Browsing by Subject "Toxoplasma"

Now showing 1 - 10 of 65
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    A decade of epigenetic research in Toxoplasma gondii
    (Elsevier, 2010) Dixon, Stacy E.; Stilger, Krista L.; Elias, Eliana V.; Naguleswaran, Arunasalam; Sullivan, William J., Jr.; Pharmacology and Toxicology, School of Medicine
    In the past 10 years, the field of parasitology has witnessed an explosion of studies investigating gene regulation. In this review, we will describe recent advances largely stemming from the study of Toxoplasma gondii, a significant opportunistic pathogen and useful model for other apicomplexan protozoa. Surprising findings have emerged, including the discovery of a wealth of epigenetic machinery in these primitive eukaryotes, unusual histone variants, and a battery of plant-like transcription factors. We will elaborate on how these unusual features impact parasite physiology and potential therapeutics as we summarize some of the key discoveries from the last decade. We will close by proposing a few questions to address in the next 10 years.
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    A GCN2-Like Eukaryotic Initiation Factor 2 Kinase Increases the Viability of Extracellular Toxoplasma gondii Parasites
    (American Society for Microbiology, 2011) Konrad, Christian; Wek, Ronald C.; Sullivan, William J., Jr.; Pharmacology and Toxicology, School of Medicine
    Toxoplasmosis is a significant opportunistic infection caused by the protozoan parasite Toxoplasma gondii, an obligate intracellular pathogen that relies on host cell nutrients for parasite proliferation. Toxoplasma parasites divide until they rupture the host cell, at which point the extracellular parasites must survive until they find a new host cell. Recent studies have indicated that phosphorylation of Toxoplasma eukaryotic translation initiation factor 2-alpha (TgIF2α) plays a key role in promoting parasite viability during times of extracellular stress. Here we report the cloning and characterization of a TgIF2α kinase designated TgIF2K-D that is related to GCN2, a eukaryotic initiation factor 2α (eIF2α) kinase known to respond to nutrient starvation in other organisms. TgIF2K-D is present in the cytosol of both intra- and extracellular Toxoplasma parasites and facilitates translational control through TgIF2α phosphorylation in extracellular parasites. We generated a TgIF2K-D knockout parasite and demonstrated that loss of this eIF2α kinase leads to a significant fitness defect that stems from an inability of the parasite to adequately adapt to the environment outside host cells. This phenotype is consistent with that reported for our nonphosphorylatable TgIF2α mutant (S71A substitution), establishing that TgIF2K-D is the primary eIF2α kinase responsible for promoting extracellular viability of Toxoplasma. These studies suggest that eIF2α phosphorylation and translational control are an important mechanism by which vulnerable extracellular parasites protect themselves while searching for a new host cell. Additionally, TgIF2α is phosphorylated when intracellular parasites are deprived of nutrients, but this can occur independently of TgIF2K-D, indicating that this activity can be mediated by a different TgIF2K.
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    A transcriptional network required for bradyzoite development in Toxoplasma gondii is dispensable for recrudescent disease
    (Springer Nature, 2023-09-28) Sokol-Borrelli, Sarah L.; Reilly, Sarah M.; Holmes, Michael J.; Orchanian, Stephanie B.; Massmann, Mackenzie D.; Sharp, Katherine G.; Cabo, Leah F.; Alrubaye, Hisham S.; Martorelli Di Genova, Bruno; Lodoen, Melissa B.; Sullivan, William J., Jr.; Boyle, Jon P.; Pharmacology and Toxicology, School of Medicine
    Identification of regulators of Toxoplasma gondii bradyzoite development and cyst formation is the most direct way to address the importance of parasite development in long-term persistence and reactivation of this parasite. Here we show that a T. gondii gene (named Regulator of Cystogenesis 1; ROCY1) is sufficient for T. gondii bradyzoite formation in vitro and in vivo. ROCY1 encodes an RNA binding protein that has a preference for 3' regulatory regions of hundreds of T. gondii transcripts, and its RNA-binding domains are required to mediate bradyzoite development. Female mice infected with ΔROCY1 parasites have reduced (>90%) cyst burden. While viable parasites can be cultivated from brain tissue for up to 6 months post-infection, chronic brain-resident ΔROCY1 parasites have reduced oral infectivity compared to wild type. Despite clear defects in bradyzoite formation and oral infectivity, ΔROCY1 parasites were able to reactivate with similar timing and magnitude as wild type parasites for up to 5 months post-infection. Therefore while ROCY1 is a critical regulator of the bradyzoite developmental pathway, it is not required for parasite reactivation, raising new questions about the persisting life stage responsible for causing recrudescent disease.
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    Autophagy participates in the unfolded protein response in Toxoplasma gondii
    (Oxford University Press, 2017-08-15) Nguyen, Hoa Mai; Berry, Laurence; Sullivan, William J., Jr.; Besteiro, Sébastien; Pharmacology and Toxicology, School of Medicine
    Environmental and genetic perturbations of endoplasmic reticulum (ER) function can lead to the accumulation of unfolded proteins. In these conditions, eukaryotic cells can activate a complex signaling network called the unfolded protein response (UPR) to reduce ER stress and restore cellular homeostasis. Autophagy, a degradation and recycling process, is part of this response. The parasitic protist Toxoplasma gondii is known to be able to activate the UPR upon ER stress, and we now show that this pathway leads to autophagy activation, supporting the idea of a regulated function for canonical autophagy as part of an integrated stress response in the parasites.
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    Azurin-Like Protein Blocks Invasion of Toxoplasma gondii through Potential Interactions with Parasite Surface Antigen SAG1
    (American Society for Microbiology, 2008) Naguleswaran, Arunasalam; Fialho, Arsenio M.; Chaudhari, Anita; Hong, Chang Soo; Chakrabarty, Ananda M.; Sullivan, William J., Jr.; Pharmacology and Toxicology, School of Medicine
    Some pathogenic bacteria produce factors that have evolved a capacity to neutralize competing microbes. The cupredoxin family protein azurin, produced by Pseudomonas aeruginosa, exhibits a remarkable ability to impede invasion of a number of diverse intracellular pathogens, including the human AIDS virus human immunodeficiency virus type 1 and the protozoan parasite Plasmodium falciparum (which causes malaria). Here we report that azurin and an azurin-like protein (Laz) from gonococci/meningococci have activity against Toxoplasma, an apicomplexan parasite that causes opportunistic infection in immunocompromised individuals. We demonstrate that the mechanism of action for Laz involves interfering with the ability of Toxoplasma to adhere to host cells. Computer structural analysis reveals that azurin shares structural features with the predominant surface antigen SAG1, which is known to play an important role in parasite attachment. Interestingly, azurin also has structural similarities to a monoclonal antibody to SAG1. Surface plasmon resonance binding studies validate that SAG1 interacts strongly with Laz and, to lesser extent, azurin. Moreover, Toxoplasma mutants lacking SAG1 are not as susceptible to the growth-inhibitory effects of Laz. Collectively, our data show that Toxoplasma adhesion can be significantly impaired by Laz, and to some extent by azurin, via interactions with SAG1. These observations indicate that Laz can serve as an important tool in the study of host-pathogen interactions and is worthy of further study for development into potential therapeutic agents.
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    Bromodomains in Protozoan Parasites: Evolution, Function, and Opportunities for Drug Development
    (American Society for Microbiology, 2017-01-11) Jeffers, Victoria; Yang, Chunlin; Huang, Sherri; Sullivan, William J., Jr.; Pharmacology and Toxicology, School of Medicine
    Parasitic infections remain one of the most pressing global health concerns of our day, affecting billions of people and producing unsustainable economic burdens. The rise of drug-resistant parasites has created an urgent need to study their biology in hopes of uncovering new potential drug targets. It has been established that disrupting gene expression by interfering with lysine acetylation is detrimental to survival of apicomplexan (Toxoplasma gondii and Plasmodium spp.) and kinetoplastid (Leishmania spp. and Trypanosoma spp.) parasites. As "readers" of lysine acetylation, bromodomain proteins have emerged as key gene expression regulators and a promising new class of drug target. Here we review recent studies that demonstrate the essential roles played by bromodomain-containing proteins in parasite viability, invasion, and stage switching and present work showing the efficacy of bromodomain inhibitors as novel antiparasitic agents. In addition, we performed a phylogenetic analysis of bromodomain proteins in representative pathogens, some of which possess unique features that may be specific to parasite processes and useful in future drug development.
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    Canonical histone H2Ba and H2A.X dimerize in an opposite genomic localization to H2A.Z/H2B.Z dimers in Toxoplasma gondii
    (Elsevier, 2014-10) Bogado, Silvina S.; Dalmasso, Carolina; Ganuza, Agustina; Kim, Kami; Sullivan, William J., Jr.; Angel, Sergio O.; Vanagas, Laura; Department of Pharmacology and Toxicology, IU School of Medicine
    Histone H2Ba of Toxoplasma gondii was expressed as recombinant protein (rH2Ba) and used to generate antibody in mouse that is highly specific. Antibody recognizing rH2Ba detects a single band in tachyzoite lysate of the expected molecular weight (12kDa). By indirect immunofluorescence (IFA) in in vitro grown tachyzoites and bradyzoites, the signal was detected only in the parasite nucleus. The nucleosome composition of H2Ba was determined through co-immunoprecipitation assays. H2Ba was detected in the same immunocomplex as H2A.X, but not with H2A.Z. Through chromatin immunoprecipitation (ChIP) assays and qPCR, it was observed that H2Ba is preferentially located at promoters of inactive genes and silent regions, accompanying H2A.X and opposed to H2A.Z/H2B.Z dimers.
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    Characterization of Plasmodium Atg3-Atg8 Interaction Inhibitors Identifies Novel Alternative Mechanisms of Action in Toxoplasma gondii
    (American Society for Microbiology, 2018-01-25) Varberg, Joseph M.; LaFavers, Kaice A.; Arrizabalaga, Gustavo; Sullivan, William J., Jr.; Pharmacology and Toxicology, School of Medicine
    Protozoan parasites, including the apicomplexan pathogens Plasmodium falciparum (which causes malaria) and Toxoplasma gondii (which causes toxoplasmosis), infect millions of people worldwide and represent major human disease burdens. Despite their prevalence, therapeutic strategies to treat infections caused by these parasites remain limited and are threatened by the emergence of drug resistance, highlighting the need for the identification of novel drug targets. Recently, homologues of the core autophagy proteins, including Atg8 and Atg3, were identified in many protozoan parasites. Importantly, components of the Atg8 conjugation system that facilitate the lipidation of Atg8 are required for both canonical and parasite-specific functions and are essential for parasite viability. Structural characterization of the P. falciparum Atg3-Atg8 (PfAtg3-Atg8) interaction has led to the identification of compounds that block this interaction. Additionally, many of these compounds inhibit P. falciparum growth in vitro, demonstrating the viability of this pathway as a drug target. Given the essential role of the Atg8 lipidation pathway in Toxoplasma, we sought to determine whether three PfAtg3-Atg8 interaction inhibitors identified in the Medicines for Malaria Venture Malaria Box exerted a similar inhibitory effect in Toxoplasma While all three inhibitors blocked Toxoplasma replication in vitro at submicromolar concentrations, they did not inhibit T. gondii Atg8 (TgAtg8) lipidation. Rather, high concentrations of two of these compounds induced TgAtg8 lipidation and fragmentation of the parasite mitochondrion, similar to the effects seen following starvation and monensin-induced autophagy. Additionally, we report that one of the PfAtg3-Atg8 interaction inhibitors induces Toxoplasma egress and provide evidence that this is mediated by an increase in intracellular calcium in response to drug treatment.
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    Characterization of TgPuf1, a member of the Puf family RNA-binding proteins from Toxoplasma gondii
    (Springer Nature, 2014-03-31) Liu, Min; Miao, Jun; Liu, Tingkai; Sullivan, William J., Jr.; Cui, Liwang; Chen, Xiaoguang; Pharmacology and Toxicology, School of Medicine
    Background: Puf proteins act as translational regulators and affect many cellular processes in a wide range of eukaryotic organisms. Although Puf proteins have been well characterized in many model systems, little is known about the structural and functional characteristics of Puf proteins in the parasite Toxoplasma gondii. Methods: Using a combination of conventional molecular approaches, we generated endogenous TgPuf1 tagged with hemagglutinin (HA) epitope and investigated the TgPuf1 expression levels and localization in the tachyzoites and bradyzoites. We used RNA Electrophoretic Mobility Shfit Assay (EMSA) to determine whether the recombination TgPuf1 has conserverd RNA binding activity and specificity. Results: TgPuf1 was expressed at a significantly higher level in bradyzoites than in tachyzoites. TgPuf1 protein was predominantly localized within the cytoplasm and showed a much more granular cytoplasmic staining pattern in bradyzoites. The recombinant Puf domain of TgPuf1 showed strong binding affinity to two RNA fragments containing Puf-binding motifs from other organisms as artificial target sequences. However, two point mutations in the core Puf-binding motif resulted in a significant reduction in binding affinity, indicating that TgPuf1 also binds to conserved Puf-binding motif. Conclusions: TgPuf1 appears to exhibit different expression levels in the tachyzoites and bradyzoites, suggesting that TgPuf1 may function in regulating the proliferation or/and differentiation that are important in providing parasites with the ability to respond rapidly to changes in environmental conditions. This study provides a starting point for elucidating the function of TgPuf1 during parasite development.
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