Abstract
Toxoplasma gondii maintains its intracellular life cycle using an extraordinary arsenal of parasite-specific organelles including the inner membrane complex (IMC), rhoptries, micronemes, and dense granules. While these unique compartments play critical roles in pathogenesis, many of their protein constituents have yet to be identified. We exploited the Vicia villosa lectin (VVL) to identify new glycosylated proteins that are present in these organelles. Purification of VVL-binding proteins by lectin affinity chromatography yielded a number of novel proteins that were subjected to further study, resulting in the identification of proteins from the dense granules, micronemes, rhoptries and IMC. We then chose to focus on three proteins identified by this approach, the SAG1 repeat containing protein SRS44, the rhoptry neck protein RON11 as well as a novel IMC protein we named IMC25. To assess function, we disrupted their genes by homologous recombination or CRISPR/Cas9. The knockouts were all successful, demonstrating that these proteins are not essential for invasion or intracellular survival. We also show that IMC25 undergoes substantial proteolytic processing that separates the C-terminal domain from the predicted glycosylation site. Together, we have demonstrated that lectin affinity chromatography is an efficient method of identifying new glycosylated parasite-specific proteins.
Highlights
Toxoplasma gondii is an obligate intracellular parasite in the phylum Apicomplexa that is capable of infecting any mammal and causes serious disease in immunocompromised individuals and congenitally infected neonates [1, 2]
To evaluate what structures Vicia villosa lectin (VVL) is labeling, we co-stained the parasites with markers for various organelles, including the inner membrane complex (IMC3), the micronemes (MIC2), the golgi (GRASP55), and the dense granules/ parasitophorous vacuole (GRA14) (Fig 1C)
In the apical end of the parasites, consistent with the rhoptries (Fig 1D). This was confirmed via co-localization with the rhoptry body protein ROP7, demonstrating that VVL does label this compartment under methanol fixation conditions
Summary
Toxoplasma gondii is an obligate intracellular parasite in the phylum Apicomplexa that is capable of infecting any mammal and causes serious disease in immunocompromised individuals and congenitally infected neonates [1, 2]. Other apicomplexans of medical importance in humans include Plasmodium falciparum, the causative agent of malaria and Cryptosporidium spp, which cause diarrhea in immunocompromised patients [3, 4]. The phylum Apicomplexa contains a wide array of animal pathogens such as Neospora caninum GL was the recipient of a PhD fellowship of the FRM (Fondation pour la Recherche Médicale). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript
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