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Tag: Christopher West

The nucleocytosolic O-fucosyltransferase Spindly affects protein expression and virulence in Toxoplasma gondii

Donna Huber on November 9, 2020

Once considered unusual, nucleocytoplasmic glycosylation is now recognized as a conserved feature of eukaryotes. While in animals O-GlcNAc transferase (OGT) modifies thousands of intracellular proteins, the human pathogen Toxoplasma gondii transfers a different sugar, fucose, to proteins involved in transcription, mRNA processing and signaling. Knockout experiments showed that TgSPY, an ortholog of plant SPINDLY and paralog of host OGT, is required for nuclear O-fucosylation. Here we verify that TgSPY is the nucleocytoplasmic O-fucosyltransferase (OFT) by 1) complementation with TgSPY-MYC3, 2) its functional dependence on amino acids critical for OGT activity, and 3) its ability to O-fucosylate itself and a model substrate and to specifically hydrolyze GDP-Fuc. While many of the endogenous proteins modified by O-Fuc are important for tachyzoite fitness, O-fucosylation by TgSPY is not essential. Growth of Δspy tachyzoites in fibroblasts is modestly affected, despite marked reductions in the levels of ectopically-expressed proteins normally modified with O-fucose. Intact TgSPY-MYC3 localizes to the nucleus and cytoplasm, whereas catalytic mutants often displayed reduced abundance. Δspy tachyzoites of a luciferase-expressing type II strain exhibited infection kinetics in mice similar to wild type but increased persistence in the chronic brain phase, potentially due to an imbalance of regulatory protein levels. The modest changes in parasite fitness in vitro and in mice, despite profound effects on reporter protein accumulation, and the characteristic punctate localization of O-fucosylated proteins, suggest that TgSPY controls the levels of proteins to be held in reserve for response to novel stresses.

Giulia Bandini, Carolina Agop-Nersesian, Hanke van der Wel, Msano Mandalasi , Hyun W Kim, Christopher M West, John Samuelson. J Biol Chem. 2020 Nov 6;jbc.RA120.015883. doi: 10.1074/jbc.RA120.015883.

Biochemical and biophysical analyses of hypoxia sensing prolyl hydroxylases from Dictyostelium discoideum and Toxoplasma gondii

Donna Huber on September 15, 2020

In animals, the response to chronic hypoxia is mediated by prolyl-hydroxylases (PHDs) that regulate the levels of hypoxia inducible transcription factor a (HIFα). PHD homologues exist in other types of eukaryotes and prokaryotes where they act on non-HIF substrates. To gain insight into the factors underlying different PHD substrates and properties, we carried out biochemical and biophysical studies on PHD homologues from the slime mold, Dictyostelium discoideum, and the protozoan parasite, Toxoplasma gondii, both lacking HIF. The respective prolyl-hydroxylases (DdPhyA and TgPhyA) catalyze prolyl-hydroxylation of S-Phase Kinase Associated Protein 1 (Skp1), a reaction enabling adaptation to different dioxygen availability. Assays with full length Skp1 substrates reveal substantial differences in the kinetic properties of DdPhyA and TgPhyA, both with respect to each other and compared with human PHD2; consistent with cellular studies TgPhyA is more active at low dioxygen concentrations than DdPhyA. TgSkp1 is a DdPhyA substrate and DdSkp1 is a TgPhyA substrate. No cross-reactivity was detected between DdPhyA/TgPhyA substrates and human PHD2. The human Skp1 E147P variant is a DdPhyA and TgPhyA substrate, suggesting some retention of ancestral interactions. Crystallographic analysis of DdPhyA enables comparisons with homologues from humans, Trichoplax adhaerens, and prokaryotes, TgPhyA informing on differences in mobile elements involved in substrate binding and catalysis. In DdPhyA, two mobile loops that enclose substrates in the PHDs are conserved, but the C-terminal helix of the PHDs is strikingly absent. The combined results support the proposal that PHD homologues have evolved kinetic and structural features suited to their specific sensing roles.

Tongri Liu, Martine I Abboud, Rasheduzzaman Chowdhury, Anthony Tumber, Adam P Hardy, Kerstin Lippl, Christopher T Lohans, Elisabete Pires, James Wickens, Michael A McDonough, Christopher M West, Christopher J Schofield. J Biol Chem. 2020 Sep 15;jbc.RA120.013998. doi: 10.1074/jbc.RA120.013998.

Toxoplasma F-box protein 1 is required for daughter cell scaffold function during parasite replication

Donna Huber on July 26, 2019

By binding to the adaptor protein SKP1 and serving as substrate receptors for the Skp1, Cullin, F-box E3 ubiquitin ligase complex, F-box proteins regulate critical cellular processes including cell cycle progression and membrane trafficking. While F-box proteins are conserved throughout eukaryotes and are well studied in yeast, plants, and animals, studies in parasitic protozoa are lagging. We have identified eighteen putative F-box proteins in the Toxoplasmagenome of which four have predicted homologs in Plasmodium. Two of the conserved F-box proteins were demonstrated to be important for Toxoplasma fitness and here we focus on an F-box protein, named TgFBXO1, because it is the most highly expressed by replicative tachyzoites and was also identified in an interactome screen as a Toxoplasma SKP1 binding protein. TgFBXO1 interacts with Toxoplasma SKP1 confirming it as a bona fide F-box protein. In interphase parasites, TgFBXO1 is a component of the Inner Membrane Complex (IMC), which is an organelle that underlies the plasma membrane. Early during replication, TgFBXO1 localizes to the developing daughter cell scaffold, which is the site where the daughter cell IMC and microtubules form and extend from. TgFBXO1 localization to the daughter cell scaffold required centrosome duplication but before kinetochore separation was completed. Daughter cell scaffold localization required TgFBXO1 N-myristoylation and was dependent on the small molecular weight GTPase, TgRab11b. Finally, we demonstrate that TgFBXO1 is required for parasite growth due to its function as a daughter cell scaffold effector. TgFBXO1 is the first F-box protein to be studied in apicomplexan parasites and represents the first protein demonstrated to be important for daughter cell scaffold function.

Carlos Gustavo Baptista, Agnieszka Lis, Bowen Deng, Elisabet Gas-Pascual, Ashley Dittmar, Wade Sigurdson, Christopher M. West, Ira J. Blader. PLoS Pathog. 2019 Jul 26;15(7):e1007946. doi: 10.1371/journal.ppat.1007946.

Skp1 isoforms are differentially modified by a dual function prolyl 4-hydroxylase/N-acetylglucosaminyltransferase in a plant pathogen

Donna Huber on July 8, 2019

Skp1 is hydroxylated by an O2-dependent prolyl hydroxylase (PhyA) that contributes to O2-sensing in the social amoeba Dictyostelium and the mammalian pathogen Toxoplasma gondii. HO-Skp1 is subject to glycosylation and the resulting pentasaccharide affects Skp1 conformation in a way that influences association of Skp1 with F-box proteins, and potentially the assembly of E3(SCF) ubiquitin ligase complexes that mediate the poly-ubiquitination of target proteins that are degraded in the 26S-proteasome. To investigate the conservation and specificity of these modifications, we analyzed proteins from the oomycete Pythium ultimum, an important crop plant pathogen. Putative coding sequences for Pythium’s predicted PhyA and first glycosyltransferase in the predicted five-enzyme pathway, a GlcNAc-transferase (Gnt1), predict a bifunctional enzyme (Phgt) that, when expressed in Dictyostelium, rescued knockouts of phyA but not gnt1. Though recombinant Phgt was also unable to glycosylate Dictyostelium HO-Skp1, it could hydrolyze UDP-GlcNAc and modify a synthetic hydroxypeptide from Dictyostelium Skp1. Pythium encodes two highly similar Skp1 isoforms, but only Skp1A was efficiently modified in vitro. While kinetic analysis revealed no evidence for processive processing of Skp1, the physical linkage of the two activities implies dedication to Skp1 in vivo. These findings indicate a widespread occurrence of the Skp1 modification pathway across protist phylogeny, suggest that both Gnt1 and PhyA are specific for Skp1, and indicate that the second Skp1 provides a bypass mechanism for O2-regulation in Pythium and other protists that conserve this gene.

Hanke van der Wel, Elisabet Gas-Pascual, Christopher M West. Glycobiology. 2019 Jul 8. pii: cwz049. doi: 10.1093/glycob/cwz049.

A Toxoplasma Prolyl Hydroxylase Mediates Oxygen Stress Responses by Regulating Translation Elongation

Donna Huber on March 26, 2019

As the protozoan parasite Toxoplasma gondii disseminates through its host, it responds to environmental changes by altering its gene expression, metabolism, and other processes. Oxygen is one variable environmental factor, and properly adapting to changes in oxygen levels is critical to prevent the accumulation of reactive oxygen species and other cytotoxic factors. Thus, oxygen-sensing proteins are important, and among these, 2-oxoglutarate-dependent prolyl hydroxylases are highly conserved throughout evolution. Toxoplasmaexpresses two such enzymes, TgPHYa, which regulates the SCF-ubiquitin ligase complex, and TgPHYb. To characterize TgPHYb, we created a Toxoplasma strain that conditionally expresses TgPHYb and report that TgPHYb is required for optimal parasite growth under normal growth conditions. However, exposing TgPHYb-depleted parasites to extracellular stress leads to severe decreases in parasite invasion, which is likely due to decreased abundance of parasite adhesins. Adhesin protein abundance is reduced in TgPHYb-depleted parasites as a result of inactivation of the protein synthesis elongation factor eEF2 that is accompanied by decreased rates of translational elongation. In contrast to most other oxygen-sensing proteins that mediate cellular responses to low O2, TgPHYb is specifically required for parasite growth and protein synthesis at high, but not low, O2 tensions as well as resistance to reactive oxygen species. In vivo, reduced TgPHYb expression leads to lower parasite burdens in oxygen-rich tissues. Taken together, these data identify TgPHYb as a sensor of high O2 levels, in contrast to TgPHYa, which supports the parasite at low O2

IMPORTANCE Because oxygen plays a key role in the growth of many organisms, cells must know how much oxygen is available. O2-sensing proteins are therefore critical cellular factors, and prolyl hydroxylases are the best-studied type of O2-sensing proteins. In general, prolyl hydroxylases trigger cellular responses to decreased oxygen availability. But, how does a cell react to high levels of oxygen? Using the protozoan parasite Toxoplasma gondii, we discovered a prolyl hydroxylase that allows the parasite to grow at elevated oxygen levels and does so by regulating protein synthesis. Loss of this enzyme also reduces parasite burden in oxygen-rich tissues, indicating that sensing both high and low levels of oxygen impacts the growth and physiology of Toxoplasma.

Celia Florimond, Charlotte Cordonnier, Rahil Taujale, Hanke van der Wel, Natarajan Kannan, Christopher M. West, Ira J. Blader. 2019. MBio.;10(2). pii: e00234-19. doi: 10.1128/mBio.00234-19.

Trypanosoma cruzi 13C-labeled O-Glycan standards for mass spectrometry

Donna Huber on January 12, 2019

Abstract

Trypanosoma cruzi is a protozoan parasite that causes Chagas disease, a debilitating condition that affects over 10 million humans in the American continents. In addition to its traditional mode of human entry via the ‘kissing bug’ in endemic areas, the infection can also be spread in non-endemic countries through blood transfusion, organ transplantation, eating food contaminated with the parasites, and from mother to fetus. Previous NMR-based studies established that the parasite expresses a variety of strain-specific and developmentally-regulated O-glycans that may contribute to virulence. In this report, we describe five synthetic O-glycan analytical standards and show their potential to enable a more facile analysis of native O-glycan isomers based on mass spectrometry.

M. Osman Sheikh, Elisabet Gas-Pascual, John N. Glushka, Juan M. Bustamante, Lance Wells, Christopher M. West. 2019. Glycobiology. https://doi.org/10.1093/glycob/cwy111

CRISPR/Cas9 and glycomics tools for Toxoplasma glycobiology

Donna Huber on November 21, 2018

Abstract

Infection with the protozoan parasite Toxoplasma gondii is a major health risk owing to birth defects, its chronic nature, ability to reactivate to cause blindness and encephalitis, and high prevalence in human populations. Unlike most eukaryotes, Toxoplasma propagates in intracellular parasitophorous vacuoles, but as for nearly all other eukaryotes, Toxoplasma glycosylates many cellular proteins and lipids and assembles polysaccharides. Toxoplasma glycans resemble those of other eukaryotes but species-specific variations have prohibited deeper investigations into their roles in parasite biology and virulence. The Toxoplasma genome encodes a suite of likely glycogenes expected to assemble N-glycans, O-glycans, a C-glycan, GPI-anchors, and polysaccharides, along with their precursors and membrane transporters. To investigate the roles of specific glycans in Toxoplasma, here we coupled genetic and glycomics approaches to map the connections between 67 glycogenes, their enzyme products, the glycans to which they contribute, and cellular functions. We applied a double-CRISPR/Cas9 strategy, in which two guide RNAs promote replacement of a candidate gene with a resistance gene; adapted MS-based glycomics workflows to test for effects on glycan formation; and infected fibroblast monolayers to assess cellular effects. By editing 17 glycogenes, we discovered novel Glc0-2-Man6-GlcNAc2–type N-glycans, a novel HexNAc-GalNAc–mucin-type O-glycan, and Tn-antigen, identified the glycosyltransferases for assembling a novel nuclear O-Fuc–type and cell surface Glc-Fuc–type O-glycans, and showed that they are important for in vitro growth. The guide sequences, editing constructs, and mutant strains are freely available to researchers to investigate the roles of glycans in their favorite biological processes.

Elisabet Gas-Pascual, Hiroshi Travis Ichikawa, Mohammed Osman Sheikh, Mariam Isabella Serji, Bowen Deng, Msano Mandalasi, Giulia Bandini, John Samuelson, Lance Wells and Christopher M. West. 2018. Journal of Biological Chemistry. 294: 1104-1125. doi: 10.1074/jbc.RA118.006072