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Protein composition of Toxoplasma endocytic and exocytic structures by proximity-dependent biotinylation

ABSTRACT: 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.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human) Bos Taurus (bovine) Toxoplasma Gondii Rh

TISSUE(S): Tachyzoite, Foreskin Fibroblast Cell Line, Cell Culture

SUBMITTER: Konstantin Barylyuk

LAB HEAD: Ross F. Waller

PROVIDER: PXD034193 | Pride | 2023-04-17

REPOSITORIES: Pride

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Project description:Toxoplasma gondii is an obligate intracellular parasite. The ability to invade cells and proliferate successfully is given by the machinery found in the pellicle of the parasite, the pellicle is a complex of three membranes. Despite the importance of the pellicle in the biology of Toxoplasma, its protein composition had not been reported. The proteins of the isolated pellicle fraction were digested with trypsin and chymotrypsin, for their identification and quantification in the case of those digested with trypsin and only identification in the case of those digested with chymotrypsin. Samples were resolved, and peptides were separated using a UHPLC ACQUITY M-Class. Spectral data were acquired in an MS with electrospray ionization and ion mobility separation Synapt G2-Si operated with data-independent acquisition and ion mobility spectrometry using the high-definition multiplexed MS/MS mode. We used the Hi3 method to quantify absolutely every protein detected. In the isolate of pellicle digested with trypsin a total of 548 proteins were identified and absolutely quantified using the internal standard and the intensity of the most abundant peptides per protein with Progenesis QI software and the database UP000002494. Among identified proteins, 11 corresponded to IMC, 11 were related to SAG (SRS), 15 to plasma membrane and glideosome and 13 to cytoskeleton (CK). In the chymotrypsin-digested pellicle sample a total of 137 proteins were identified and 22 of them were proteins that were not identified in the trypsin-digested sample. Due to the characteristics of the SRS proteins, they have a role in generating the host's immune response or are involved in adhesion, motility and invasion. The 11 proteins found with the analysis of E. mass. Study of these SRS proteins to determine their role in the dynamic events of the parasite, such as adhesion and motility as well as in the generation of the immune response, is important in the search for therapies against toxoplasmosis

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	F002843.dat	Other
	LK01.raw	Raw
	LK02.raw	Raw
	LK03.raw	Raw
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Protein composition of Toxoplasma endocytic and exocytic structures by proximity-dependent biotinylation

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