Cryptosporidium uses multiple distinct secretory organelles to interact with and modify its host cell

Guérin, Amandine; Strelau, Katherine M.; Barylyuk, Konstantin; Wallbank, Bethan A.; Berry, Laurence; Crook, Oliver M.; Lilley, Kathryn S.; Waller, Ross F.; Striepen, Boris

doi:10.1016/j.chom.2023.03.001

Cited by 35 publications

(23 citation statements)

References 70 publications

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“…These secretory vesicles are key in the formation of the parasitophorous vacuole in which the parasite resides. It was recently discovered that the related parasite Cryptosporidium boasts a small granule as a subset of the dense granules [112] and the question remains of whether in Toxoplasma dense granules also form a heterogeneous population of secretory compartments [113]. We consider the jointly inferred clusters of the transcripts corresponding to these proteins in the transcriptomic data, and find that the majority of these proteins are highly expressed towards the G/S transition stage of the cell cycle.…”

Section: Resultsmentioning

confidence: 99%

Semi-supervised Bayesian integration of multiple spatial proteomics datasets

Coleman,

Breckels,

Waller

et al. 2024

Preprint

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The subcellular localisation of proteins is a key determinant of their function. High-throughput analyses of these localisations can be performed using mass spectrometry-based spatial proteomics, which enables us to examine the localisation and relocalisation of proteins. Furthermore, complementary data sources can provide additional sources of functional or localisation information. Examples include protein annotations and other high-throughput `omic assays. Integrating these modalities can provide new insights as well as additional confidence in results, but existing approaches for integrative analyses of spatial proteomics datasets are limited in the types of data they can integrate and do not quantify uncertainty. Here we propose a semi-supervised Bayesian approach to integrate spatial proteomics datasets with other data sources, to improve the inference of protein sub-cellular localisation. We demonstrate our approach outperforms other transfer-learning methods and has greater flexibility in the data it can model. To demonstrate the flexibility of our approach, we apply our method to integrate spatial proteomics data generated for the parasite Toxoplasma gondii with time-course gene expression data generated over its cell cycle. Our findings suggest that proteins linked to invasion organelles are associated with expression programs that peak at the end of the first cell-cycle. Furthermore, this integrative analysis divides the dense granule proteins into heterogeneous populations suggestive of potentially different functions. Our method is disseminated via the mdir R package available on the lead author's Github.

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Section: Resultsmentioning

confidence: 99%

Semi-supervised Bayesian integration of multiple spatial proteomics datasets

Coleman,

Breckels,

Waller

et al. 2024

Preprint

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“…These include CpGP900 (cgd7_4020), TRAPC1 (cgd1_3500), CpTSP8 (cgd6_780), CpROM1 (cgd3_980), cgd1_3550 (APPLE domain-containing protein), cgd2_1590 (APPLE/EGF-like domain-containing protein), and cgd7_1960 (WD40/YVTN repeat-like domain-containing protein) (9)(10)(11)23). Among them, cgd7_1960 was more recently discovered as one of the proteins in the "microneme cluster" by HyperLOPIT technology and confirmed by IFA of hemagglutinin (HA)-tagged transgenic C. parvum (11). This HyperLOPIT-based study identifies several new candidate micronemal proteins based on their clustering with known microneme markers (e.g., CpGP900).…”

Section: Discussionmentioning

confidence: 99%

“…Toxoplasma and Plasmodium possess an array of cortical/subpellicular microtubules (SPMTs) for maintaining the shape of zoites (16), while currently there is no evidence that SPMTs are involved in intracellular trafficking of secretory molecules. The two short intraconoidal microtubules, unseen in Cryptosporidium , are implied for transporting “microtubule-associated vesicles” to the apical tip during the rhoptry docking and exocytosis (6, 8, 11). The F-actin, which is part of the glideosome in gliding zoites and the moving junction formed during the parasite invasion, is implied in apical positioning of rhoptries (17).…”

Section: Introductionmentioning

confidence: 99%

Requirement of microtubules for secretion of a micronemal protein CpTSP4 in the invasive stage of the apicomplexanCryptosporidium parvum

Wang,

Jiang,

et al. 2023

Preprint

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The zoonoticCryptosporidium parvumis a global contributor to infantile diarrheal diseases and opportunistic infections in immunocompromised/weakened individuals. Like other apicomplexans, it possesses several specialized secretory organelles, including micronemes, rhoptry and dense granules. However, the understanding of cryptosporidial micronemal composition and secretory pathway remains limited. Here we report a new micronemal protein inC. parvum, namely thrombospondin repeat domain-containing protein-4 (CpTSP4), providing insights into these ambiguities. Immunostaining and enzyme-linked assays show that CpTSP4 is prestored in the micronemes of unexcysted sporozoites but secreted during sporozoite excystation, gliding and invasion. In excysted sporozoites, CpTSP4 is also distributed on the two central microtubules unique toCryptosporidium. The secretion and microtubular distribution could be completely blocked by selective kinesin-5 inhibitors, SB-743921 and SB-715992, resulting in the accumulation of CpTSP4 in micronemes. These support the kinesin-dependent microtubular trafficking of CpTSP4 for secretion. We also localize γ-tubulin, consistent with kinesin-dependent anterograde trafficking. Additionally, recombinant CpTSP4 displays nanomolar binding affinity to host cell surface, for which heparin acts as one of the host ligands. A novel heparin-binding motif is identified and biochemically validated for its contribution to the adhesive property of CpTSP4 by peptide competition assays and site-directed mutagenesis. These findings shed light on the mechanisms of intracellular trafficking and secretion of a cryptosporidial micronemal protein and the interaction of a TSP-family protein with host cells.ImportanceCryptosporidium parvumis a globally distributed apicomplexan parasite infecting humans and/or animals. Like other apicomplexans, it possesses specialized secretory organelles in the zoites, in which micronemes discharge molecules to facilitate the movement and invasion of zoites. Although past and recent studies have identified several proteins in cryptosporidial micronemes, our understanding on the composition, secretory pathways and domain-ligand interactions of micronemal proteins remains limited. This study identifies a new micronemal protein, namely CpTSP4, that is discharged during excystation, gliding and invasion ofC. parvumsporozoites. The CpTSP4 secretion depends on the intracellular trafficking on the twoCryptosporidium-unique microtubes that could be blocked by kinesin-5/Eg5 inhibitors. Additionally, a novel heparin-binding motif is identified and biochemically validated, which contributes to the nanomolar binding affinity of CpTSP4 to host cells. These findings indicate that kinesin-dependent microtubular trafficking is critical to CpTSP4 secretion and heparin/heparan sulfate is one of the ligands for this micronemal protein.

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“…The insertion of the selected gRNA sequences into the vector was performed by VectorBuilder. The gRNA library (200 μg) was linearized with NotI and transfected into approximately 1-2×10 8 RHΔhxgprt parasites divided between four separate cuvettes. Then, transfected parasites were grown in 4×150-mm dishes with confluent Vero cell monolayers.…”

Section: In Vitro and In Vivo Pooled Crispr Screensmentioning

confidence: 99%

In vivoCRISPR screens identify novel virulence genes among proteins of unassigned subcellular localization inToxoplasma

Tachibana,

Sasai,

Yamamoto

2024

Preprint

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The research field to identify and characterize virulence genes in Toxoplasma gondii has been dramatically advanced by a series of in vivo CRISPR screens. Although subcellular localizations of thousands of proteins were predicted by the spatial proteomic method called hyperLOPIT, those of more than 1000 proteins remained unassigned and their essentiality in virulence was also unknown. In this study, we generated two small-scale gRNA libraries targeting approximately 600 hyperLOPIT-unassigned proteins and performed in vivo CRISPR screens. As a result, we identified several in vivo fitness-conferring genes that were previously unreported. We further characterized two candidates, TgGTPase and TgRimM, which are localized in the cytoplasm and the apicoplast, respectively. Both genes are essential for parasite virulence and widely conserved in the phylum Apicomplexa. Collectively, our current study provides a resource for estimating the in vivo essentiality of Toxoplasma proteins with previously unknown localizations.

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Cryptosporidium uses multiple distinct secretory organelles to interact with and modify its host cell

Cited by 35 publications

References 70 publications

Semi-supervised Bayesian integration of multiple spatial proteomics datasets

Semi-supervised Bayesian integration of multiple spatial proteomics datasets

Requirement of microtubules for secretion of a micronemal protein CpTSP4 in the invasive stage of the apicomplexanCryptosporidium parvum

In vivoCRISPR screens identify novel virulence genes among proteins of unassigned subcellular localization inToxoplasma

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