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Item Histone-Modifying Complexes Regulate Gene Expression Pertinent to the Differentiation of the Protozoan Parasite Toxoplasma gondii(Taylor & Francis, 2005) Saksouk, Nehmé; Bhatti, Micah M.; Kieffer, Sylvie; Smith, Aaron T.; Musset, Karine; Garin, Jérôme; Sullivan, William J., Jr.; Cesbron-Delauw, Marie-France; Hakimi, Mohamed-Ali; Pharmacology and Toxicology, School of MedicinePathogenic apicomplexan parasites like Toxoplasma and Plasmodium (malaria) have complex life cycles consisting of multiple stages. The ability to differentiate from one stage to another requires dramatic transcriptional changes, yet there is a paucity of transcription factors in these protozoa. In contrast, we show here that Toxoplasma possesses extensive chromatin remodeling machinery that modulates gene expression relevant to differentiation. We find that, as in other eukaryotes, histone acetylation and arginine methylation are marks of gene activation in Toxoplasma. We have identified mediators of these histone modifications, as well as a histone deacetylase (HDAC), and correlate their presence at target promoters in a stage-specific manner. We purified the first HDAC complex from apicomplexans, which contains novel components in addition to others previously reported in eukaryotes. A Toxoplasma orthologue of the arginine methyltransferase CARM1 appears to work in concert with the acetylase TgGCN5, which exhibits an unusual bias for H3 [K18] in vitro. Inhibition of TgCARM1 induces differentiation, showing that the parasite life cycle can be manipulated by interfering with epigenetic machinery. This may lead to new approaches for therapy against protozoal diseases and highlights Toxoplasma as an informative model to study the evolution of epigenetics in eukaryotic cells.Item MYST Family Histone Acetyltransferases in the Protozoan Parasite Toxoplasma gondii(American Society for Microbiology, 2005) Smith, Aaron T.; Tucker-Samaras, Samantha D.; Fairlamb, Alan H.; Sullivan, William J., Jr.; Pharmacology and Toxicology, School of MedicineThe restructuring of chromatin precedes tightly regulated events such as DNA transcription, replication, and repair. One type of chromatin remodeling involves the covalent modification of nucleosomes by histone acetyltransferase (HAT) complexes. The observation that apicidin exerts antiprotozoal activity by targeting a histone deacetyltransferase has prompted our search for more components of the histone modifying machinery in parasitic protozoa. We have previously identified GNAT family HATs in the opportunistic pathogen Toxoplasma gondii and now describe the first MYST (named for members MOZ, Ybf2/Sas3, Sas2, and Tip60) family HATs in apicomplexa (TgMYST-A and -B). The TgMYST-A genomic locus is singular and generates a approximately 3.5-kb transcript that can encode two proteins of 411 or 471 amino acids. TgMYST-B mRNA is approximately 7.0 kb and encodes a second MYST homologue. In addition to the canonical MYST HAT catalytic domain, both TgMYST-A and -B possess an atypical C2HC zinc finger and a chromodomain. Recombinant TgMYST-A exhibits a predilection to acetylate histone H4 in vitro at lysines 5, 8, 12, and 16. Antibody generated to TgMYST-A reveals that both the long and short (predominant) versions are present in the nucleus and are also plentiful in the cytoplasm. Moreover, both TgMYST-A forms are far more abundant in rapidly replicating parasites (tachyzoites) than encysted parasites (bradyzoites). A bioinformatics survey of the Toxoplasma genome reveals numerous homologues known to operate in native MYST complexes. The characterization of TgMYST HATs represents another important step toward understanding the regulation of gene expression in pathogenic protozoa and provides evolutionary insight into how these processes operate in eukaryotic cells in general.Item Quinoline Derivative MC1626, a Putative GCN5 Histone Acetyltransferase (HAT) Inhibitor, Exhibits HAT-Independent Activity against Toxoplasma gondii(American Society for Microbiology, 2007) Smith, Aaron T.; Livingston, Meredith R.; Mai, Antonello; Filetici, Patrizia; Queener, Sherry F.; Sullivan, William J., Jr.; Pharmacology and Toxicology, School of MedicineWe report that quinoline derivative MC1626, first described as an inhibitor of the histone acetyltransferase (HAT) GCN5, is active against the protozoan parasite Toxoplasma gondii in vitro. However, MC1626 does not inhibit Toxoplasma GCN5 HATs or reduce HAT-mediated activity; rather, this quinoline may target the plastid organelle called the apicoplast.Item The protozoan parasite Cryptosporidium parvum possesses two functionally and evolutionarily divergent replication protein A large subunits(Elsevier, 2005) Rider, S. Dean, Jr.; Cai, Xiaomin; Sullivan, William J., Jr.; Smith, Aaron T.; Radke, Jay; White, Michael; Zhu, Guan; Pharmacology and Toxicology, School of MedicineVery little is known about protozoan replication protein A (RPA), a heterotrimeric complex critical for DNA replication and repair. We have discovered that in medically and economically important apicomplexan parasites, two unique RPA complexes may exist based on two different types of large subunit RPA1. In this study, we characterized the single-stranded DNA binding features of two distinct types (i.e. short and long) of RPA1 subunits from Cryptosporidium parvum (CpRPA1A and CpRPA1B). These two proteins differ from human RPA1 in their intrinsic single-stranded DNA binding affinity (K) and have significantly lower cooperativity (omega). We also identified the RPA2 and RPA3 subunits from C. parvum, the latter of which had yet to be reported to exist in any protozoan. Using fluorescence resonance energy transfer technology and pull-down assays, we confirmed that these two subunits interact with each other and with CpRPA1A and CpRPA1B. This suggests that the heterotrimeric structure of RPA complexes may be universally conserved from lower to higher eukaryotes. Bioinformatic analyses indicate that multiple types of RPA1 are present in the other apicomplexans Plasmodium and Toxoplasma. Apicomplexan RPA1 proteins are phylogenetically more related to plant homologues and probably arose from a single gene duplication event prior to the expansion of the apicomplexan lineage. Differential expression during the life cycle stages in three apicomplexan parasites suggests that the two RPA1 types exercise specialized biological functions.Item The transcription of bradyzoite genes in Toxoplasma gondii is controlled by autonomous promoter elements(Wiley, 2008) Behnke, Michael S.; Radke, Josh B.; Smith, Aaron T.; Sullivan, William J., Jr.; White, Michael W.; Pharmacology and Toxicology, School of MedicineExperimental evidence suggests that apicomplexan parasites possess bipartite promoters with basal and regulated cis-elements similar to other eukaryotes. Using a dual luciferase model adapted for recombinational cloning and use in Toxoplasma gondii, we show that genomic regions flanking 16 parasite genes, which encompass examples of constitutive and tachyzoite- and bradyzoite-specific genes, are able to reproduce the appropriate developmental stage expression in a transient luciferase assay. Mapping of cis-acting elements in several bradyzoite promoters led to the identification of short sequence spans that are involved in control of bradyzoite gene expression in multiple strains and under different bradyzoite induction conditions. Promoters that regulate the heat shock protein BAG1 and a novel bradyzoite-specific NTPase during bradyzoite development were fine mapped to a 6-8 bp resolution and these minimal cis-elements were capable of converting a constitutive promoter to one that is induced by bradyzoite conditions. Gel-shift experiments show that mapped cis-elements are bound by parasite protein factors with the appropriate functional sequence specificity. These studies are the first to identify the minimal sequence elements that are required and sufficient for bradyzoite gene expression and to show that bradyzoite promoters are maintained in a 'poised' chromatin state throughout the intermediate host life cycle in low passage strains. Together, these data demonstrate that conventional eukaryotic promoter mechanisms work with epigenetic processes to regulate developmental gene expression during tissue cyst formation.Item Translation Regulation by Eukaryotic Initiation Factor-2 Kinases in the Development of Latent Cysts in Toxoplasma gondi(American Society for Biochemistry and Molecular Biology, 2008) Narasimhan, Jana; Joyce, Bradley R.; Naguleswaran, Arunasalam; Smith, Aaron T.; Livingston, Meredith R.; Dixon, Stacy E.; Coppens, Isabelle; Wek, Ronald C.; Sullivan, William J., Jr.; Pharmacology and Toxicology, School of MedicineA key problem in the treatment of numerous pathogenic eukaryotes centers on their development into latent forms during stress. For example, the opportunistic protist Toxoplasma gondii converts to latent cysts (bradyzoites) responsible for recrudescence of disease. We report that Toxoplasma eukaryotic initiation factor-2alpha (TgIF2alpha) is phosphorylated during stress and establish that protozoan parasites utilize translation control to modulate gene expression during development. Importantly, TgIF2alpha remains phosphorylated in bradyzoites, explaining how these cells maintain their quiescent state. Furthermore, we have characterized novel eIF2 kinases; one in the endoplasmic reticulum and a likely regulator of the unfolded protein response (TgIF2K-A) and another that is a probable responder to cytoplasmic stresses (TgIF2K-B). Significantly, our data suggest that 1) the regulation of protein translation through eIF2 kinases is associated with development, 2) eIF2alpha phosphorylation is employed by cells to maintain a latent state, and 3) endoplasmic reticulum and cytoplasmic stress responses evolved in eukaryotic cells before the early diverging Apicomplexa. Given its importance to pathogenesis, eIF2 kinase-mediated stress responses may provide opportunities for novel therapeutics.Item Understanding mechanisms and the role of differentiation in pathogenesis of Toxoplasma gondii: a review(SciELO, 2009) Sullivan, William J., Jr.; Smith, Aaron T.; Joyce, Bradley R.; Pharmacology and Toxicology, School of MedicineParasite differentiation from proliferating tachyzoites into latent bradyzoites is central to pathogenesis and transmission of the intracellular protozoan pathogen Toxoplasma gondii. The presence of bradyzoite-containing cysts in human hosts and their subsequent rupture can cause life-threatening recrudescence of acute infection in the immuno-compromised and cyst formation in other animals contributes to zoonotic transmission and widespread dissemination of the parasite. In this review, we discuss the evidence showing how the clinically relevant process of bradyzoite differentiation is regulated at both transcriptional and post-transcriptional levels. Specific regulatory factors implicated in modulating bradyzoite differentiation include promoter-based cis-elements, epigenetic modifications and protein translation control through eukaryotic initiation factor -2 (eIF2). In addition to a summary of the current state of knowledge in these areas we discuss the pharmacological ramifications and pose some questions for future research.