Abstract
A characteristic of the latent cyst stage of Toxoplasma gondii is a thick cyst wall that forms underneath the membrane of the bradyzoite vacuole. Previously, our laboratory group published a proteomic analysis of purified in vitro cyst wall fragments that identified an inventory of cyst wall components. To further refine our understanding of the composition of the cyst wall, several cyst wall proteins were tagged with a promiscuous biotin ligase (BirA*), and their interacting partners were screened by streptavidin affinity purification. Within the cyst wall pulldowns, previously described cyst wall proteins, dense granule proteins, and uncharacterized hypothetical proteins were identified. Several of the newly identified hypothetical proteins were validated to be novel components of the cyst wall and tagged with BirA* to expand the model of the cyst wall interactome. Community detection of the cyst wall interactome model revealed three distinct clusters: a dense granule, a cyst matrix, and a cyst wall cluster. Characterization of several of the identified cyst wall proteins using genetic strategies revealed that MCP3 affects in vivo cyst sizes. This study provides a model of the potential protein interactions within the cyst wall and the groundwork to understand cyst wall formation.IMPORTANCE A model of the cyst wall interactome was constructed using proteins identified through BioID. The proteins within this cyst wall interactome model encompass several proteins identified in a prior characterization of the cyst wall proteome. This model provides a more comprehensive understanding of the composition of the cyst wall and may lead to insights on how the cyst wall is formed.
Highlights
A characteristic of the latent cyst stage of Toxoplasma gondii is a thick cyst wall that forms underneath the membrane of the bradyzoite vacuole
Under bradyzoite-inducing conditions without supplemental biotin, the BirA* tag fused to BPK1, MCP4, MAG1, or GRA6 showed no activity (Fig. S1B and C); the CST1-BirA* strain showed selfbiotinylation of the CST1 protein (Fig. S1C)
When exogenous 150 M biotin was supplemented to the bradyzoite-inducing medium, the BirA* tag fused to MAG1, MCP4, BPK1, CST1, and GRA6 displayed biotinylation activity as an increase in streptavidin signal that was observed at the cyst wall compared to that in the Pru strain under immunofluorescence assays (IFAs) (Fig. 1B)
Summary
A characteristic of the latent cyst stage of Toxoplasma gondii is a thick cyst wall that forms underneath the membrane of the bradyzoite vacuole. The cyst wall is an electrondense structure that consists of an outer condensed sponge-like layer that transitions into a looser layer extending into the cyst matrix [3] Proteins such as MAG1 [4], MCP4 [5], BCP1 [6], and various dense granule proteins [7] have been shown to localize to the cyst wall. (Pru) was completed using tandem mass spectrometry of immunoprecipitated in vitro cyst wall membrane fragments [14] This analysis yielded an inventory of previously undescribed cyst wall proteins (CST2/GRA50, CST3/GRA51, CST4, CST5/GRA52, and CST6/GRA53) that were validated to localize to the cyst wall by immunofluorescence. Two of these novel cyst wall proteins were further characterized, which revealed that CST2 plays a role in parasite virulence. The molecular interactions of the components at the cyst wall remain unclear
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