A New Conditional Transcription Factor for Use in Toxoplasma Gondii.

The phylogenetic tree segregates into three major groups, which probably descended from a common ancestor: the bacteria, the archaea, and the eucarya (1–3). While bacteria and archaea lack a true nucleus and intracellular organelles, eucarya possess these. The origin of the ancestor remains elusive, but it emerged about 4 billion years ago. The bacteria probably started life 3.5 billion years ago and eukaryotes emerged 1.75 billion years thereafter. Sometime in between archaea appeared. Eukaryotes began as unicellular organisms but soon also developed multicellular forms. Bacteria and archaea had sufficient time to exploit all niches of the abiotic environment, ranging from ice cold glacial lakes to fiery geysers – from the highest mountain peaks to the deepest sea beds. They also quickly developed forms of mutualism by forming colonies composed of mixed populations. With the evolution of eucarya, new environments could be exploited. Today prokaryotes are the most prevalent form of life on earth, making up the majority of our biomass. It has been estimated that more than 10 prokaryotic organisms live on our globe. Thus, archaea and bacteria are far more prevalent than eucarya. Even human beings comprise more prokaryotic cells than mammalian cells, which range on the order of 10 human cells and 10 prokaryotes mostly concentrated in our intestinal system (4–7).

Immunophilins are protein chaperones with peptidylprolyl isomerase activity that belong to one of two large families, the cyclosporin-binding cyclophilins (CyPs) and the FK506-binding proteins (FKBPs). Each family displays characteristic and conserved sequence features that differ between the two families. We report a novel group of dual-family immunophilins that contain both CyP and FKBP domains for which we propose the name FCBP (FK506- and cyclosporin-binding protein). The FCBP of Toxoplasma gondii, a protozoan parasite, contained N-terminal FKBP and C-terminal CyP domains joined by tetratricopeptide repeats. Structure-function analysis revealed that both domains were functional and exhibited family-specific drug sensitivity. The individual domains of FCBP inhibited calcineurin (protein phosphatase 2B) in the presence of the appropriate drugs. In binding studies, FCBP recruited calcineurin in the presence of FK506 and a putative target of rapamycin homolog in the presence of rapamycin. Two additional FCBP sequences in Flavobacterium and one in Treponema (spirochete) were also identified in which the CyP and FKBP domains were in the reverse order. T. gondii growth was inhibited by cyclosporin and FK506 in a moderately synergistic manner. The knockdown of FCBP by RNA interference revealed its essentiality for T. gondii growth. Clearly, the FCBPs are novel chaperones and potential targets of multiple immunosuppressant drugs.

Invariant NKT (iNKT) cells constitute a versatile T cell subset with important regulatory functions, which are thought to result essentially from their capacity to promptly produce cytokines that influence the Th1/Th2 balance. In this study, we report that these cells can also express Foxp3, an important transcriptional regulator associated with suppressive activity, once they have been exposed to TGF-β. Foxp3 was expressed by iNKT cells from both peripheral and cord blood. CD4(+) iNKT cells acquired Foxp3 expression preferentially, although a lower proportion of their CD4(-) counterpart also became positive. All Foxp3(+) iNKT cells displayed CD25 but not necessarily CTLA4 or GITR, regardless of the upregulation of these markers in the presence of TGF-β. Exposure to TGF-β decreased IL-4 and IFN-γ production while increasing IL-10, independently from Foxp3 expression. IL-17 was not detected. TGF-β induced high levels of Foxp3, but no suppressor activity, which emerged only in the presence of rapamycin. Peripheral and cord blood Foxp3(+) iNKT cells suppressed the proliferation of conventional autologous and heterologous CD4(+) T cells equally, in a cell contact-dependent and Ag-independent manner. Our findings demonstrate that human iNKT cells become suppressive in the presence of TGF-β plus rapamycin, thus adding a new facet to their complex functional properties.

We have used drugs to examine the role(s) of the actin and microtubule cytoskeletons in the intracellular growth and replication of the intracellular protozoan parasite, Toxoplasma gondii. By using a 5 minute infection period and adding the drugs shortly after entry we can treat parasites at the start of intracellular development and 6-8 hours prior to the onset of daughter cell budding. Using this approach we found, somewhat surprisingly, that reagents that perturb the actin cytoskeleton in different ways (cytochalasin D, latrunculin A and jasplakinolide) had little effect on parasite replication although they had the expected effects on the host cells. These actin inhibitors did, however, disrupt the orderly turnover of the mother cell organelles leading to the formation of a large residual body at the posterior end of each pair of budding parasites. Treating established parasite cultures with the actin inhibitors blocked ionophore-induced egression of tachyzoites from the host cells, demonstrating that intracellular parasites were susceptible to the effects of these inhibitors. In contrast, the anti-microtubule drugs oryzalin and taxol, and to a much lesser extent nocodazole, which affect microtubule dynamics in different ways, blocked parasite replication by disrupting the normal assembly of the apical conoid and the microtubule inner membrane complex (IMC) in the budding daughter parasites. Centrosome replication and assembly of intranuclear spindles, however, occurred normally. Thus, daughter cell budding per se is dependent primarily on the parasite microtubule system and does not require a dynamic actin cytoskeleton, although disruption of actin dynamics causes problems in the turnover of parasite organelles.

The apicomplexan parasite Toxoplasma gondii recognizes, binds, and penetrates virtually any kind of mammalian cell using a repertoire of proteins released from late secretory organelles and a unique form of gliding motility (also named glideosome) that critically depends on actin filaments and myosin. How T. gondii glycosylated proteins mediate host-parasite interactions remains elusive. To date, only limited evidence is available concerning N-glycosylation in apicomplexans. Here we report comprehensive proteomics and glycomics analyses showing that several key components required for host cell-T. gondii interactions are N-glycosylated. Detailed structural characterization confirmed that N-glycans from T. gondii total protein extracts consist of oligomannosidic (Man(5-8)(GlcNAc)2) and paucimannosidic (Man(3-4)(GlcNAc)2) sugars, which are rarely present on mature eukaryotic glycoproteins. In situ fluorescence using concanavalin A and Pisum sativum agglutinin predominantly stained the entire parasite body. Visualization of Toxoplasma glycoproteins purified by affinity chromatography followed by detailed proteomics and glycan analyses identified components involved in gliding motility, moving junction, and other additional functions implicated in intracellular development. Importantly tunicamycin-treated parasites were considerably reduced in motility, host cell invasion, and growth. Collectively these results indicate that N-glycosylation probably participates in modifying key proteins that are essential for host cell invasion by T. gondii.

An Antibody Delivery System for Regulated Expression of Therapeutic Levels of Monoclonal Antibodies In Vivo

An Antibody Delivery System for Regulated Expression of Therapeutic Levels of Monoclonal Antibodies In Vivo

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Toxoplasmosis is an infectious disease caused by Toxoplasma gondii, a protozoan, intracellular parasite that infects up to a third of the World’s population. Infection occurs when food or water is consumed with oocysts secreted from cat feces, or by eating undercooked or raw meat containing cysts. Primary infection is usually subclinical, but some patients may have lymphadenopathy or eye disease such as chorioretinitis. An infection acquired during pregnancy can cause serious damage to the fetus. In immunocompromised patients, reactivation of latent disease can cause life-threatening encephalitis. The diagnosis of toxoplasmosis can be established by direct detection of the parasite or using serological methods. The most commonly used and probably most effective therapeutic treatment is a combination of pyrimethamine with sulfadiazine and folic acid. The review contains information on the management of patients with acute Toxoplasma infection, as well as pregnant women infected during pregnancy. The issues of fetal toxoplasmosis and neonatal toxoplasmosis, eye diseases associated with toxoplasmosis, and the course of toxoplasmosis in immunocompromised individuals are discussed. Contradictions concerning the effectiveness of primary and secondary prevention in pregnant women are considered.

Evaluation of anti-parasitic activities of new quinolones containing nitrofuran moiety against Toxoplasma gondii

Apicomplexan parasites like Toxoplasma gondii are distinctive in their utilization of para site encoded motor systems to invade cells. Invasion results in the establishment of the parasitophorous vacuole (PV) within the infected cell. Most apicomplexans complete their intracellular tenure within the infected cell in the PV that is demarcated from the host cytoplasm by the parasitophorous vacuole membrane (PVM). In this chapter I focus on the events surrounding the formation of the PVM and selected activities attributed to it. Its central role as the interface between the parasite and its immediate environment, the host cytoplasm, is validated by the diversity of functions attributed to it. While functions in structural organization, nutrient acquisitions and signaling have been defined their molecular bases remain largely unknown. Several recent studies and the decoding of the Toxoplasma genome have set the stage for a rapid expansion in our understanding of the role of the PVM in parasite biology. Toxoplasma gondii, like all apicomplexan parasites are obligate intracellular pathogens. This family of parasites utilize their own actin-myosin based motor systems to gain entry into susceptible cells establishing themselves, in some cases transiently (e.g., Theileria spp) in specialized vacuolar compartment, the parasitophorous vacuole (PV). The T. gondii PV is highly dynamic compartment defining the replication permissive niche for the parasite. The delimiting membrane defining the parasitophorous vacuole, the parasitophorous vacuole membrane or PVM is increasingly being recognized as a specialized "organelle" that in the context of the infected cell is extracorporeal to the parent organism, the parasite. A systematic study of this enigmatic organelle has been severely limited by several issues. Primary among these is the fact that it is formed only in the context of the infected cell thereby limiting the amount of material. Secondly, unlike other cellular organelles that can often be purified by conventional approaches, the PVM, cannot be purified away from host cell organelles (see below). In spite of these significant obstacles considerable progress has been made in recent years toward understanding the biogenesis of the PVM, identification of its protein complement and the characterization of activities within it. These studies demonstrate that the PVM, on its own and by virtue of its interactions with cellular components, plays critical functions in the structural integrity of the vacuole, nutrient acquisition and the manipulation of cellular functions. In addition it appears that the repertoire of activities at the PVM is likely to be plastic reflecting temporal changes associated with the replicative phase of parasite growth. Finally, the PVM likely forms the foundation for the cyst wall as the parasite differentiates in the establishment of latent infection. As the critical border crossing between the parasite and invaded cell the study of the PVM provides a fertile area for new investigation aided by the recent decoding of the Toxoplasma genome (available at wwww.ToxoDB.org) and the application of proteomic analyses to basic questions in parasite biology.

Background: Toxoplasmosis represents a neglected equatorial poverty disorder triggered by an intracellular mandatory protozoan parasite, known as Toxoplasma gondii. Aim of the work: Purpose of this study was to assess the Effectiveness of Toxoplasma Surface Antigen Grade I for Diagnosis of Human Toxoplasma gondii in Egypt by Sandwich ELISA Technique. Subjects and Methods: This study was conducted on 94 individuals, divided to 3 categories, category I: Toxoplasma gondii, category II: Other parasites, It encompassed 10 infected patients that have E. histolytica and 14 infected with G. lamblia and category III: Healthy control group. Result: The cutoff value was 0.233 when detecting Toxoplasma (SAG1). The serum findings appear positive in 43 cases (86 per cent) of category I, whilst 7 cases (14 per cent) appear negative. In category II (patient with different parasites): 10 positive cases of Entamoeba histolytica have been confirmed, while other 14 cases were positive with G. lamblia. All healthy control cases (category III) were negative. The sensitivity was 86% However; the specificity was observed 81.81%. Finally from the obtained results, we can conclude that: to evaluate the different immunodiagnostic antigens detection assays, choice and purification of the suitable antigen, accompanied by the manufacture and purification of its particular antibodies, are mandatory. The employment of rabbit anti-Toxoplasma gondii IgG polyclonal antibodies in sandwich ELISA techniques for identification of SAG1 in human serum provide a sensitive and specific tool for immunodiagnosis in human toxoplasmosis. Keywords: Toxoplasma gondii; diagnosis; ELISA; Human toxoplasmosis; blood; patients.

Protein kinases in Toxoplasma gondii

To defend against intracellular pathogens such as Toxoplasma gondii, the host generates a robust type 1 immune response. Specifically, host defense against T. gondii is defined by an IL-12–dependent IFN-γ response that is critical for host resistance. Previously, we demonstrated that host resistance is mediated by T-bet–dependent ILC-derived IFN-γ by maintaining IRF8+ conventional type 1 dendritic cells during parasitic infection. Therefore, we hypothesized that innate lymphoid cells are indispensable for host survival. Surprisingly, we observed that T-bet–deficient mice succumb to infection quicker than do mice lacking lymphocytes, suggesting an unknown T-bet-dependent–mediated host defense pathway. Analysis of parasite-mediated inflammatory myeloid cells revealed a novel subpopulation of T-bet+ myeloid cells (TMCs). Our results reveal that TMCs have the largest intracellular parasite burden compared with other professional phagocytes, suggesting they are associated with active killing of T. gondii. Mechanistically, we established that IL-12 is necessary for the induction of inflammatory TMCs during infection and these cells are linked to a role in host survival.

Intracellular parasites like Toxoplasma gondii scavenge host nutrients, particularly lipids, to support their growth and survival. Although Toxoplasma is known to adjust its metabolism based on nutrient availability, the mechanisms that mediate lipid sensing and metabolic adaptation remain poorly understood. Here, we perform a genome-wide CRISPR screen under lipid-rich (10% Fetal Bovine Serum (FBS)) and lipid-limited (1% FBS) conditions to identify genes critical for lipid-responsive fitness. We identify the Toxoplasma protein GRA38 as a lipid-dependent regulator of parasite fitness. GRA38 exhibits phosphatidic acid (PA) phosphatase (PAP) activity in vitro, which is significantly reduced by mutation of its conserved DxDxT/V catalytic motif. Disruption of GRA38 leads to the accumulation of PA species and widespread alterations in lipid composition, consistent with impaired PAP activity. These lipid imbalances correlate with reduced parasite virulence in mice. Our findings identify GRA38 as a metabolic regulator important for maintaining lipid homeostasis and pathogenesis in Toxoplasma gondii.