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Tag: Toxoplasma gondii

The role of potassium and host calcium signaling in Toxoplasma gondii egress

Toxoplasma gondii is an obligate intracellular parasite and replicates inside a parasitophorous vacuole (PV) within the host cell. The membrane of the PV (PVM) contains pores that permits for equilibration of ions and small molecules between the host cytosol and the PV lumen. Ca2+ signaling is universal and both T. gondii and its mammalian host cell utilize Ca2+ signals to stimulate diverse cellular functions. Egress of T. gondii from host cells is an essential step for the infection cycle of T. gondii, and a cytosolic Ca2+ increase initiates a Ca2+ signaling cascade that culminates in the stimulation of motility and egress. In this work we demonstrate that intracellular T. gondii tachyzoites are able to take up Ca2+ from the host cytoplasm during host cell signaling events. Both intracellular and extracellular Ca2+ sources are important in reaching a threshold of parasite cytosolic Ca2+ needed for successful egress. Two peaks of Ca2+ were observed in egressing single parasites with the second peak resulting from Ca2+ entry. We patched infected host cells to allow the delivery of precise concentrations of Ca2+ for the stimulation of motility and egress. Using this approach of patching infected host cells, allowed us to determine that increasing the host cytosolic Ca2+ to a specific concentration can trigger egress, which is further accelerated by diminishing the concentration of potassium (K+).

Stephen A Vella, Christina A Moore, Zhu-Hong Li, Miryam A Hortua Triana, Evgeniy Potapenko, Silvia N J Moreno. Cell Calcium. 2021 Jan 19;94:102337. doi: 10.1016/j.ceca.2020.102337

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

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.

Trainee Spotlight: Megna Tiwari

Megna Tiwari 

Megna Tiwari is a second-year Ph.D. trainee in the laboratory of Diego Huet. She is originally from Newport Beach, California and completed her undergraduate degree in Cell, Molecular and Developmental Biology at the University of California, Riverside (UCR). While at UCR, she worked as an undergraduate researcher in the fungal genomics lab of Dr. Jason Stajich for 2 years and co-founded Women in STEM Engaging Riverside (WISER). After graduation, she worked as a blood bank lab technician at LifeStream Blood Bank where she screened for and routinely found blood samples positive for understudied pathogenic parasites. Her fascination with pathogenic parasites led her to seek a thesis-based Master of Science in Biology at California State University, Fullerton under the supervision of Dr. Veronica Jimenez. During this period, Megna worked on understanding the functional and structural relationship of mechanosensitive ion channels found in T. cruzi and cemented her passion for molecular parasitology.

Megna has been awarded a CTEGD T32 Training Fellowship. She currently serves as Vice-president of CTEGD’s Graduate Student Association and New Student Liaison for the Department of Cellular Biology’s Graduate Student Association.

Why did you choose UGA? 

My master’s research in parasitology reaffirmed my passion for research in unconventional parasitic pathogens. Therefore, I applied for doctoral programs that would allow me to remain in the field of cell and molecular parasitology and the CTEGD at UGA was the perfect place for me to obtain the best possible training as a parasitologist.

What is your research focus/project and why are you interested in the topic? 

The over-reaching research goal of the Huet lab is the investigation of the highly divergent metabolic adaptations of apicomplexans. My research interests in the lab have led me to study the role of the ATP synthase in the apicomplexan Toxoplasma gondii, the causative agent of toxoplasmosis. For my project, I am examining the role of apicomplexan-specific ATP synthase subunits and how they might contribute to the regulation of the ATP synthase function in the parasite.

What are your future professional plans?  

Following graduation from UGA, I hope to continue on for a postdoctoral research position in parasitology.

What do you hope to do for your capstone experience? 

For my capstone experience, I want to gain an outside perspective and understanding of foreign research culture that I can apply to my own research when I return to the CTEGD. 

What is your favorite thing about UGA and/or Athens? 

At the CTEGD, I love the collaborative nature. If I am trying to learn a new technique or understand new concepts, I am able to easily walk down the hall to a neighboring lab and get advice. In Athens, for entertainment, I love the endless craft beer scene and I love all the greenery and being able to hike gaps of the Appalachian trail!


Support trainees like Megna by giving today to the Center for Tropical & Emerging Global Diseases.

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

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.

Potent Tetrahydroquinolone Eliminates Apicomplexan Parasites

Apicomplexan infections cause substantial morbidity and mortality, worldwide. New, improved therapies are needed. Herein, we create a next generation anti-apicomplexan lead compound, JAG21, a tetrahydroquinolone, with increased sp3-character to improve parasite selectivity. Relative to other cytochrome b inhibitors, JAG21 has improved solubility and ADMET properties, without need for pro-drug. JAG21 significantly reduces Toxoplasma gondii tachyzoites and encysted bradyzoites in vitro, and in primary and established chronic murine infections. Moreover, JAG21 treatment leads to 100% survival. Further, JAG21 is efficacious against drug-resistant Plasmodium falciparum in vitro. Causal prophylaxis and radical cure are achieved after P. berghei sporozoite infection with oral administration of a single dose (2.5 mg/kg) or 3 days treatment at reduced dose (0.625 mg/kg/day), eliminating parasitemia, and leading to 100% survival. Enzymatic, binding, and co-crystallography/pharmacophore studies demonstrate selectivity for apicomplexan relative to mammalian enzymes. JAG21 has significant promise as a pre-clinical candidate for prevention, treatment, and cure of toxoplasmosis and malaria.

Martin J. McPhillie, Ying Zhou, Mark R. Hickman, James A. Gordon, Christopher R. Weber, Qigui Li, Patty J. Lee, Kangsa Amporndanai, Rachel M. Johnson, Heather Darby, Stuart Woods, Zhu-hong Li, Richard S. Priestley, Kurt D. Ristroph, Scott B. Biering, Kamal El Bissati, Seungmin Hwang, Farida Esaa Hakim, Sarah M. Dovgin, Joseph D. Lykins, Lucy Roberts, Kerrie Hargrave, Hua Cong, Anthony P. Sinai, Stephen P. Muench, Jitender P. Dubey, Robert K. Prud’homme, Hernan A. Lorenzi, Giancarlo A. Biagini, Silvia N. Moreno, Craig W. Roberts, Svetlana V. Antonyuk, Colin W. G. Fishwick, and Rima McLeod. Front. Cell. Infect. Microbiol., 09 June 2020 |

A terminal α3-galactose modification regulates an E3 ubiquitin ligase subunit in Toxoplasma gondii

Skp1, a subunit of E3 Skp1/Cullin-1/F-box protein ubiquitin ligases, is modified by a prolyl hydroxylase that mediates O2-regulation of the social amoeba Dictyostelium and the parasite Toxoplasma gondii. The full effect of hydroxylation requires modification of the hydroxyproline by a pentasaccharide that, in Dictyostelium, influences Skp1 structure to favor assembly of Skp1/F-box protein subcomplexes. In Toxoplasma, the presence of a contrasting penultimate sugar assembled by a different glycosyltransferase enables testing of the conformational control model. To define the final sugar and its linkage, here we identified the glycosyltransferase that completes the glycan and found that it is closely related to glycogenin, an enzyme that may prime glycogen synthesis in yeast and animals. However, the Toxoplasma enzyme catalyzes formation of a Galα1,3Glcα- rather than the Glcα1,4Glcα- linkage formed by glycogenin. Kinetic and crystallographic experiments showed that the glycosyltransferase Gat1 is specific for Skp1 in Toxoplasma and also in another protist, the crop pathogen Pythium ultimum. The fifth sugar is important for glycan function as indicated by the slow-growth phenotype of gat1Δ parasites. Computational analyses indicated that, despite the sequence difference, the Toxoplasma glycan still assumes an ordered conformation that controls Skp1 structure and revealed the importance of non-polar packing interactions of the fifth sugar. The substitution of glycosyltransferases in Toxoplasma and Pythium by an unrelated bifunctional enzyme that assembles a distinct but structurally compatible glycan in Dictyostelium is a remarkable case of convergent evolution, that emphasizes the importance of the terminal α-galactose and establishes the phylogenetic breadth of Skp1 glycoregulation.

Msano MandalasiHyun W. KimDavid ThiekerM. Osman SheikhElisabet Gas-PascualKazi RahmanPeng ZhaoNitin G. DanielHanke van der WelH. Travis IchikawaJohn N. GlushkaLance Wells, Robert J. Woods, Zachary A. Wood, and Christopher M. West. J Biol Chem. 2020 May 15. pii: jbc.RA120.013792. doi: 10.1074/jbc.RA120.013792.

Trainee Spotlight: Edwin Pierre Louis

Trainee Edwin Pierre Louis


Edwin Pierre Louis is a pre-doctoral trainee in the laboratory of Dr. Drew Etheridge. Originally from Haiti, he immigrated to the US to attend the University of Florida (UF), where he graduated with a Bachelor of Science in Biochemistry Molecular Biology. After earning his degree at UF, Edwin accepted a position as a biological scientist in the UF Center of Excellence for Regenerative Health Biotechnology, with a focus on gene and cell based therapeutic development, where he worked for three years. There, he first discovered his love of host-pathogen interactions as a biological scientist working under the supervision of Dr. Richard Snyder for the component Florida Biologix at this center and later merged to create Brammer Bio which was subsequently acquired by Thermo Fisher Scientific. During this time in industry, he realized that to improve his scientific capacities he would need to continue his studies by pursuing a graduate degree. As part of his preparations to apply to a graduate program, he joined the UGA post-baccalaureate PREP program whose mission is to prepare students interested in a graduate degree for the application process. During this time, he was granted the opportunity to join Dr. Michael Terns’ laboratory for a year where he investigated the molecular mechanism of CRISPR-Cas based viral defense in Streptococcus thermophilus as well as prime adaptation events in the type II-A CRISPR-Cas system.

Since attending UGA, Edwin has been awarded both the Gateway to Graduate School Bridge Program and the Graduate Scholars Leadership, Engagement and Development Program (GS LEAD) scholarships sponsored by the National Science Foundation (NSF).

What is your research focus and why are you interested in the topic?

Broadly, my key research interests center around how organisms like viruses and parasites manipulate their host cell in order to grow and propagate. My current project is focused on elucidating how the protozoan pathogen Toxoplasma gondii is able to use secreted protein effectors to manipulate its host cells functions.

Why did you choose UGA?

I chose to study at the University of Georgia, in part, because of my excellent post-baccalaureate experience in the PREP program. It was evident from my interactions that UGA excels at fostering a productive relationship between students and faculty. Regardless of any faculty member’s relationship to the students, there was a sustained willingness for faculty to give of their time in order to see the students succeed.  I also decided to pursue my PhD at UGA because of the cutting-edge research and in particular the collection of outstanding parasitologists that is uniquely found in the Center for Tropical and Emerging Global Diseases (CTEGD).

What are your future professional plans?

As I continue my graduate studies on host pathogen interaction, I plan to do some post-doctoral trainings to augment my apprenticeship and ultimately become an independent scientist to lead my own research group.  I also hope to be able to give back to the local community that has contributed so much to my own personal success by donating my time and knowledge to mentor young budding scientists especially those from underprivileged homes and/or underdeveloped countries.


Support trainees like Edwin by giving today to the Center for Tropical & Emerging Global Diseases.

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Genetic Indicators for Calcium Signaling Studies in Toxoplasma gondii

Fluctuations of the cytosolic calcium ion (Ca2+) concentration regulate a variety of cellular functions in all eukaryotes. Cells express a sophisticated set of mechanisms to balance the cytosolic Ca2+ levels and the signals that elevate Ca2+ in the cytosol are compensated by mechanisms that reduce it. Alterations in Ca2+-dependent homeostatic mechanisms are the cause of many prominent diseases in humans, such as heart failure or neuronal death.

The genetic tractability of Toxoplasma gondii and the availability of genetic tools enabled the use of Genetically Encoded Calcium Indicators (GECIs) expressed in the cytoplasm, which started a new era in the studies of Toxoplasma calcium signaling. It was finally possible to see Ca2+ oscillations prior to exit of the parasite from host cells. Years after Endo et al showed that ionophores triggered egress, the assumption that oscillations occur prior to egress from host cells has been validated by experiments using GECIs. GECIs allowed the visualization of specific Ca2+ signals in live intracellular parasites and to distinguish these signals from host cell calcium fluctuations. In this chapter we present an overview describing “tried and true” methods of our lab who pioneered the first use of GECI’s in Toxoplasma, including GECI choice, methodology for transfection and selection of ideal clones, their characterization, and the use of GECI-expressing parasites for fluorometric and microscopic analysis.

Stephen A. Vella, Abigail Calixto, Beejan Asady, Zhu-Hong Li, Silvia N. J. Moreno. Methods Mol Biol. 2020;2071:187-207. doi: 10.1007/978-1-4939-9857-9_11.

The Vacuolar Zinc Transporter TgZnT Protects Toxoplasma gondii from Zinc Toxicity

Zinc (Zn2+) is the most abundant biological metal ion aside from iron and is an essential element in numerous biological systems, acting as a cofactor for a large number of enzymes and regulatory proteins. Zn2+ must be tightly regulated, as both the deficiency and overabundance of intracellular free Zn2+ are harmful to cells. Zn2+ transporters (ZnTs) play important functions in cells by reducing intracellular Zn2+ levels by transporting the ion out of the cytoplasm. We characterized a Toxoplasma gondii gene (TgGT1_251630, TgZnT), which is annotated as the only ZnT family Zn2+ transporter in T. gondii. TgZnT localizes to novel vesicles that fuse with the plant-like vacuole (PLV), an endosome-like organelle. Mutant parasites lacking TgZnT exhibit reduced viability in in vitro assays. This phenotype was exacerbated by increasing zinc concentrations in the extracellular media and was rescued by media with reduced zinc. Heterologous expression of TgZnT in a Zn2+-sensitive Saccharomyces cerevisiae yeast strain partially restored growth in media with higher Zn2+ concentrations. These results suggest that TgZnT transports Zn2+ into the PLV and plays an important role in the Zn2+tolerance of T. gondii extracellular tachyzoites.

IMPORTANCE Toxoplasma gondii is an intracellular pathogen of human and animals. T. gondii pathogenesis is associated with its lytic cycle, which involves invasion, replication, egress out of the host cell, and invasion of a new one. T. gondii must be able to tolerate abrupt changes in the composition of the surrounding milieu as it progresses through its lytic cycle. We report the characterization of a Zn2+ transporter of T. gondii (TgZnT) that is important for parasite growth. TgZnT restored Zn2+ tolerance in yeast mutants that were unable to grow in media with high concentrations of Zn2+. We propose that TgZnT plays a role in Zn2+ homeostasis during the T. gondii lytic cycle.

Nathan M. Chasen, Andrew J. Stasic, Beejan Asady, Isabelle Coppens, Silvia N. J. Moreno. 2019. mSphere.; 4(3). pii: e00086-19. doi: 10.1128/mSphere.00086-19.

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