Browsing by Subject "Parasite"

Now showing 1 - 10 of 13
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    Cap-independent translation directs stress-induced differentiation of the protozoan parasite Toxoplasma gondii
    (Elsevier, 2024) Dey, Vishakha; Holmes, Michael J.; Bastos, Matheus S.; Wek, Ronald C.; Sullivan, William J., Jr.; Pharmacology and Toxicology, School of Medicine
    Translational control mechanisms modulate the microbial latency of eukaryotic pathogens, enabling them to evade immunity and drug treatments. The protozoan parasite Toxoplasma gondii persists in hosts by differentiating from proliferative tachyzoites to latent bradyzoites, which are housed inside tissue cysts. Transcriptional changes facilitating bradyzoite conversion are mediated by a Myb domain transcription factor called BFD1, whose mRNA is present in tachyzoites but not translated into protein until stress is applied to induce differentiation. We addressed the mechanisms by which translational control drives BFD1 synthesis in response to stress-induced parasite differentiation. Using biochemical and molecular approaches, we show that the 5'-leader of BFD1 mRNA is sufficient for preferential translation upon stress. The translational control of BFD1 mRNA is maintained when ribosome assembly near its 5'-cap is impaired by insertion of a 5'-proximal stem-loop and upon knockdown of the Toxoplasma cap-binding protein, eIF4E1. Moreover, we determined that a trans-acting RNA-binding protein called BFD2/ROCY1 is necessary for the cap-independent translation of BFD1 through its binding to the 5'-leader. Translation of BFD2 mRNA is also suggested to be preferentially induced under stress but by a cap-dependent mechanism. These results show that translational control and differentiation in Toxoplasma proceed through cap-independent mechanisms in addition to canonical cap-dependent translation. Our identification of cap-independent translation in protozoa underscores the antiquity of this mode of gene regulation in cellular evolution and its central role in stress-induced life-cycle events.
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    The common parasite Toxoplasma gondii induces prostatic inflammation and microglandular hyperplasia in a mouse model
    (Wiley, 2017-07) Colinot, Darrelle L.; Garbuz, Tamila; Bosland, Maarten C.; Wang, Liang; Rice, Susan E.; Sullivan, William J., Jr.; Arrizabalaga, Gustavo; Jerde, Travis J.; Pharmacology and Toxicology, School of Medicine
    BACKGROUND: Inflammation is the most prevalent and widespread histological finding in the human prostate, and associates with the development and progression of benign prostatic hyperplasia and prostate cancer. Several factors have been hypothesized to cause inflammation, yet the role each may play in the etiology of prostatic inflammation remains unclear. This study examined the possibility that the common protozoan parasite Toxoplasma gondii induces prostatic inflammation and reactive hyperplasia in a mouse model. METHODS: Male mice were infected systemically with T. gondii parasites and prostatic inflammation was scored based on severity and focality of infiltrating leukocytes and epithelial hyperplasia. We characterized inflammatory cells with flow cytometry and the resulting epithelial proliferation with bromodeoxyuridine (BrdU) incorporation. RESULTS: We found that T. gondii infects the mouse prostate within the first 14 days of infection and can establish parasite cysts that persist for at least 60 days. T. gondii infection induces a substantial and chronic inflammatory reaction in the mouse prostate characterized by monocytic and lymphocytic inflammatory infiltrate. T. gondii-induced inflammation results in reactive hyperplasia, involving basal and luminal epithelial proliferation, and the exhibition of proliferative inflammatory microglandular hyperplasia in inflamed mouse prostates. CONCLUSIONS: This study identifies the common parasite T. gondii as a new trigger of prostatic inflammation, which we used to develop a novel mouse model of prostatic inflammation. This is the first report that T. gondii chronically encysts and induces chronic inflammation within the prostate of any species. Furthermore, T. gondii-induced prostatic inflammation persists and progresses without genetic manipulation in mice, offering a powerful new mouse model for the study of chronic prostatic inflammation and microglandular hyperplasia.
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    Effects of PERK eIF2α Kinase Inhibitor against Toxoplasma gondii
    (American Society for Microbiology, 2018-10-24) Augusto, Leonardo; Martynowicz, Jennifer; Staschke, Kirk A.; Wek, Ronald C.; Sullivan, William J., Jr.; Biochemistry and Molecular Biology, School of Medicine
    Toxoplasma gondii is an obligate intracellular parasite that has infected one-third of the population. Upon infection of warm-blooded vertebrates, the replicating form of the parasite (tachyzoite) converts into a latent form (bradyzoite) present in tissue cysts. During immune deficiency, bradyzoites can reconvert into tachyzoites and cause life-threatening toxoplasmosis. We previously reported that translational control through phosphorylation of the α subunit of T. gondii eukaryotic initiation factor 2 (eIF2α) (TgIF2α) is a critical component of the parasite stress response. Diverse stresses can induce the conversion of tachyzoites to bradyzoites, including those disrupting the parasite's endoplasmic reticulum (ER) (ER stress). Toxoplasma possesses four eIF2α kinases, one of which (TgIF2K-A) localizes to the parasite ER analogously to protein kinase R-like endoplasmic reticulum kinase (PERK), the eIF2α kinase that responds to ER stress in mammalian cells. Here, we investigated the effects of a PERK inhibitor (PERKi) on Toxoplasma Our results show that the PERKi GSK2606414 blocks the enzymatic activity of TgIF2K-A and reduces TgIF2α phosphorylation specifically in response to ER stress. PERKi also significantly impeded multiple steps of the tachyzoite lytic cycle and sharply lowered the frequency of bradyzoite differentiation in vitro Pretreatment of host cells with PERKi prior to infection did not affect parasite infectivity, and PERKi still impaired parasite replication in host cells lacking PERK. In mice, PERKi conferred modest protection from a lethal dose of Toxoplasma Our findings represent the first pharmacological evidence supporting TgIF2K-A as an attractive new target for the treatment of toxoplasmosis.
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    Elongator protein 3 (Elp3) lysine acetyltransferase is a tail-anchored mitochondrial protein in Toxoplasma gondii
    (Elsevier, 2013) Stilger, Krista L.; Sullivan, William J., Jr.; Pharmacology and Toxicology, School of Medicine
    Background: Protein acetylation is prevalent in mitochondria, yet acetyltransferases mediating this activity are unknown. Results: Toxoplasma Elongator protein 3 (Elp3) possesses a unique C-terminal transmembrane domain necessary and sufficient to target it to the mitochondria. Conclusion: Elp3 is an essential tail-anchored mitochondrial acetyltransferase in Toxoplasma. Significance: Elp3 has conserved functions involving mitochondria that may predate its established role in transcription.
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    A forward genetic approach to identifying novel calcium regulators in Toxoplasma Gondii
    (2017-07-25) LaFavers, Kaice Arminda; Arrizabalaga, Gustavo; Brustovetsky, Nickolay; Cummins, Theodore; Gilk, Stacey; Sullivan, William J., Jr.
    Toxoplasma gondii is an obligate intracellular eukaryotic pathogen that causes severe neurologic disease in immunocompromised adults and congenitally infected neonates. Events critical to the propagation of T. gondii, such as invasion and egress, are regulated by calcium-dependent signaling. In order to identify unique components of the parasite’s calcium signaling networks, members of the Arrizabalaga laboratory have used a forward genetics approach to isolate mutants with altered sensitivity to the calcium ionophore A23187. Exposing extracellular parasites to A23187 induces protein secretion, motility and cytoskeletal rearrangements and prolonged treatment causes exhaustion of factors required for invasion, which results in what is referred to as ionophore induced death (iiDeath). Mutants capable of surviving this treatment were isolated from a chemically mutagenized population. Whole genome sequencing of one such mutant, MBD2.1, identified a nonsense mutation in a protein of unknown function (TGGT1_069070, ToxoDBv7.2) Complementation of MBD 2.1 with a wild-type copy of TGGT1_069070 restored sensitivity to iiDeath treatment. Endogenous tagging of this locus revealed that the encoded protein is secreted from a unique parasite secretory organelle known as the dense granule into the parasitophorous vacuole, leading to its designation as TgGRA41. Complete knockout of TgGRA41 recapitulates the resistance to iiDeath observed in MBD2.1 but also exhibits a dramatic decrease in propagation in tissue culture not seen in the original mutant. The knockout shows defects in multiple steps of the lytic including compromised invasion efficiency and premature egress of parasites from host cells. Cytosolic calcium measurements of extracellular parasites show enhanced uptake of calcium in the knockout strain as compared to parental and complemented, suggesting that the loss of TgGra41 results in calcium dysregulation. Together, these results provide a novel insight into the role that the parasitophorous vacuole of T. gondii plays in calcium homeostasis and calcium-dependent signaling processes.
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    Lysine acetyltransferase Gcn5-B regulates the expression of crucial genes in Toxoplasma and its function is regulated through lysine acetylation
    (2014-04-02) Wang, Jiachen; Sullivan, William J., Jr.; Queener, Sherry F.; Arrizabalaga, Gustavo; Nass, Richard M.; Lu, Tao
    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 form of GCN5b containing a point mutation that ablates enzymatic activity (E703G). Stabilization of this dominant-negative form of 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). We also demonstrate that GCN5b interacts with AP2-domain proteins, which are plant-like transcription factors in Apicomplexa. The interactions between GCN5b, AP2IX-7, and AP2X-8 were confirmed by reciprocal co-immunoprecipitation and revealed 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.
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    Mechanisms of Toxoplasma gondii persistence and latency
    (Oxford University Press, 2012) Sullivan, William J., Jr.; Jeffers, Victoria; Pharmacology and Toxicology, School of Medicine
    Toxoplasma gondii is an obligate intracellular protozoan parasite that causes opportunistic disease, particularly in immunocompromised individuals. Central to its transmission and pathogenesis is the ability of the proliferative stage (tachyzoite) to convert into latent tissue cysts (bradyzoites). Encystment allows Toxoplasma to persist in the host, and affords the parasite a unique opportunity to spread to new hosts without proceeding through its sexual stage, which is restricted to felids. Bradyzoite tissue cysts can cause reactivated toxoplasmosis if host immunity becomes impaired. A greater understanding of the molecular mechanisms orchestrating bradyzoite development is needed to better manage the disease. Here we will review key studies that have contributed to our knowledge about this persistent form of the parasite and how to study it, with a focus on how cellular stress can signal for the reprogramming of gene expression needed during bradyzoite development.
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    mRNA cap-binding protein eIF4E1 is a novel regulator of Toxoplasma gondii latency
    (American Society for Microbiology, 2024) Holmes, Michael J.; Bastos, Matheus S.; Dey, Vishakha; Severo, Vanessa; Wek, Ronald C.; Sullivan, William J., Jr.; Pharmacology and Toxicology, School of Medicine
    The protozoan parasite Toxoplasma gondii causes serious opportunistic disease due to its ability to persist in patients as latent tissue cysts. The molecular mechanisms coordinating conversion between proliferative parasites (tachyzoites) and latent cysts (bradyzoites) are not fully understood. We previously showed that phosphorylation of eIF2α accompanies bradyzoite formation, suggesting that this clinically relevant process involves regulation of mRNA translation. In this study, we investigated the composition and role of eIF4F multi-subunit complexes in translational control. Using CLIPseq, we find that the cap-binding subunit, eIF4E1, localizes to the 5'-end of all tachyzoite mRNAs, many of which show evidence of stemming from heterogeneous transcriptional start sites. We further show that eIF4E1 operates as the predominant cap-binding protein in two distinct eIF4F complexes. Using genetic and pharmacological approaches, we found that eIF4E1 deficiency triggers efficient spontaneous formation of bradyzoites without stress induction. Consistent with this result, we also show that stress-induced bradyzoites exhibit reduced eIF4E1 expression. Overall, our findings establish a novel role for eIF4F in translational control required for parasite latency and microbial persistence. Importance: Toxoplasma gondii is an opportunistic pathogen important to global human and animal health. There are currently no chemotherapies targeting the encysted form of the parasite. Consequently, a better understanding of the mechanisms controlling encystation is required. Here we show that the mRNA cap-binding protein, eIF4E1, regulates the encystation process. Encysted parasites reduce eIF4E1 levels, and depletion of eIF4E1 decreases the translation of ribosome-associated machinery and drives Toxoplasma encystation. Together, these data reveal a new layer of mRNA translational control that regulates parasite encystation and latency.
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    mRNA cap-binding protein eIF4E1 is a novel regulator of Toxoplasma gondii latency
    (bioRxiv, 2023-10-09) Holmes, Michael J.; Bastos, Matheus S.; Dey, Vishakha; Severo, Vanessa; Wek, Ronald C.; Sullivan, William J., Jr.; Pharmacology and Toxicology, School of Medicine
    The protozoan parasite Toxoplasma gondii causes serious opportunistic disease due to its ability to persist in patients as latent tissue cysts. The molecular mechanisms coordinating conversion between proliferative parasites (tachyzoites) and dormant cysts (bradyzoites) are not fully understood. We previously showed that phosphorylation of eIF2α accompanies bradyzoite formation, suggesting that this clinically relevant process involves regulation of mRNA translation. In this study, we investigated the composition and role of eIF4F multi-subunit complexes in translational control. Using CLIPseq, we find that the cap-binding subunit, eIF4E1, localizes to the 5’-end of all tachyzoite mRNAs, many of which show evidence of stemming from heterogenous transcriptional start sites. We further show that eIF4E1 operates as the predominant cap-binding protein in two distinct eIF4F complexes. Using genetic and pharmacological approaches, we found that eIF4E1 deficiency triggers efficient spontaneous formation of bradyzoites without stress induction. Consistent with this result, we also show that stress-induced bradyzoites exhibit reduced eIF4E1 expression. Overall, our findings establish a novel role for eIF4F in translational control required for parasite latency and microbial persistence.
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    Protein intrinsic disorder in the acetylome of intracellular and extracellular Toxoplasma gondii
    (Royal Society of Chemistry, 2013) Xue, Bin; Jeffers, Victoria; Sullivan, William J., Jr.; Uversky, Vladimir N.; Pharmacology and Toxicology, School of Medicine
    Toxoplasma gondii is an obligate intracellular parasite of the phylum Apicomplexa, which includes a number of species of medical and veterinary importance. Inhibitors of lysine deacetylases (KDACs) exhibit potent antiparasitic activity, suggesting that interference with lysine acetylation pathways hold promise for future drug targeting. Using high resolution LC-MS/MS to identify parasite peptides enriched by immunopurification with acetyl-lysine antibody, we recently produced an acetylome of the proliferative intracellular stage of Toxoplasma. In this study, we used similar approaches to greatly expand the Toxoplasma acetylome by identifying acetylated proteins in non-replicating extracellular tachyzoites. The functional breakdown of acetylated proteins in extracellular parasites is similar to intracellular parasites, with an enrichment of proteins involved in metabolism, translation, and chromatin biology. Altogether, we have now detected over 700 acetylation sites on a wide variety of parasite proteins of diverse function in multiple subcellular compartments. We found 96 proteins uniquely acetylated in intracellular parasites, 216 uniquely acetylated in extracellular parasites, and 177 proteins acetylated in both states. Our findings suggest that dramatic changes occur at the proteomic level as tachyzoites transition from the intracellular to extracellular environment, similar to reports documenting significant changes in gene expression during this transition. The expanded dataset also allowed a thorough analysis of the degree of protein intrinsic disorder surrounding lysine residues targeted for this post-translational modification. These analyses indicate that acetylated lysines in proteins from extracellular and intracellular tachyzoites are largely located within similar local environments, and that lysine acetylation preferentially occurs in intrinsically disordered or flexible regions.