A 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-2␣ (TgIF2␣) is phosphorylated during stress and establish that protozoan parasites utilize translation control to modulate gene expression during development. Importantly, TgIF2␣ 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) eIF2␣ 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.A well characterized mechanism by which eukaryotic cells respond to environmental stress involves phosphorylation of eukaryotic initiation factor-2 (eIF2) 3 (1-3). The eIF2 combined with GTP delivers Met-tRNA i Met to the translational machinery during initiation of protein synthesis. In mammalian cells, four eIF2 kinases have been described that are each activated by unique stress arrangements. For example, in response to accumulation of malfolded protein in the lumen of the endoplasmic reticulum (so-called ER stress), PEK/Perk (EIF2KA3) phosphorylates the ␣ subunit of eIF2 at serine 51, causing this translation factor to become an inhibitor of its own guanine nucleotide exchange factor, eIF2B. The resulting repression in general translation prevents further synthesis of secretory proteins that would further overload the ER and allows cells sufficient time to trigger the unfolded protein response (UPR) (2). The UPR is a program of mRNA expression involving genes that function in the assembly and transport of secretory proteins (4). In addition to ER stress, three other eIF2 kinases have been described that recognize different forms of cytoplasmic stress in mammalian cells. These include: GCN2 (EIF2KA4), which responds to nutrient deprivation and is well conserved among eukaryotes (3, 5), HRI (EIF2KA1), which is reported to be activated by heme deficiency, oxidative stress induced by arsenite treatment, and heat shock (6, 7), and PKR (EIF2KA2), which is involved in the antiviral defenses (8, 9).Very little research has been performed on eIF2 kinase and related stress response pathways in early-diverging eukaryotes, including pathogenic eukaryotes. However, viability, pathogenesis, and transmission of many parasites hinges on their ability to recogn...

Histone acetylation has been linked to developmental changes in gene expression and is a validated drug target of apicomplexan parasites, but little is known about the roles of individual histone modifying enzymes and how they are recruited to target genes. The protozoan parasite Toxoplasma gondii (phylum Apicomplexa) is unusual among invertebrates in possessing two GCN5-family lysine acetyltransferases (KATs). While GCN5a is required for gene expression in response to alkaline stress, this KAT is dispensable for parasite proliferation in normal culture conditions. In contrast, GCN5b cannot be disrupted, suggesting it is essential for Toxoplasma viability. To further explore the function of GCN5b, we generated clonal parasites expressing an inducible HA-tagged dominant-negative form of GCN5b containing a point mutation that ablates enzymatic activity (E703G). Stabilization of this dominant-negative GCN5b was mediated through ligand-binding to a destabilization domain (dd) fused to the protein. Induced accumulation of the ddHAGCN5b(E703G) protein led to a rapid arrest in parasite replication. Growth arrest was accompanied by a decrease in histone H3 acetylation at specific lysine residues as well as reduced expression of GCN5b target genes in GCN5b(E703G) parasites, which were identified using chromatin immunoprecipitation coupled with microarray hybridization (ChIP-chip). Proteomics studies revealed that GCN5b interacts with AP2-domain proteins, apicomplexan plant-like transcription factors, as well as a “core complex” that includes the co-activator ADA2-A, TFIID subunits, LEO1 polymerase-associated factor (Paf1) subunit, and RRM proteins. The dominant-negative phenotype of ddHAGCN5b(E703G) parasites, considered with the proteomics and ChIP-chip data, indicate that GCN5b plays a central role in transcriptional and chromatin remodeling complexes. We conclude that GCN5b has a non-redundant and indispensable role in regulating gene expression required during the Toxoplasma lytic cycle.

In the past ten 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 ten years.

The opportunistic apicomplexan parasite Toxoplasma gondii damages fetuses in utero and threatens immunocompromised individuals. The toxicity associated with standard antitoxoplasmal therapies, which target the folate pathway, underscores the importance of examining alternative pharmacological strategies. Parasitic protozoa cannot synthesize purines de novo; consequently, targeting purine salvage enzymes is a plausible pharmacological strategy. Several enzymes critical to purine metabolism have been studied in T. gondii, but IMP dehydrogenase (IMPDH), which catalyzes the conversion of IMP to XMP, has yet to be characterized. Thus, we have cloned the gene encoding this enzyme in T. gondii. Northern blot analysis shows that two IMPDH transcripts are present in T. gondii tachyzoites. The larger transcript contains an open reading frame of 1,656 nucleotides whose deduced protein sequence consists of 551 amino acids (TgIMPDH). The shorter transcript is an alternative splice product that generates a 371-amino-acid protein lacking the active-site flap (TgIMPDH-S). When TgIMPDH is expressed as a recombinant protein fused to a FLAG tag, the fusion protein localizes to the parasite cytoplasm. Immunoprecipitation with anti-FLAG was employed to purify recombinant TgIMPDH, which converts IMP to XMP as expected. Mycophenolic acid is an uncompetitive inhibitor relative to NAD ؉ , with a intercept inhibition constant (K ii ) of 0.03 ؎ 0.004 M. Tiazofurin and its seleno analog were not inhibitory to the purified enzyme, but adenine dinucleotide analogs such as TAD and the nonhydrolyzable ␤-methylene derivatives of TAD or SAD were inhibitory, with K ii values 13-to 60-fold higher than that of mycophenolic acid.IMP dehydrogenase (IMPDH) converts IMP to XMP in the presence of NAD, a critical rate-limiting step in the biosynthesis of guanine nucleotides. Increased IMPDH activity is associated with actively dividing cells; consequently, IMPDH has been exploited as a target for anticancer, antiviral, immunosuppressive, and antimicrobial drug therapies (9, 15). Significant differences in IMDPH enzyme kinetics and inhibitor sensitivities between human and microbial homologues suggest that IMPDH may be an exploitable target for antimicrobials (25,36,40,42). Purine metabolic enzymes hold particular promise as targets to combat intracellular parasites while minimizing side effects to host cells. Parasites are inherently more reliant on these enzymes to maintain growth and are incapable of synthesizing purine precursors de novo (34).Toxoplasma gondii is an obligate intracellular protozoan responsible for fetal damage when the infection is acquired in utero (24), heart transplant complications (37), and opportunistic infections in immunocompromised individuals (39). T. gondii, as a member of the phylum Apicomplexa, is related to other parasites such as Plasmodium spp. (malaria) and Cryptosporidium spp. (26). A wide variety of molecular genetic tools have been developed for T. gondii (27,28). In particular, the generation of reagents for rever...

We have previously shown that protozoan parasites, such as Toxoplasma gondii, contain a high prevalence of intrinsically disordered regions in their predicted proteins. Here, we determine that both TgGCN5-family histone acetyltransferases (HATs) contain unusually high levels of intrinsic disorder. A previously identified basic-rich nuclear localization signal (NLS) in the N-terminus of TgGCN5-A is located within such a region of predicted disorder, but this NLS is not conserved in TgGCN5-B. We therefore analyzed the intrinsically disordered regions of TgGCN5-B for basicrich sequences that could be indicative of a functional NLS, and this led to the identification of a novel NLS for TgGCN5-B, RPAENKKRGR. The functionality of the GCN5-B NLS was validated experimentally and has predictive value. These studies demonstrate that basic-rich sequences within regions predicted to be intrinsically disordered constitute criteria for a candidate NLS. KeywordsApicomplexa; parasite; cellular trafficking; GCN5; chromatin; epigenetics The obligate intracellular protozoan Toxoplasma gondii (Apicomplexa) is a serious opportunist pathogen. Completion of genome sequencing revealed that ~58% of predicted Toxoplasma genes encode hypothetical proteins of unknown function (ToxoDB.org). The discovery of new protein motifs is essential for improving predictions about the location and function of unknown proteins. We have previously determined that the genomes of earlybranching eukaryotic protozoa contain a large proportion of predicted proteins with Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access Author ManuscriptMol Biochem Parasitol. Author manuscript; available in PMC 2012 February 1. . Disordered regions are characterized by moderate to low amino acid sequence complexity with very few bulky, hydrophobic amino acids and with an enrichment of polar and charged amino acids and the structure-breaking proline, and can be predicted using computational methods [2]. Determining the degree of disorder in a protein can assist in predicting the biological relevance of a given domain, as many regions of disorder map to areas of protein-protein interaction or post-translational modification [3-6].We have previously described the presence of lengthy (600-800 amino acids), unconserved N-terminal extensions on the two GCN5-family member histone acetyltransferases (HATs), . These extensions are not present on the GCN5 homologues in other lower eukaryotes, save the fellow apicomplexan parasite Plasmodium falciparum [9], and they have no currently known protein motifs that would help in...