuses with the PV prior to vacuolar disruption. On the basis of these results, we postulate a mechanism by which ER/PV fusion is a crucial event in PV disruption. Fusion of the ER with the PV, releasing calcium into the vacuole, may also be the mechanism by which intravacuolar parasite movement and IFN-γ-induced parasite egression occur.

Presentation of Toxoplasma gondii Antigens via the Endogenous Major Histocompatibility Complex Class I Pathway in Nonprofessional and Professional Antigen-Presenting Cells

ABSTRACT Challenge with the intracellular protozoan parasite Toxoplasma gondii induces a potent CD8+ T-cell response that is required for resistance to infection, but many questions remain about the factors that regulate the presentation of major histocompatibility complex class I (MHC-I)-restricted parasite antigens and about the role of professional and nonprofessional accessory cells. In order to address these issues, transgenic parasites expressing ovalbumin (OVA), reagents that track OVA/MHC-I presentation, and OVA-specific CD8+ T cells were exploited to compare the abilities of different infected cell types to stimulate CD8+ T cells and to define the factors that contribute to antigen processing. These studies reveal that a variety of infected cell types, including hematopoietic and nonhematopoietic cells, are capable of activating an OVA-specific CD8+ T-cell hybridoma, and that this phenomenon is dependent on the transporter associated with antigen processing and requires live T. gondii. Several experimental approaches indicate that T-cell activation is a consequence of direct presentation by infected host cells rather than cross-presentation. Surprisingly, nonprofessional antigen-presenting cells (APCs) were at least as efficient as dendritic cells at activating this MHC-I-restricted response. Studies to assess whether these cells are involved in initiation of the CD8+ T-cell response to T. gondii in vivo show that chimeric mice expressing MHC-I only in nonhematopoietic compartments are able to activate OVA-specific CD8+ T cells upon challenge. These findings associate nonprofessional APCs with the initial activation of CD8+ T cells during toxoplasmosis.

Chapter 8 - Toxoplasmosis

Toxoplasma gondii, an Apicomplexan, is a pathogic protozoan that can infect the central nervous system. Infection during pregnancy can result in a congenial infection with severe neurological sequelae. In immunocompromisedindividuals reactivation of latent neurological foci can result in encephalitis. Immunocompetent individuals infected with T. gondii are typically asymptomatic and maintain this infection for life. However, recent studies suggest that these asymptomatic infections may have effects on behavior and other physiological processes. Toxoplasma gondii infects approximately one-third of the world population, making it one of the most successful parasitic organisms. Cats and other felidae serve as the definite host producing oocysts, an environmentally resistant life cycle stage found in cat feces, which can transmit the infection when ingested orally. A wide variety of warm-blooded animals, including humans, can serve as the intermediate host in which tissue cysts (containing bradyzoites) develop. Transmission also occurs due to ingestion of the tissue cysts. There are three predominant clonal lineages, termed Types I, II and III, and an association with higher pathogenicity with the Type I strains in humans has emerged. This chapter presents a review of the biology of this infection including the life cycle, transmission, epidemiology, parasite strains, and the host immune response. The major clinical outcomes of congenital infection, chorioretinitis and encephalitis, and the possible association of infection of toxoplasmosis with neuropsychiatric disorders such as schizophrenia, are reviewed.

Investigation into the Mechanism of Gamma Interferon-Mediated Inhibition of Toxoplasma gondii in Murine Astrocytes

ABSTRACT Toxoplasma gondii is an obligate intracellular parasite that is a common opportunistic pathogen of the central nervous system in AIDS patients. Gamma interferon (IFN-γ) alone or in combination with interleukin-1 (IL-1), IL-6, or tumor necrosis factor alpha significantly inhibits the growth of T. gondii in murine astrocytes, suggesting these are important nonimmune effector cells in the brain. Inhibition was found to be independent of a nitric oxide-mediated or tryptophan starvation mechanism. Both reactive oxygen intermediates and iron deprivation are IFN-γ-mediated mechanisms known to operate against intracellular parasites in other cell types. Astrocytes generated from mice genetically deficient in the production of reactive oxygen intermediates (phox−/−mice) were found to inhibit growth of T. gondii when stimulated with IFN-γ alone or in combination with other cytokines. The reactive oxygen inhibitor catalase and the reactive oxygen scavengers mannitol and thiourea failed to reverse the IFN-γ-induced inhibition of T. gondii in astrocytes. These data indicate that IFN-γ-induced inhibition in astrocytes is independent of reactive oxygen intermediates. IFN-γ-induced inhibition could not be reversed by the addition of iron salts, ferric citrate, ferric nitrate, or ferric transferrin. Pretreatment of astrocytes with desferrioxamine also did not induce the inhibition of T. gondii. These data indicate that the mechanism of IFN-γ inhibition was not due to iron deprivation. IFN-γ had no effect on T. gondii invasion of astrocytes, but inhibition of growth and loss of tachyzoite vacuoles were evident in IFN-γ-treated astrocytes by 24 h after invasion. Overall, these data suggest that IFN-γ-activated astrocytes inhibitT. gondii by an as-yet-unknown mechanism.

Role of autophagy in the host defense against Toxoplasma gondii in astrocytes

Autophagy has recently been implicated in the host defense against the intracellular protozoan pathogen, Toxoplasma gondii, a major opportunistic pathogen of the central nervous system in immunosuppressed individuals. In both IFNγ-activated macrophages and astrocytes, the p47 GTPases traffic to the T. gondii parasitophorous vacuole, followed by vacuolar disruption, parasite killing, and clearance of the dead parasites. In macrophages, it is relatively well established that autophagy is involved in parasite elimination and killing. The role of autophagy in parasite elimination in astrocytes, a dominant host cell in the central nervous system, is much less clear. Our studies indicate that in IFNγ-stimulated astrocytes, autophagy of disrupted vacuoles and/or dead parasites does not occur but rather that degradation of the parasite occurs in the host cytoplasm. However, recent studies indicate autophagy may be involved in the elimination of the degraded parasite material from the astrocyte host cell cytoplasm and suggest that autophagous removal of degraded parasite material may be necessary for survival of the host cell. Delivery of parasite antigen from the cytosol to the endolysosomal compartments in astrocytes is of importance as it suggests a pathway by which astrocytes could present Toxoplasma antigens via the MHC Class II pathway and function as an antigen-presenting cell for the parasite in the brain.

Toxoplasma gondii Presentations at the 10th International Workshops on Opportunistic Protists: 100 Years and Counting

Toxoplasma gondii, an apicomplexan parasite of mammals, was first identified over 100 years ago (in 1908) by Nicolle and Manceaux, who isolated tachyzoites from the gundi, a North African rodent (34). Splendore also identified this parasite in the tissue of a rabbit in 1908 (46). The genus Toxoplasma was named for its bow-like shape (from the Greek “toxo,” for bow or arc, and “plasma,” for creature). The presence of a tissue cyst (bradyzoite) life stage was rapidly recognized, but it was not until almost 60 years later that this organism was recognized to be a coccidian and that felines were identified as being the definitive hosts by several groups working independently, including Dubey and Frenkel in 1970 (16). The association of T. gondii with food-borne and waterborne transmission has resulted in its classification as a National Institute of Allergy and Infectious Diseases (NIAID) category B priority agent. Due to the extensive repertoire of applicable experimental techniques available for this pathogen, it has become a model organism for the study of intracellular pathogens. Research on T. gondii continues to move rapidly, and this review will address information related to recent advances in our understanding of the biology of T. gondii presented at the IWOP-10 held in Boston, MA, 28 to 31 May 2008, and the symposium entitled “Centenary Celebration of Toxoplasma Discovery” held at that meeting. History, epidemiology, and life cycle. The details of the history of the discovery of T. gondii were reviewed at IWOP-10 by Dubey (15) and were also described in recent reviews (1, 14). T. gondii is estimated to infect about one-third of the world’s human population and is a significant zoonotic and veterinary pathogen. In humans and veterinary hosts, T. gondii is frequently associated with congenital infection and abortion. This parasite can be transmitted by the vertical transmission of the rapidly growing tachyzoite form if an immunologically naive mother acquires a new infection during pregnancy. In addition, T. gondii is an opportunistic pathogen associated with encephalitis or systemic infections in immunocompromised hosts such as individuals with advanced human immunodeficiency virus infection (i.e., AIDS). Tachyzoites divide rapidly within host cells and are thought to be responsible for the clinical manifestations of infection. In humans, T. gondii is most commonly acquired by the oral

Cerebral Toxoplasmosis. Pathogenesis, Host Resistance and Behavioural Consequences.

Abstract Following infection with Toxoplasma gondii, tachyzoites proliferate within a variety of nucleated cells in various organs during the acute stage of infection. Interferon-gamma (IFN-γ)-dependent cell-mediated immune responses, and humoral immune responses to a lesser extent, control the tachyzoite proliferation, but the parasite establishes a chronic infection by forming tissue cysts, especially in the brain, heart and skeletal muscle. IFN-γ can activate microglia, astrocytes, and brain microvascular endothelial cells to prevent tachyzoite growth by using different mechanisms depending on the cell types. T cells are an essential producer of IFN-γ to control tachyzoites, and interleukin (IL)-12 plays a crucial role in inducing their IFN-γ production. Many other cytokines such as TNF-α and IL-6 are involved in the IFN-γ-mediated protective immune responses against tachyzoites in the brain. In addition, multiple cytokines and molecules such as IL-10, IL-27, and lipoxin A4 play important downregulatory roles on the immune responses to prevent development of immunopathology. T. gondii can form cysts in multiple cell types in the brain, including neurons and astrocytes. Recently, CD8+ T cells were revealed to have an activity to remove tissue cysts from the brains of chronically infected mice. This anti-cyst activity of the T cells is does not require their IFN-γ but requires perforin. Therefore, the immune system appears to use two different mechanisms, one is mediated by IFN-γ and another is mediated by perforin, depending on the stage of the parasite that it targets. Therefore, cerebral infection with T. gondii is controlled by well-organized and orchestrated functions of the immune system. Genetic factors in both the host and the parasite also affect the susceptibility to the cerebral infection. Although chronic infection with T. gondii has been considered as “latent”, recent studies indicated a correlation of the infection with cryptogenic epilepsy and neuropsychiatric disorders such as schizophrenia. T. gondii can also cause congenital infection to the fetus, in which the brain is the major organ affected.

Differential gene expression in murine astrocytes infected with virulent (type I)vs. a virulent (type II) strains of Toxoplasma gondii

Toxoplasma gondii is a ubiquitous parasite infecting up to 30% of the population worldwide. Most individuals infected with T. gondii harbor a chronic infection that persists for the lifetime of the host with the parasite located predominantly in neural and muscular tissue. In some immune competent hosts however, chronic infection is associated with neurological disorders including schizophrenia, depression, suicidal behavior, headaches and cryptogenic epilepsy. There are three strains of T. gondii, designated type I, type II and type III, with evidence indicating type I strains are more virulent in humans, causing more severe clinical outcomes. Astrocytes are the predominant glial cell in the brain and can serve as a host cell for tachyzoite and bradyzoite stages in the brain. In this study we have used a transcriptional approach to dissect the host response to virulent type I RH strain vs. a virulent type II Me49 strain, in murine astrocytes. The results showed the type I strain induces a distinctly different host cell response in astrocytes inducing larger changes in gene expression levels and affecting different host cell pathways as compared to the type II strain. Type I vs. type II strains showed some common responses that may represent the ‘core’ response to parasite infection with host immune response genes the dominant pathway affected by both strains. However Toxoplasma strains show clear differences in modulation of some of these core host responses and importantly in some pathways related to pathogenesis of the nervous system. Neurobiological processes significantly affected by type I strain infection included effects on neurodevelopmental processes and nerve impulse transmission, including many gene homologues associated with Schizophrenia. The strain-specific effects could help explain the different clinical outcomes of Toxoplasma infections in humans and specifically, the distinct neurological complications such as Schizophrenia that occurs in the latent infection in some immune competent individuals.