Dataset Information

Mass spectrometry-based characterization of Toxoplasma gondii MORC interactome

ABSTRACT: T. gondii has a complex life cycle typified by an asexual development taking place in vertebrate, and a sexual reproduction which occurs exclusively in felids and thereby is less studied. The developmental transitions rely on changes in gene expression patterns, and recent studies have assigned roles for chromatin shapers, including histone modifications, in establishing specific epigenetic programs for each given stage. Here, we identified T. gondii microrchidia (MORC) protein as an upstream transcriptional repressor of sexual commitment. We performed affinity purification coupled to tandem mass spectrometry analyses to identify its binding partners.

INSTRUMENT(S): LTQ Orbitrap Velos, Q Exactive

ORGANISM(S): Toxoplasma Gondii Pru Toxoplasma Gondii Rh

SUBMITTER: Yohann Couté

LAB HEAD: Virginie Brun

PROVIDER: PXD016846 | Pride | 2020-01-09

REPOSITORIES: Pride

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Publications

A MORC-driven transcriptional switch controls Toxoplasma developmental trajectories and sexual commitment.

Farhat Dayana C DC Swale Christopher C Dard Céline C Cannella Dominique D Ortet Philippe P Barakat Mohamed M Sindikubwabo Fabien F Belmudes Lucid L De Bock Pieter-Jan PJ Couté Yohann Y Bougdour Alexandre A Hakimi Mohamed-Ali MA

Nature microbiology 20200224 4

Toxoplasma gondii has a complex life cycle that is typified by asexual development that takes place in vertebrates, and sexual reproduction, which occurs exclusively in felids and is therefore less studied. The developmental transitions rely on changes in the patterns of gene expression, and recent studies have assigned roles for chromatin shapers, including histone modifications, in establishing specific epigenetic programs for each given stage. Here, we identified the T. gondii microrchidia (M ...[more]

PMID: 32094587

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Project description:The phylum Apicomplexa comprises an ancient group of early divergent eukaryotes, including some of the most deadly pathogens of medical and veterinary importance. Plasmodium species are responsible for malaria, which causes as many as 700,000 deaths per year, while Toxoplasma gondii chronically infects up to 30% of the human population, with immunocompromised patients and pregnant women at risk for adverse outcomes, such as toxoplasmic encephalitis and spontaneous abortion, respectively. T. gondii is considered a model system not only for its pathogenic relatives but also for intracellular parasitism and infection biology in general. T. gondii has common eukaryotic organelles, including the nucleus, endoplasmic reticulum (ER), and a single Golgi stack, but also specific secretory organelles named dense granules, micronemes, and rhoptries that contain parasite-derived factors required for host infection. Rhoptries and micronemes are formed de novo during parasite replication, and this process requires significant protein and lipid trafficking through the secretory pathway. The trafficking mechanisms employed by T. gondii retain several typical eukaryote components as well as evolving divergent features. Protein trafficking of this parasite is mediated by entry into a canonical ER followed by vesicle packaging through a single Golgi complex. Post-Golgi protein sorting to specific organelles requires the function of dynamin-related protein B, which is involved in fission events. Downstream Rab-GTPases function throughout the parasite secretory pathway. T. gondii soluble N-ethylmaleimide-sensitive-factor attachment protein receptor (SNARE) proteins in docking and fusion at target membranes have also been described. However, unlike in mammalian cells, T. gondii ER is reduced so that the nuclear envelope itself contributes to a substantial proportion of its total volume. Whereas in mammalian cells hundreds of Golgi stacks occupy the perinuclear area, the Golgi apparatus is limited to a single discrete structure in T.gondii. The post-Golgi system also named the endosome-like compartment (ELC) is involved in the trafficking of microneme proteins. The ELC is decorated by the small GTPases, Rab5 and Rab7, which are typically associated with the endosomal system. Nevertheless, classical endocytosis has not yet been validated in T. gondii. This parasite has no lysosomes; rather the parasite harbors acidic vesicles that were thought to be precursors of the rhoptry organelles. The parasite lacks most components of endosomal sorting complexes (ESCRT), which are known for their roles in forming multivesicular bodies that deliver ubiquitinated membrane proteins and lipids to lysosomes for degradation. The machinery required for caveogenesis and caveola-dependent invaginations have not yet been identified in the parasite. Furthermore, while evidence of conventional clathrin-dependent endocytosis by T.gondii is lacking, clathrin is present exclusively in post-Golgi compartments where its function is restricted to post-Golgi trafficking, and the uptake of cytosol proteins by the tachyzoites of T. gondii has recently been described using an endocytosis assay. However, the mechanisms underlying the events of this unconventional endocytosis in the parasite remain to be determined. Clearly, the secretory pathway of T. gondii can be considered a stripped-down version of the more complex trafficking machinery that characterizes higher eukaryotes. Despite this minimal trafficking machinery, the parasites actively rely on a membrane vesicle formation and transport during its intracellular lifecycle; however, to date, comparatively little is known about the mechanisms involved in trafficking pathways in T. gondii. We previously reported a T. gondii sortilin-like receptor (TgSORTLR) that regulates protein transport and is essential for apical secretory organelle biogenesis and host infection17. Moreover, the C-terminal tail of TgSORTLR was shown to be involved in recruiting many cytosolic cargo proteins including two homologues of the core retromer components, Vps26 and Vps35, which are known to regulate retrograde transport from endosomes to the trans-Golgi Network (TGN) in yeast and mammals. Here, we report that a singular architecture with a trimer Vps35-Vps26-Vps29 core complex acts as the major cargo endosomal recycling machinery and is required for parasite integrity and more specifically for secretory organelle biogenesis and maintenance of a multiple ligand-binding transporter at the T. gondii membrane. Our findings provide strong evidence that the unconventional TgSORTLR-containing endosome-like compartment is involved in distinct mechanisms for the delivery of major retromer-dependent cargo. In this current work, we demonstrate a definitive role for the endocytic recycling pathway in T. gondii pathogenesis.

Project description:Phosphorylation is a regulatory mechanism by which intracellular apicomplexan parasites control the process of invading and modifying host cells. However, the sporulated oocysts’ phosphoprotein repertoires between virulent and avirulent strains of Toxoplasma gondii and the underlying mechanisms contributing to virulence is still a mystery. A quantitative analysis of the sporulated oocysts’ phosphoproteome profile of T. gondii virulent and avirulent strains belonging to different genotypes was conducted to reveal phosphoprotein expression patterns that contribute to the virulence of a particular phenotype. Phosphopeptides from the sporulated oocysts of the virulent strain PYS (Chinese ToxoDB#9) and the avirulent strain type II (PRU strain) were enriched by titanium dioxide (TiO2) affinity chromatography and quantified using iBT technology. A total of 10,645 unique phosphopeptides, 8,181 nonredundant phosphorylation sites and 2,792 phosphoproteins were identified. The quantitative analysis identified 4,129 differentially expressed phosphopeptides (DEPs) between sporulated oocysts of the PYS strain and PRU strain (|log1.5 fold change| > 1 and P < 0.05), including 2,485 upregulated and 1,644 downregulated phosphopeptides. Motif analysis identified 24 motifs from the upregulated phosphorylated peptides including 22 serine motifs and two threonine motifs (TPE and TP), and 15 motifs from the downregulated phosphorylated peptides including 12 serine motifs and three threonine motifs (TP, RxxT and KxxT) in PYS strain when comparing PYS/PRU. Several kinases were consistent with motifs of overrepresented phosphopeptides, such as PKA, PKG, CKII, IKK, MAPK, EGFR, INSR, Jak, Syk, Src, Ab1. GO analysis, KEGG pathway analysis and STRING analysis revealed differentially expressed phosphoproteins (DEPs) exhibiting significant functional properties. Kinase associated network analysis showed that AGC kinase had the greatest connected peptides. Our findings reveal significant difference in phosphopeptides of sporulated oocysts between virulent and avirulent T. gondii strains, which provides solid foundation for further exploring the mechanisms contributing to the virulence of T. gondii.

Dataset Information

Mass spectrometry-based characterization of Toxoplasma gondii MORC interactome

Publications

A MORC-driven transcriptional switch controls Toxoplasma developmental trajectories and sexual commitment.

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