Dataset Information

MS-based proteomic characterization of Toxoplasma gondii BCC7 interactome

ABSTRACT: Toxoplasma gondii is an obligate intracellular protozoan parasite of a wide range of homeotherm animals worldwide. T. gondii belongs to the ancient phylum of Apicomplexa and shares with the other companion groups from the Alveolata infrakingdom, a featuring trilamellar pellicle outlining the cell while a permanent apico-basal polarity also characterizes the T. gondii zoites. In addition to the plasma membrane bilayer, the pellicle also encompasses the underneath inner membrane complex (IMC) itself derived from flattened and fused vesicles identified as alveoli and made of a IMC and IMC-like protein’s repertoire, yet to complete. We identified a 500 kDa protein encoded by TGGT1_311230, the basal complex component BCC7, exhibiting a posterior localization. To gain insight about the role of BCC7 in T. gondii, we characterized its interactome.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human) Toxoplasma Gondii Rh

SUBMITTER: Yohann Couté

LAB HEAD: Yohann Couté

PROVIDER: PXD032362 | Pride | 2022-07-25

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

Items per page:

1 - 5 of 14

Publications

The BCC7 Protein Contributes to the <i>Toxoplasma</i> Basal Pole by Interfacing between the MyoC Motor and the IMC Membrane Network.

Vigetti Luis L Labouré Tatiana T Roumégous Chloé C Cannella Dominique D Touquet Bastien B Mayer Claudine C Couté Yohann Y Frénal Karine K Tardieux Isabelle I Renesto Patricia P

International journal of molecular sciences 20220526 11

<i>T. gondii</i> is a eukaryotic parasite that has evolved a stage called tachyzoite which multiplies in host cells by producing two daughter cells internally. These nascent tachyzoites bud off their mother and repeat the division process until the expanding progenies escape to settle and multiply in other host cells. Over these intra- and extra-cellular phases, the tachyzoite maintains an essential apicobasal polarity that emerges through a unique bidirectional budding process of the elongating ...[more]

PMID: 35682673

Similar Datasets

Project description:The population structure of Toxoplasma gondii includes three highly prevalent clonal lineages, types I, II, and III, which differ greatly in virulence in the mouse model. Previous studies have implicated a family of serine threonine protein kinases found in rhoptries (ROPs) as important in mediating virulence differences between types I vs. III and II vs. III. Here, we explored the genetic basis of differences in virulence between the highly virulent type I lineage and moderately virulent type II based on a new genetic cross and linkage mapping. Genome-wide association revealed a single quantitative trait locus controls the > 4 log difference in lethality between these strains. Neither ROP16 nor ROP18, previously implicated in virulence differences in T. gondii, were found to contribute to differences between types I and II. Instead, the major virulence locus contained a cluster of pseudokinases denoted as rhoptry protein 5 (ROP5); this locus contains a tandem cluster of polymorphic alleles that differed in expression levels between strains. ROP5 alleles contained only part of the catalytic triad of canonical S/T kinases, and consistent with this they lack demonstrable kinase activity in vitro. Genetic disruption of the rop5 locus in the type I lineage lead to a > 5 log increase in the lethal dose, and surviving mice developed lasting immunity and were protected from an otherwise lethal challenge. These findings reveal that amplification of a polymorphic cluster of pseudokinases plays an important role in pathogenesis of toxoplasmosis in the mouse model. We used a new genotyping protocol based on hybridization of parental and recombinant progeny genomic DNA (gDNA) to Affymetrix gene arrays constructed for T. gondii (http://ancillary.toxodb.org/docs/Array-Tutorial.html). We identified 1,603 single feature polymorphisms (SFP) based on probes that successfully discriminated perfect match (type II) from mismatch (type I) hybridization across all clones and provided reliable SFP markers for this cross.

Project description:Toxoplasma gondii is a widely prevalent parasite of animals and humans that causes a broad spectrum of conditions ranging from asymptomatic or mild cold-like infections to abortion, severe ocular disease, encephalitis, and sometimes death. It is a member of the phylum Apicomplexa that also includes such significant human pathogens as Plasmodium spp. and Cryptosporidium spp. causing malaria and diarrheal disease, respectively. The toxoplasma cell has a multilayered pellicle: under the plasma membrane lies the inner membrane complex (IMC) comprised of flat membrane sacks supported by cortical microtubules and a meshwork of cytoskeletal filaments. The IMC acts as a mechanical barrier for membrane vesicles trafficking to and from the plasma membrane, necessitating the existence of dedicated cites for endo- and exocytosis where the IMC is interrupted. One such site is located at the apical end of the cell where the contents of the apical secretory organelles is exocytosed upon invasion. However, endocytosis in Toxoplasma and other apicomplexans is poorly understood, although conspicuous invaginations of the plasma membrane observed microscopically have been dubbed the micropore and postulated to be involved in endocytosis. We found that the endocytic markers including the subunits α and μ of the AP2 adaptor complex, dynamin-related protein C (DrpC), an Eps15 homology domain-containing protein, and the kelch-domain protein K13 localise to distinct persistent foci in the pellicle of Toxoplasma. Another type of persistent punctate structures found in the IMC are the enigmatic apical annuli whose function is completely unknown. We have discovered a new component of the annuli which is a putative LMBR1 family region protein embedded into the plasma membrane. Our experiments indicate that this protein – and annuli by extension – has a role in exocytosis. To better understand the molecular composition of these endocytic and exocytic structures and gain an insight into their function, we genetically fused them with a promiscuous bacterial biotin-ligase BirA* and used them as baits in proximity-dependent biotin identification experiments (BioID). We employed TMT10plex labelling and MS3-level tandem mass spectrometry with synchronous precursor selection (SPS-MS3) to achieve accurate quantification of multiple samples. Several hundred proteins have been identified and quantified in three biological replicates for every bait and the parental cell line control. Proteins significantly enriched in the BirA*-tagged cells relative to the control have been identified using linear modelling with LIMMA.

Project description:The outcome of infections with Toxoplasma gondii in humans is dependent in part on the genetic makeup of the infecting organism. Recent studies have indicated that most infecting Toxoplasma organisms fall into 1 of 3 canonical lineages. Previous studies have investigated the effects of Toxoplasma on its host cell transcriptome. Little is known, however, about the effects of three canonical lineages on brain cells, the principal site of parasite lifelong persistence. In this study, we examined the transcriptional profile of human neuroepithelioma cells in response to T. gondii infection using microarray analysis to characterize the strain-specific host cell response to 3 canonical T. gondii strains. We found that the extent of the expression changes varied considerably among the three strains. Neuroepithelial cells infected with type I exhibited the most differential gene expression, whereas type II infected cells had a substantially smaller number of genes which were differentially expressed. Cells infected with type III exhibited intermediate effects on gene expression. The three strains also differed in the individual genes and gene pathways which were altered following cellular infection. For example, gene ontology (GO) analysis indicated that type I infection largely affects genes related to central nervous system while type III infection largely alters genes which affect nucleotide metabolism; type II infection does not alter expression of a clearly defined set of genes. Moreover, Ingenuity pathway analysis (IPA) revealed the sophistication of different strain in its interactions with the host. These differences may explain some of the variation in the neurobiological effects of different strains of Toxoplasma on infected individuals. We infected SK-N-MC cells with 3 canonical Toxoplasma strains and assessed their gene expression in RNA at 20 hours post-infection using Affymetrix GeneChip. Infections and controls (no tachyzoites) were performed in duplicate and experiments for each strain were carried out on three separate occasions in order to include both technical replicates and biological replicates.

Project description:Intracellular pathogens including the apicomplexan and opportunistic parasite Toxoplasma gondii profoundly modify their host cells in order to establish infection. We have shown previously that intracellular T. gondii inhibit up-regulation of regulatory and effector functions in murine macrophages (MΦ) stimulated with interferon (IFN)-γ, which is the cytokine crucial for controlling the parasites’ replication. Using genome-wide transcriptome analysis we show herein that infection with T. gondii leads to global unresponsiveness of murine macrophages to IFN-γ. More than 61% and 89% of the transcripts, which were induced or repressed by IFN-γ in non-infected MΦ, respectively, were not altered after stimulation of T. gondii-infected cells with IFN-γ. These genes are involved in a variety of biological processes, which are mostly but not exclusively related to immune responses. Analyses of the underlying mechanisms revealed that IFN-γ-triggered nuclear translocation of STAT1 still occurred in Toxoplasma-infected MΦ. However, STAT1 bound aberrantly to oligonucleotides containing the IFN-γ-responsive gamma-activated site (GAS) consensus sequence. Conversely, IFN-γ did not induce formation of active GAS-STAT1 complexes in nuclear extracts from infected MΦ. Mass spectrometry of protein complexes bound to GAS oligonucleotides showed that T. gondii-infected MΦ are unable to recruit non-muscle actin to IFN-γ-responsive DNA sequences, which appeared to be independent of stimulation with IFN-γ and of STAT1 binding. IFN-γ-induced recruitment of BRG-1 and acetylation of core histones at the IFN-γ-regulated CIITA promoter IV, but not β-actin was diminished by >90% in Toxoplasma-infected MΦ as compared to non-infected control cells. Remarkably, treatment with histone deacetylase inhibitors restored the ability of infected macrophages to express the IFN-γ regulated genes H2-A/E and CIITA. Taken together, these results indicate that Toxoplasma-infected MΦ are unable to respond to IFN-γ due to disturbed chromatin remodelling, but can be rescued using histone deacetylase inhibitors. Comparison of 4 different RNA pools with a 2-Color-Loop Design including 10 microarrays: [1] T. gondii infected and IFN-gamma treated, [2] T. gondii infected and untreated, [3] Non-infected and IFN-gamma treated, and [4] Non-infected and untreated.

			Action	DRS
	BCC7-R1.mgf	Mgf
	BCC7-R1.raw	Raw
	BCC7-R2.mgf	Mgf
	BCC7-R2.raw	Raw
	BCC7-R3.mgf	Mgf