POFUT2-mediated O-glycosylation of MIC2 is dispensable for Toxoplasma gondii tachyzoites

AbstractToxoplasma gondii is a ubiquitous obligate intracellular eukaryotic parasite that causes congenital birth defects, disease of the immunocompromised and blindness. Protein glycosylation plays an important role in the infectivity and evasion of immune response of many eukaryotic parasites and is also of great relevance to vaccine design. Here, we demonstrate that MIC2, the motility-associated adhesin of T. gondii, has highly glycosylated thrombospondin repeat domains (TSR). At least seven C-linked and three O-linked glycosylation sites exist within MIC2, with >95% occupancy at O-glycosylation sites. We demonstrate that the addition of O-glycans to MIC2 is mediated by a protein O-fucosyltransferase 2 homologue (TgPOFUT2) encoded by TGGT1_273550. While POFUT2 homologues are important for stabilizing motility associated adhesins and host infection in other apicomplexan parasites, in T. gondii loss of TgPOFUT2 has only a modest impact on MIC2 levels and the wider proteome. Consistent with this, both plaque formation and tachyzoite infectivity are broadly similar in the presence or absence of TgPOFUT2. These findings demonstrate that TgPOFUT2 O-glycosylates MIC2 and that this glycan is dispensable in T. gondii tachyzoites.

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Protein O-fucosyltransferase 2–mediated O-glycosylation of the adhesin MIC2 is dispensable for Toxoplasma gondii tachyzoite infection

Journal of Biological Chemistry ◽

10.1074/jbc.ra118.005357 ◽

2018 ◽

Vol 294 (5) ◽

pp. 1541-1553 ◽

Cited By ~ 11

Author(s):

Sachin Khurana ◽

Michael J. Coffey ◽

Alan John ◽

Alessandro D. Uboldi ◽

My-Hang Huynh ◽

...

Keyword(s):

Toxoplasma Gondii ◽

Immune Responses ◽

Enzymatic Digestion ◽

Protein Glycosylation ◽

Apicomplexan Parasites ◽

Obligate Intracellular ◽

Congenital Birth Defects ◽

Glycosylation Sites ◽

Great Relevance ◽

Host Infection

Toxoplasma gondii is a ubiquitous, obligate intracellular eukaryotic parasite that causes congenital birth defects, disease in immunocompromised individuals, and blindness. Protein glycosylation plays an important role in the infectivity and evasion of immune responses of many eukaryotic parasites and is also of great relevance to vaccine design. Here we demonstrate that micronemal protein 2 (MIC2), a motility-associated adhesin of T. gondii, has highly glycosylated thrombospondin repeat (TSR) domains. Using affinity-purified MIC2 and MS/MS analysis along with enzymatic digestion assays, we observed that at least seven C-linked and three O-linked glycosylation sites exist within MIC2, with >95% occupancy at these O-glycosylation sites. We found that addition of O-glycans to MIC2 is mediated by a protein O-fucosyltransferase 2 homolog (TgPOFUT2) encoded by the TGGT1_273550 gene. Even though POFUT2 homologs are important for stabilizing motility-associated adhesins and for host infection in other apicomplexan parasites, loss of TgPOFUT2 in T. gondii had only a modest impact on MIC2 levels and the wider parasite proteome. Consistent with this, both plaque formation and tachyzoite invasion were broadly similar in the presence or absence of TgPOFUT2. These findings indicate that TgPOFUT2 O-glycosylates MIC2 and that this glycan, in contrast to previous findings in another study, is dispensable in T. gondii tachyzoites and for T. gondii infectivity.

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Untargeted Metabolomics Uncovers the Essential Lysine Transporter in Toxoplasma gondii

Metabolites ◽

10.3390/metabo11080476 ◽

2021 ◽

Vol 11 (8) ◽

pp. 476

Author(s):

Joachim Kloehn ◽

Matteo Lunghi ◽

Emmanuel Varesio ◽

David Dubois ◽

Dominique Soldati-Favre

Keyword(s):

Amino Acids ◽

Toxoplasma Gondii ◽

Rna Degradation ◽

Major Facilitator Superfamily ◽

Untargeted Metabolomics ◽

Apicomplexan Parasites ◽

Obligate Intracellular ◽

Intracellular Parasites ◽

Major Facilitator ◽

Plasma Membrane Transporter

Apicomplexan parasites are responsible for devastating diseases, including malaria, toxoplasmosis, and cryptosporidiosis. Current treatments are limited by emerging resistance to, as well as the high cost and toxicity of existing drugs. As obligate intracellular parasites, apicomplexans rely on the uptake of many essential metabolites from their host. Toxoplasma gondii, the causative agent of toxoplasmosis, is auxotrophic for several metabolites, including sugars (e.g., myo-inositol), amino acids (e.g., tyrosine), lipidic compounds and lipid precursors (cholesterol, choline), vitamins, cofactors (thiamine) and others. To date, only few apicomplexan metabolite transporters have been characterized and assigned a substrate. Here, we set out to investigate whether untargeted metabolomics can be used to identify the substrate of an uncharacterized transporter. Based on existing genome- and proteome-wide datasets, we have identified an essential plasma membrane transporter of the major facilitator superfamily in T. gondii—previously termed TgApiAT6-1. Using an inducible system based on RNA degradation, TgApiAT6-1 was depleted, and the mutant parasite’s metabolome was compared to that of non-depleted parasites. The most significantly reduced metabolite in parasites depleted in TgApiAT6-1 was identified as the amino acid lysine, for which T. gondii is predicted to be auxotrophic. Using stable isotope-labeled amino acids, we confirmed that TgApiAT6-1 is required for efficient lysine uptake. Our findings highlight untargeted metabolomics as a powerful tool to identify the substrate of orphan transporters.

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Vitamin and cofactor acquisition in apicomplexans: Synthesis versus salvage

Journal of Biological Chemistry ◽

10.1074/jbc.aw119.008150 ◽

2019 ◽

Vol 295 (3) ◽

pp. 701-714 ◽

Cited By ~ 3

Author(s):

Aarti Krishnan ◽

Joachim Kloehn ◽

Matteo Lunghi ◽

Dominique Soldati-Favre

Keyword(s):

Toxoplasma Gondii ◽

Nutrient Availability ◽

Metabolic Reconstruction ◽

Mammalian Host ◽

Free Living ◽

Apicomplexan Parasites ◽

Obligate Intracellular ◽

Functional Studies ◽

Intracellular Parasites ◽

Large Repertoire

The Apicomplexa phylum comprises diverse parasitic organisms that have evolved from a free-living ancestor. These obligate intracellular parasites exhibit versatile metabolic capabilities reflecting their capacity to survive and grow in different hosts and varying niches. Determined by nutrient availability, they either use their biosynthesis machineries or largely depend on their host for metabolite acquisition. Because vitamins cannot be synthesized by the mammalian host, the enzymes required for their synthesis in apicomplexan parasites represent a large repertoire of potential therapeutic targets. Here, we review recent advances in metabolic reconstruction and functional studies coupled to metabolomics that unravel the interplay between biosynthesis and salvage of vitamins and cofactors in apicomplexans. A particular emphasis is placed on Toxoplasma gondii, during both its acute and latent stages of infection.

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Protein O- and C-Glycosylation pathways in Toxoplasma gondii and Plasmodium falciparum

Parasitology ◽

10.1017/s0031182019000040 ◽

2019 ◽

Vol 146 (14) ◽

pp. 1755-1766 ◽

Cited By ~ 5

Author(s):

Giulia Bandini ◽

Andreia Albuquerque-Wendt ◽

Jan Hegermann ◽

John Samuelson ◽

Françoise H. Routier

Keyword(s):

Plasmodium Falciparum ◽

Toxoplasma Gondii ◽

Protein Glycosylation ◽

Specific Gene ◽

Post Translational Modifications ◽

Apicomplexan Parasites ◽

Causative Agents ◽

Thrombospondin Type 1 Repeats ◽

Great Public Health

AbstractApicomplexan parasites are amongst the most prevalent and morbidity-causing pathogens worldwide. They are responsible for severe diseases in humans and livestock and are thus of great public health and economic importance. Until the sequencing of apicomplexan genomes at the beginning of this century, the occurrence of N- and O-glycoproteins in these parasites was much debated. The synthesis of rudimentary and divergent N-glycans due to lineage-specific gene loss is now well established and has been recently reviewed. Here, we will focus on recent studies that clarified classical O-glycosylation pathways and described new nucleocytosolic glycosylations in Toxoplasma gondii, the causative agents of toxoplasmosis. We will also review the glycosylation of proteins containing thrombospondin type 1 repeats by O-fucosylation and C-mannosylation, newly discovered in Toxoplasma and the malaria parasite Plasmodium falciparum. The functional significance of these post-translational modifications has only started to emerge, but the evidence points towards roles for these protein glycosylation pathways in tissue cyst wall rigidity and persistence in the host, oxygen sensing, and stability of proteins involved in host invasion.

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The Skull’s Girder: A Brief Review of the Cranial Base

Journal of Developmental Biology ◽

10.3390/jdb9010003 ◽

2021 ◽

Vol 9 (1) ◽

pp. 3

Author(s):

Shankar Rengasamy Venugopalan ◽

Eric Van Otterloo

Keyword(s):

Birth Defects ◽

Vertebral Column ◽

Endochondral Ossification ◽

Cranial Base ◽

Facial Skeleton ◽

The Core ◽

Skull Vault ◽

Congenital Birth Defects ◽

Intimate Association ◽

The Brain

The cranial base is a multifunctional bony platform within the core of the cranium, spanning rostral to caudal ends. This structure provides support for the brain and skull vault above, serves as a link between the head and the vertebral column below, and seamlessly integrates with the facial skeleton at its rostral end. Unique from the majority of the cranial skeleton, the cranial base develops from a cartilage intermediate—the chondrocranium—through the process of endochondral ossification. Owing to the intimate association of the cranial base with nearly all aspects of the head, congenital birth defects impacting these structures often coincide with anomalies of the cranial base. Despite this critical importance, studies investigating the genetic control of cranial base development and associated disorders lags in comparison to other craniofacial structures. Here, we highlight and review developmental and genetic aspects of the cranial base, including its transition from cartilage to bone, dual embryological origins, and vignettes of transcription factors controlling its formation.

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Recent African strains of Zika virus display higher transmissibility and fetal pathogenicity than Asian strains

Nature Communications ◽

10.1038/s41467-021-21199-z ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Fabien Aubry ◽

Sofie Jacobs ◽

Maïlis Darmuzey ◽

Sebastian Lequime ◽

Leen Delang ◽

...

Keyword(s):

Birth Defects ◽

Zika Virus ◽

Public Health Surveillance ◽

Fetal Loss ◽

Surveillance Systems ◽

Apparent Paradox ◽

Congenital Birth Defects ◽

Fetal Mice ◽

Puzzling Aspect ◽

Low Passage

AbstractThe global emergence of Zika virus (ZIKV) revealed the unprecedented ability for a mosquito-borne virus to cause congenital birth defects. A puzzling aspect of ZIKV emergence is that all human outbreaks and birth defects to date have been exclusively associated with the Asian ZIKV lineage, despite a growing body of laboratory evidence pointing towards higher transmissibility and pathogenicity of the African ZIKV lineage. Whether this apparent paradox reflects the use of relatively old African ZIKV strains in most laboratory studies is unclear. Here, we experimentally compare seven low-passage ZIKV strains representing the recently circulating viral genetic diversity. We find that recent African ZIKV strains display higher transmissibility in mosquitoes and higher lethality in both adult and fetal mice than their Asian counterparts. We emphasize the high epidemic potential of African ZIKV strains and suggest that they could more easily go unnoticed by public health surveillance systems than Asian strains due to their propensity to cause fetal loss rather than birth defects.

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Toxoplasma gondii myosins B/C

The Journal of Cell Biology ◽

10.1083/jcb.200012116 ◽

2001 ◽

Vol 155 (4) ◽

pp. 613-624 ◽

Cited By ~ 66

Author(s):

Frédéric Delbac ◽

Astrid Sänger ◽

Eva M. Neuhaus ◽

Rolf Stratmann ◽

James W. Ajioka ◽

...

Keyword(s):

Cell Division ◽

Toxoplasma Gondii ◽

Daughter Cell ◽

Gliding Motility ◽

Apicomplexan Parasites ◽

Daughter Cells ◽

Membrane Complex ◽

Alternatively Spliced ◽

Butanedione Monoxime ◽

Inner Membrane Complex

In apicomplexan parasites, actin-disrupting drugs and the inhibitor of myosin heavy chain ATPase, 2,3-butanedione monoxime, have been shown to interfere with host cell invasion by inhibiting parasite gliding motility. We report here that the actomyosin system of Toxoplasma gondii also contributes to the process of cell division by ensuring accurate budding of daughter cells. T. gondii myosins B and C are encoded by alternatively spliced mRNAs and differ only in their COOH-terminal tails. MyoB and MyoC showed distinct subcellular localizations and dissimilar solubilities, which were conferred by their tails. MyoC is the first marker selectively concentrated at the anterior and posterior polar rings of the inner membrane complex, structures that play a key role in cell shape integrity during daughter cell biogenesis. When transiently expressed, MyoB, MyoC, as well as the common motor domain lacking the tail did not distribute evenly between daughter cells, suggesting some impairment in proper segregation. Stable overexpression of MyoB caused a significant defect in parasite cell division, leading to the formation of extensive residual bodies, a substantial delay in replication, and loss of acute virulence in mice. Altogether, these observations suggest that MyoB/C products play a role in proper daughter cell budding and separation.

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The Plastid of Toxoplasma gondii Is Divided by Association with the Centrosomes

The Journal of Cell Biology ◽

10.1083/jcb.151.7.1423 ◽

2000 ◽

Vol 151 (7) ◽

pp. 1423-1434 ◽

Cited By ~ 163

Author(s):

Boris Striepen ◽

Michael J. Crawford ◽

Michael K. Shaw ◽

Lewis G. Tilney ◽

Frank Seeber ◽

...

Keyword(s):

Cell Division ◽

Toxoplasma Gondii ◽

Genetic Manipulation ◽

Fluorescent Proteins ◽

Recombinant Expression ◽

Molecular Genetic ◽

Time Lapse ◽

Microtubule Organization ◽

Apicomplexan Parasites ◽

Daughter Cells

Apicomplexan parasites harbor a single nonphotosynthetic plastid, the apicoplast, which is essential for parasite survival. Exploiting Toxoplasma gondii as an accessible system for cell biological analysis and molecular genetic manipulation, we have studied how these parasites ensure that the plastid and its 35-kb circular genome are faithfully segregated during cell division. Parasite organelles were labeled by recombinant expression of fluorescent proteins targeted to the plastid and the nucleus, and time-lapse video microscopy was used to image labeled organelles throughout the cell cycle. Apicoplast division is tightly associated with nuclear and cell division and is characterized by an elongated, dumbbell-shaped intermediate. The plastid genome is divided early in this process, associating with the ends of the elongated organelle. A centrin-specific antibody demonstrates that the ends of dividing apicoplast are closely linked to the centrosomes. Treatment with dinitroaniline herbicides (which disrupt microtubule organization) leads to the formation of multiple spindles and large reticulate plastids studded with centrosomes. The mitotic spindle and the pellicle of the forming daughter cells appear to generate the force required for apicoplast division in Toxoplasma gondii. These observations are discussed in the context of autonomous and FtsZ-dependent division of plastids in plants and algae.

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