Toxoplasma gondii-infected cells are resistant to multiple inducers of apoptosis

Infection with certain intracellular pathogens, including viruses and bacteria, may induce host cell apoptosis. On the other hand, infection with some viruses inhibits apoptosis. Complex protozoan parasites, including Toxoplasma gondii and members of Plasmodium, Leishmania, and Microsporidia, are also obligate intracellular pathogens, yet relatively little is known regarding their subversion of host cell functions. We now report that cells infected with T. gondii are resistant to multiple inducers of apoptosis, including Fas-dependent and Fas-independent CTL-mediated cytotoxicity, IL-2 deprivation, gamma irradiation, UV irradiation, and the calcium ionophore beauvericin. Inhibition of such a broad array of apoptosis inducers suggests that a mechanism common to many, or perhaps all, apoptotic pathways is involved. The inhibitory activity requires live intracellular parasite and ongoing protein synthesis. Despite T. gondii-mediated inhibition of DNA fragmentation, infected cells can still be lysed by CTL.     

Regulation and function of T-cell-mediated immunity during Toxoplasma gondii infection

The intracellular protozoan Toxoplasma gondii is a widespread opportunistic parasite of humans and animals. Normally, T. gondii establishes itself within brain and skeletal muscle tissues, persisting for the life of the host. Initiating and sustaining strong T-cell-mediated immunity is crucial in preventing the emergence of T. gondii as a serious pathogen. The parasite induces high levels of gamma interferon (IFN-gamma) during initial infection as a result of early T-cell as well as natural killer (NK) cell activation. Induction of interleukin-12 by macrophages is a major mechanism driving early IFN-gamma synthesis. The latter cytokine, in addition to promoting the differentiation of Th1 effectors, is important in macrophage activation and acquisition of microbicidal functions, such as nitric oxide release. During chronic infection, parasite-specific T lymphocytes release high levels of IFN-gamma, which is required to prevent cyst reactivation. T-cell-mediated cytolytic activity against infected cells, while easily demonstrable, plays a secondary role to inflammatory cytokine production. While part of the clinical manifestations of toxoplasmosis results from direct tissue destruction by the parasite, inflammatory cytokine-mediated immunopathologic changes may also contribute to disease progression.     

Alpha beta T cell response to Toxoplasma gondii in previously unexposed individuals

The mechanisms by which T cells from previously unexposed hosts respond in vitro to certain intracellular pathogens remain to be fully understood. We report and characterize the in vitro reactivity to Toxoplasma gondii of human alpha beta T cells from T. gondii-seronegative individuals. Resting alpha beta T cells from these individuals proliferated in response to PBMC infected with T. gondii or pulsed with T. gondii lysate Ags. This was accompanied by an increase in the percentage of CD4+ alpha beta T cells. Purified CD4+ alpha beta T cells but not CD8+ alpha beta T cells proliferated in response to these T. gondii preparations. Both CD4+ alpha beta T cells with naive (CD45RA+) and memory (CD45RO+) phenotypes from adults as well as alpha beta T cells from T. gondii-seronegative newborns proliferated after incubation with T. gondii. This alpha beta T cell response to the parasite was inhibited by anti-HLA-DR mAb and to a lesser degree by anti-HLA-DQ mAb. Use of paraformaldehyde-fixed PBMC completely abrogated the proliferation of alpha beta T cells, indicating the need for processing of T. gondii Ags. Analysis of the TCR V beta expression did not show evidence for restriction in TCR V beta usage during T. gondii stimulation of alpha beta T cells. Alpha beta T cells secreted significant amounts of IFN-gamma after incubation with T. gondii-infected monocytes. This rapid and remarkable alpha beta T cell response may play an important role in the early events of the immune response to T. gondii.     

Mechanisms of innate resistance to Toxoplasma gondii infection

The interaction of protozoan parasites with innate host defences is critical in determining the character of the subsequent infection. The initial steps in the encounter of Toxoplasma gondii with the vertebrate immune system provide a striking example of this important aspect of the host-parasite relationship. In immuno-competent individuals this intracellular protozoan produces an asymptomatic chronic infection as part of its strategy for transmission. Nevertheless, T. gondii is inherently a highly virulent pathogen. The rapid induction by the parasite of a potent cell-mediated immune response that both limits its growth and drives conversion to a dormant cyst stage explains this apparent paradox. Studies with gene-deficient mice have demonstrated the interleukin-12 (IL-12)-dependent production of interferon gamma (IFN-gamma) to be of paramount importance in controlling early parasite growth. However, this seems to be independent of nitric oxide production as mice deficient in inducible nitric oxide synthase (iNOS) and tumour necrosis factor receptor were able to control early growth of T. gondii, although, they later succumbed to infection. Nitric oxide does, however, seem to be important in controlling persistent infection; treating chronic infection with iNOS metabolic inhibitors results in disease reactivation. Preliminary evidence implicates neutrophils in effector pathways against this parasite distinct from that described for macrophages. Once initiated, IL-12-dependent IFN-gamma production in synergy with other proinflammatory cytokines can positively feed back on itself to induce 'cytokine shock'. Regulatory cytokines, particularly IL-10, are essential to down-regulate inflammation and limit host pathology.     

In control of its fate: gene regulation in Toxoplasma gondii

The intracellular protozoan parasite Toxoplasma gondii is an important opportunistic pathogen in animals and man. In parallel to its clinical significance, T. gondii is also receiving considerable attention as an attractive model organism for intracellular parasitism. Regulation of gene expression at various levels underlies the intricate interplay between the parasite and its host cell, as well as the interconversions between different life-stages. In this article we will discuss some of what is currently known about gene organization and gene regulation in T. gondii as well as some of the tools available to dissect the parasite at a molecular level.     

Inhibition of in vitro replication of the oyster parasite Perkinsus marinus by the natural iron chelators transferrin, lactoferrin, and desferrioxamine

The mammalian iron-binding proteins transferrin and lactoferrin, the bactericidal peptide lactoferricin B, and the bacterial siderophore desferrioxamine were tested for their ability to inhibit the in vitro replication of the oyster parasite Perkinsus marinus. All three chelators were effective in reducing the parasite proliferation in a dose-dependent manner. Lactoferricin B, a peptide of lactoferrin that exhibits bactericidal properties unrelated to iron chelation, had no inhibitory activity on the parasite. When the chelators were partially or completely saturated with the appropriate iron equivalents, their inhibitory effects on the parasite proliferation were diminished or abolished accordingly, confirming that this activity was related to the chelator's capacity for iron sequestration. Our results indicate that the parasite has a strong requirement for soluble iron and its growth rates are correlated with iron availability. We propose that excess iron accumulation in the host Crassostrea virginica promotes parasite proliferation. P. marinus may avoid oxidative damage that would compromise its intracellular survival by exhaustion the host's intracellular selected iron pools required for superoxide and hydroxyl radical production.     

IFN-gamma is required for viral clearance from central nervous system oligodendroglia

Infection of the central nervous system (CNS) by the JHM strain of mouse hepatitis virus (JHMV) is a rodent model of the human demyelinating disease multiple sclerosis. The inability of effective host immune responses to eliminate virus from the CNS results in a chronic infection associated with ongoing recurrent demyelination. JHMV infects a variety of CNS cell types during the acute phase of infection including ependymal cells, astrocytes, microglia, oligodendroglia, and rarely in neurons. Replication within the majority of CNS cell types is controlled by perforin-dependent virus-specific CTL. However, inhibition of viral replication in oligodendroglia occurs via a perforin-independent mechanism(s). The potential role for IFN-gamma as mediator controlling JHMV replication in oligodendroglia was examined in mice deficient in IFN-gamma secretion (IFN-gamma0/0 mice). IFN-gamma0/0 mice exhibited increased clinical symptoms and mortality associated with persistent virus, demonstrating an inability to control replication. Neither antiviral Ab nor CTL responses were diminished in the absence of IFN-gamma, although increased IgG1 was detected in IFN-gamma0/0 mice. Increased virus Ag in the absence of IFN-gamma localized almost exclusively to oligodendroglia and was associated with increased CD8+ T cells localized within white matter. These data suggest that although perforin-dependent CTL control virus replication within astrocytes and microglia, which constitute the majority of infected CNS cells, IFN-gamma is critical for control of viral replication in oligodendroglia. Therefore, different mechanisms are used by the host defenses to control virus replication within the CNS, dependent upon the phenotype of the targets of virus replication.     

Leishmania donovani infection of a susceptible host results in apoptosis of Th1-like cells: rescue of anti-leishmanial CMI by providing Th1-specific bystander costimulation

A protective immune response against Leishmania donovani infection is mediated by T-helper type 1 (Th1) cells. Th1 induced cell-mediated immunity (CMI), as assessed by anti-leishmanial DTH response, is lost in a susceptible host such as BALB/c mice. Although the impaired Th1 function eventuates in unhindered parasite growth and in manifestation of the susceptible phenotype, the mechanism of down-regulation of the Th1 function is yet to be elucidated. Here, we provide evidence that the parasite down-regulates the expression of a Th1-specific costimulatory molecule, M150, on the surface of infected BALB/c mice-derived macrophages. Th cells are rendered unresponsive to anti-CD3 Ab-mediated stimulation after interaction with infected macrophages. The anergized T cells produce much less IL-2, IL-4 and IFN-gamma compared to those T cells which were costimulated using normal macrophages. The defect in proliferation, anti-CD3 Ab induced unresponsiveness and IFN-gamma but not IL-4 production can be restored by providing bystander costimulation through M150. These results not only unfold a novel immune evasion strategy used by the parasite but also clarify the mechanism of Th1 cell debilitation during the disease. Recovery of Th1 cytokine production by bystander costimulation through M150 may help in formulating a new strategy for the elimination of intracellular parasites.     

Elevated interleukin-10-to-interleukin-12 ratio in feline immunodeficiency virus-infected cats predicts loss of type 1 immunity to Toxoplasma gondii

Similar to human immunodeficiency virus, feline immunodeficiency virus (FIV) induces immunodeficiency and enhanced susceptibility to secondary pathogens. To explore cytokine alterations in lentivirus immunodeficiency, constitutive mRNA expression was measured in lymph nodes of healthy and FIV-infected cats before and after challenge with Toxoplasma gondii. Cytokine mRNA expression was similar in control and FIV-infected cats during the first 10 weeks after infection. At 16 weeks, interferon (IFN)-gamma, tumor necrosis factor-alpha, and interleukin (IL)-10 mRNA were increased in FIV-infected cats. Challenge with T. gondii induced an increase in IL-2, IFN-gamma, and IL-12 in the lymph nodes of control cats, whereas IFN-gamma and IL-10 but not IL-2 or IL-12 increased in the lymph nodes of FIV-T. gondii coinfected cats. These results indicate that FIV immunodeficiency may derive from a failure to generate an IL-12-dependent type 1 response and that an elevated level of IL-10 mRNA expression is a predictor of lentivirus immunodeficiency.     

A method to obtain large quantities of Toxoplasma gondii tachyzoites with extreme purity

In order to extract genetic material from the intracellular parasite Toxoplasma gondii, large numbers of organisms free of host cells are necessary. A method is described in which the RH strain of T. gondii was cultivated in nonadherent U937 cells. The purification was done by countercurrent elutriation. The resulting population of 10(10) T. gondii contained only 0.17% host cell material. The method is simple, rapid, and minimizes the use of animals.     

The role of the cytoskeleton in host cell invasion by Toxoplasma gondii

The protozoan parasite Toxoplasma gondii provides a model system for studying invasion by intracellular parasites belonging to the phylum Apicomplexa. Taking advantage of the versatility of T. gondii for genetic and cell biological studies, we have shown that parasite motility and cell invasion are powered by an actin-myosin based motor in the parasite. Unlike bacterial cell uptake, parasite invasion does not involve significant alterations in the host cell cytoskeleton. Instead, invasion is an active process of penetration into the host cell by the parasite. The force for cell penetration is provided by a unique form of substrate-dependent motility termed gliding. Gliding motility is characterized by the rearward capping of surface membrane proteins that propels the parasite forward in a helical spiral. Both actin and myosin are localized beneath the plasma membrane in the parasite where they presumably combine to produce the force necessary for motility. During cell invasion, the rearward capping of cell surface receptors envelopes the parasite in a unique vacuole derived from the host cell plasma membrane. This system offers insights into force generation and motility in a simple organism that is also an important human pathogen.     

Toxoplasma gondii: dithiol-induced Ca2+ flux causes egress of parasites from the parasitophorous vacuole

Ca2+ is an essential activator of motility in the obligate intracellular parasite Toxoplasma gondii. Ca2+ ionophore A23187 and intracellular microinjection of Ca2+ initiate motility of parasites residing in parasitophorous vacuoles (PV). The source of Ca2+ and the mechanism by which it activates motility in vivo remain uncertain. Exposure of the parasites to dithiothreitol (DTT) can activate egress of previously nonmotile intravacuolar parasites within 60 sec. DTT is also known to activate both isoforms of the highly concentrated nucleoside triphosphate hydrolase (NTPase) produced by T. gondii. Using an adherent cell analysis system (ACAS) for Ca2+ imaging, a brief 15-50% increase in intra-PV fluorescence ratio was observed after exposure of infected fibroblasts to 5 mM DTT. Chelation of intracellular Ca2+ with BAPTA-AM and extracellular Ca2+ with EGTA blocked the DTT effect; however, this chelation did not prevent the activation of parasites nor the Ca2+ response to the Ca2+ ionophore ionomycin, suggesting that the Ca2+ that activates motility may reside near or within the parasite itself. This result demonstrates that an increase in Ca2+ within the vacuole precedes the onset of motility and the correlation of the DTT effect on motility and tachyzoite NTPase suggests that NTPase activation may be involved in the Ca2+ flux.     

A dichotomous role for nitric oxide during acute Toxoplasma gondii infection in mice

Production of nitric oxide by macrophages is believed to be an important microbicidal mechanism for a variety of intracellular pathogens, including Toxoplasma gondii. Mice with a targeted disruption of the inducible nitric oxide synthase gene (iNOS) were infected orally with T. gondii tissue cysts. Time to death was prolonged compared with parental controls. Histologic analysis of tissue from infected mice showed scattered small foci of inflammation with parasites in various tissues of iNOS-/- mice, whereas tissue from the parental C57BL/6 mice had more extensive tissue inflammation with few visible parasites. In particular, extensive ulceration and necrosis of distal small intestine and fatty degeneration of the liver was seen in the parental mice at day 7 postinfection, as compared with the iNOS-/- mice where these tissues appeared normal. Serum interferon gamma and tumor necrosis factor alpha levels postinfection were equally elevated in both mouse strains. Treatment of the parental mice with a NO synthase inhibitor, aminoguanidine, prevented early death in these mice as well as the hepatic degeneration and small bowel necrosis seen in acutely infected control parentals. These findings indicate that NO production during acute infection with T. gondii can kill intracellular parasites but can be detrimental, even lethal, to the host.     

Selection and characterization of Toxoplasma gondii mutants resistant to artemisinin

Toxoplasma gondii infection, like malaria, is sensitive to inhibition by artemisinin (ART). Mechanisms of action for ART in malaria treatment have been proposed, but little is known about its effects in T. gondii infection. To better understand its inhibitory effects on T. gondii, mutants resistant to ART were selected by progressive culture in permissive levels of the drug. Five clonal isolates were established and characterized. The isolates were approximately 65-fold less sensitive to ART than is the parental RH and showed cross-resistance to the ART derivatives dihydroartemisinin and artemether. In addition to ART resistance, 1 clone (C9) formed morphologically unusual parasitophorous vacuoles and another (A2) was avirulent for mice and protected mice from challenge with the wild type. These clonal T. gondii mutant isolates will be useful for the study of not only the mechanism of action of ART but also parasite vacuole biology and virulence factors.     

Association of HFE protein with transferrin receptor in crypt enterocytes of human duodenum

In hereditary hemochromatosis (HH), intestinal absorption of dietary iron is increased, leading to excessive iron accumulation in tissues and resultant organ damage. The HFE protein, which is defective in HH, normally is expressed in crypt enterocytes of the duodenum where it has a unique, predominantly intracellular localization. In placenta, the HFE protein colocalizes with and forms a stable association with the transferrin receptor (TfR), providing a link between the HFE protein and iron transport. In the present study, we examined the relationship of the HFE protein to the TfR in enterocytes of the human duodenum and measured the uptake of transferrin-bound iron and ionic iron by isolated crypt and villus enterocytes. Immunocytochemistry showed that the HFE protein and TfR both are expressed in the crypt enterocytes. Western blots showed that, as was the case in human placenta, the HFE protein in crypt enterocytes is physically associated with the TfR and with beta2-microglobulin. The crypt cell fraction exhibited dramatically higher transferrin-bound iron uptake than villus cells. On the other hand, the villus cells showed 2-3 times higher uptake of ionic iron than crypt cells. We propose that the HFE protein modulates the uptake of transferrin-bound iron from plasma by crypt enterocytes and participates in the mechanism by which the crypt enterocytes sense the level of body iron stores. Impairment of this function caused by HFE gene mutations in HH could provide a paradoxical signal in crypt enterocytes that programs the differentiating enterocytes to absorb more dietary iron when they mature into villus enterocytes.     

An effective analytical psychotherapy in crosscultural context. An East Asian student in the United States

Nucleoside triphosphate hydrolase is an abundant protein secreted by the obligate protozoan parasite Toxoplasma gondii. The protein has apyrase activity, degrading ATP to the di- and mono-phosphate forms. Because T. gondii is incapable of de novo synthesis of purines, it is postulated that NTPase may be used by the parasite to salvage purines from the host cell for survival and replication. To elucidate the molecular mechanisms of NTP gene expression, we isolated from the virulent RH strain of T. gondii the putative promoter region of three tandemly repeated NTP genes (NTP1, 2, 3). Using deletion constructs linked to the chloramphenicol acetyl transferase (CAT) reporter gene, we defined an active promoter within the first 220 bp. Sequence analysis of this region reveals the lack of a TATA box, but the promoter region is associated with a sequence which resembles an initiator element (Inr) in the NTP1 and NTP3 genes. This sequence which is similar to other Inrs known to regulate the expression of a wide variety of RNA polymerase II genes, is required for NTP expression. The NTP3 promoter contains sufficient information for developmentally regulated expression of CAT activity when the actively replicating stage tachyzoite differentiates into the dormant bradyzoite form.     

Improvement predictors in obsessive-compulsive disorder. An event-related potential study

The maturation of HIV-1 virions is accomplished through the proteolytic cleavage of Gag and GagPol precursor polyproteins by the viral-encoded protease (PR). Since virions are assembled from unprocessed polyproteins, the intracellular activation of PR must be limited. An experimental system was established that allows the investigation of the intracellular regulation of PR activity. By expressing Gag in trans with the GagPol precursor, downregulation of the intracellular PR activity associated with GagPol was demonstrated. Inhibition of PR activity was dependent upon the context of PR expression. Sequences capable of mediating this inhibition were localized to capsid. A mechanism through which Gag regulates PR activity is proposed whereby the disproportionate synthesis of Gag inhibits the activation of PR in the cytoplasm. Further elucidation of the mechanism of intracellular inhibition of PR activity may facilitate the development of novel PR inhibitors capable of inhibiting viral replication in vivo.