Dataset Information

Toxoplasma conoid proteome by BioID

ABSTRACT: Toxoplasma gondii, a widely prevalent human and animal pathogen and a relative of the malaria-causing Plasmodium spp., is a member of the phylum Apicomplexa encompassing a large number of single-celled eukaryotic organisms that are obligate endoparasites of animals. Apicomplexans evolved multiple adaptations to parasitism. One such distinctive feature of the apicomplexan cell, which the phylum is named after, is the apical complex comprising a battery of secretory vesicles and unusual cytoskeletal structures. The latter include the conoid, apical polar rings, and sub-pellicular microtubules. Functions of the apical cytoskeletal structures are poorly understood due to incomplete knowledge of their molecular composition. Furthermore, the conoid is believed to be heavily reduced or missing from Plasmodium species and other members of the class Aconoidasida. We have applied a spatial proteomic method called proximity-dependent biotin identification, BioID, to identify conoid-associated proteins in the model apicomplexan Toxoplasma gondii. We chose three proteins located at the apex of the T. gondii cell as BioID baits fused with the promiscuous biotin-ligase BirA*: SAS6L at the conoid, RNG2 linking the conoid and one of the apical polar rings, and MORN3 distributed at the apical subdomain of the inner membrane complex but excluded from the vicinity of the conoid. The enrichment of biotinylated proteins on streptavidin matrix in the BioID-bait cells relative to the untransformed parental cell line control was determined using a shotgun proteomic approach with label-free quantitation. The location of conoid protein candidates prioritised by BioID has been further validated by fluorescence microscopy in T. gondii tachyzoites and several life-cycle stages of P. berghei. Collectively we show that the conoid is a conserved apicomplexan element at the heart of the invasion mechanisms of these highly successful and often devastating parasites.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Toxoplasma Gondii Rh

TISSUE(S): Tachyzoite

SUBMITTER: Konstantin Barylyuk

LAB HEAD: Ross F. Waller

PROVIDER: PXD022785 | Pride | 2021-09-09

REPOSITORIES: Pride

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Publications

Molecular characterization of the conoid complex in Toxoplasma reveals its conservation in all apicomplexans, including Plasmodium species.

Koreny Ludek L Zeeshan Mohammad M Barylyuk Konstantin K Tromer Eelco C EC van Hooff Jolien J E JJE Brady Declan D Ke Huiling H Chelaghma Sara S Ferguson David J P DJP Eme Laura L Tewari Rita R Waller Ross F RF

PLoS biology 20210311 3

The apical complex is the instrument of invasion used by apicomplexan parasites, and the conoid is a conspicuous feature of this apparatus found throughout this phylum. The conoid, however, is believed to be heavily reduced or missing from Plasmodium species and other members of the class Aconoidasida. Relatively few conoid proteins have previously been identified, making it difficult to address how conserved this feature is throughout the phylum, and whether it is genuinely missing from some ma ...[more]

PMID: 33705380

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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: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
	P062_2_95.dat	Other
	P062_2_95.mgf	Mgf
	P062_2_95.mzid.gz	Mzid
	P062_2_95.mzid_P062_2_95.MGF	Mzid
	P062_2_95.raw	Raw