Project description:Apicomplexan parasites, together with ciliates and dinoflagellates, belong to the Alveolata superphylum. Apicomplexa possess secretory organelles called rhoptries that undergo regulated exocytosis during invasion. Upon injection into the host cell, rhoptry proteins support invasion, vacuole formation, and subversion of host immune function. Rhoptry exocytosis involves a “rosette” of 8 particles embedded in the plasma membrane; such peculiar structure is also conserved in Ciliata and its formation requires Alveolata-restricted “Nd” proteins. These findings point to the existence of an Alveolata-conserved mechanism for the discharge of secretory organelles, and provide proof of concept that we can decipher rhoptry exocytosis machinery using a PAN-Alveolata approach. Thus, to uncover new rhoptry secretion factors, we used the transcriptional profiles of Nd genes of the ciliate Tetrahymena thermophila to select genes that are co-regulated, and also conserved in Apicomplexa. In this way we identified two uncharacterized Tetrahymena proteins that contribute to the exocytosis of secretory organelles. They have similar architecture and show homology with Plasmodium Cysteine Repeat Modular Proteins (CRMPs), a family of proteins essential for Plasmodium transmission from the mosquito to the host. We functionally characterized the two Toxoplasma CRMPs, including their dynamic location during invasion. They are not required for rosette formation, but they are essential for rhoptry secretion, suggesting a function distinct from that of previously characterized exocytic “Nd” factors. To characterize interacting proteins we performed pull-down experiments and analyzed the eluates by liquid chromatography-tandem mass spectrometry.
Project description:Apicomplexan parasites, together with ciliates and dinoflagellates, belong to the Alveolata superphylum. Apicomplexa possess secretory organelles called rhoptries that undergo regulated exocytosis during invasion. Upon injection into the host cell, rhoptry proteins support invasion, vacuole formation, and subversion of host immune function. Rhoptry exocytosis involves a “rosette” of 8 particles embedded in the plasma membrane; such peculiar structure is also conserved in Ciliata and its formation requires Alveolata-restricted “Nd” proteins. These findings point to the existence of an Alveolata-conserved mechanism for the discharge of secretory organelles, and provide proof of concept that we can decipher rhoptry exocytosis machinery using a PAN-Alveolata approach. Thus, to uncover new rhoptry secretion factors, we used the transcriptional profiles of Nd genes of the ciliate Tetrahymena thermophila to select genes that are co-regulated, and also conserved in Apicomplexa. In this way we identified two uncharacterized Tetrahymena proteins that contribute to the exocytosis of secretory organelles. They have similar architecture and show homology with Plasmodium Cysteine Repeat Modular Proteins (CRMPs), a family of proteins essential for Plasmodium transmission from the mosquito to the host. We functionally characterized the two Toxoplasma CRMPs, including their dynamic location during invasion. They are not required for rosette formation, but they are essential for rhoptry secretion, suggesting a function distinct from that of previously characterized exocytic “Nd” factors. To characterize interacting proteins we performed pull-down experiments and analyzed the eluates by liquid chromatography-tandem mass spectrometry.
Project description:The protozoan pathogen Toxoplasma gondii relies on tight regulation of gene expression to invade and establish infection in its host. The divergent gene regulatory mechanisms of Toxoplasma and related apicomplexan pathogens rely heavily on regulators of chromatin structure and histone modifications. The important contribution of histone acetylation for Toxoplasma in both acute and chronic infection has been demonstrated, where histone acetylation increases at active gene loci. However, the direct consequences of specific histone acetylation marks and the chromatin pathway that influences transcriptional regulation in response to the modification is unclear. As a reader of lysine acetylation, the bromodomain serves as a mediator between the acetylated histone and transcriptional regulators. Here we show that the bromodomain protein TgBDP1 which is conserved amongst Apicomplexa and within the Alveolata superphylum, is essential for Toxoplasma asexual proliferation. Using CUT&TAG we demonstrate that TgBDP1 is recruited to transcriptional start sites of a large proportion of parasite genes. Transcriptional profiling during TgBDP1 knockdown revealed that loss of TgBDP1 leads to major dysregulation of gene expression, implying multiple roles for TgBDP1 in both gene activation and repression. This is supported by interactome analysis of TgBDP1 demonstrating that TgBDP1 forms a core complex with two other bromodomain proteins and an ApiAP2 factor. This core complex appears to interact with other epigenetic factors such as nucleosome remodelling complexes. We conclude that TgBDP1 interacts with diverse epigenetic regulators to exert opposing influences on gene expression in the Toxoplasma tachyzoite.
Project description:The primary objective of this prospective observational study is to characterize the gut and oral microbiome as well as the whole blood transcriptome in gastrointestinal cancer patients and correlate these findings with cancer type, treatment efficacy and toxicity. Participants will be recruited from existing clinical sites only, no additional clinical sites are needed.
Project description: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.