Conservation, abundance, glycosylation profile, and localization of the TSP protein family in Cryptosporidium parvum

John, Alan; Bader, Stefanie; Soler, Niccolay Madiedo; Wiradiputri, Kharizta; Tichkule, Swapnil; Smyth, Sean T.; Ralph, Stuart A.; Jex, Aaron R.; Scott, Nichollas E.; Tonkin, Christopher J.; Goddard‐Borger, Ethan D.

doi:10.1016/j.jbc.2023.103006

Cited by 6 publications

(7 citation statements)

References 81 publications

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“…The C. parvum genome encodes 12 TSP-family proteins (CpTSP1 to 12) (see a list of CpTSP proteins in Table S1 in the supplemental material) ( 19 , 20 ). Among them, CpTSP1 (aka TRAP-C1) and CpTSP8 were localized to the apical region and/or surface of excysted sporozoites ( 20 – 22 ).…”

Section: Resultsmentioning

confidence: 99%

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

Wang,

Jiang,

et al. 2024

mBio

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The zoonotic Cryptosporidium parvum is a global contributor to infantile diarrheal diseases and opportunistic infections in immunocompromised or 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 in C. parvum , namely, thrombospondin (TSP)-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 to Cryptosporidium . The secretion and microtubular distribution could be completely blocked by the 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 the host cell surface, for which heparin acts as one of the host ligands. A novel heparin-binding motif is identified and validated biochemically 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. IMPORTANCE Cryptosporidium parvum is 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 of 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 of C. parvum sporozoites. The CpTSP4 secretion depends on the intracellular trafficking on the two Cryptosporidium -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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Section: Resultsmentioning

confidence: 99%

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

Wang,

Jiang,

et al. 2024

mBio

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“…CpTSP4 is prestored in micronemes, but transported along the two central microtubules for secretion in sporozoites during and after excystation. The C. parvum genome encodes 12 TSP-family proteins (CpTSP1 to 12) (see Table S1 in the supplemental material) (19,20).…”

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

“…The C. parvum genome encodes 12 TSP-family proteins (CpTSP1 to 12) (see Table S1 in the supplemental material) (19, 20). Among them, CpTSP1 (aka TRAP-C1) and CpTSP8 were localized to the apical region and/or surface in excysted sporozoites (20–22). This study focused on CpTSP4 (Gene ID: cgd8_150; Genbank: XP_625479) based on its moderate size (488 aa) and relatively simple domain architecture, i.e., an N-terminal signal peptide (SP; 25 aa), a single PAN/APPLE domain, and two TSP1-like repeats, but lacking a transmembrane domain (TMD) (Fig.…”

Section: Resultsmentioning

confidence: 99%

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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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“…Apicomplexa are a phylum of largely parasitic single-celled organisms that include significant causes ofdisease in humans and livestock, such as malaria, toxoplasmosis and cryptosporidiosis. To date, U-ExM has been used to study the cell biology of Plasmodium 6,14,15 , Toxoplasma 16,17 , Cryptosporidium 7,18 , and Neospora 19 . To date, U-ExM has only been applied to parasites cultured in vitro and prepared as either isolated parasites or infected-cell monolayers.…”

Section: Introductionmentioning

confidence: 99%

Mosquito Tissue Ultrastructure-Expansion Microscopy (MoTissU-ExM) enables ultrastructural and anatomical analysis of malaria parasites and their mosquito

Liffner,

Alves e Silva,

Vega-Rodriguez

et al. 2024

Preprint

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Study of malaria parasite cell biology is challenged by their small size, which can make visualisation of individual organelles difficult or impossible using conventional light microscopy. In recent years, the field has attempted to overcome this challenge through the application of ultrastructure expansion microscopy (U-ExM), which physically expands a biological sample approximately 4.5-fold. To date, U-ExM has mostly been used to visualise blood-stage parasites and used exclusively on parasites in vitro. Here we develop Mosquito Tissue U-ExM (MoTissU-ExM), a method for preparing dissected mosquito salivary glands and midguts by U-ExM. MoTissU-ExM preserves both host and parasite ultrastructure, enabling visualisation of oocysts and sporozoites in situ. We validate that MoTissU-ExM samples expand as expected, provide a direct comparison of the same dissected tissues before and after MoTissU-ExM, and highlight some of the key host and parasite structures that can be visualised following MoTissU-ExM. Finally, we provide a point-by-point protocol for how to perform MoTissU-ExM, along with details on how best to image the expanded tissues, and how to troubleshoot common issues.

show abstract

Conservation, abundance, glycosylation profile, and localization of the TSP protein family in Cryptosporidium parvum

Cited by 6 publications

References 81 publications

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

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

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

Mosquito Tissue Ultrastructure-Expansion Microscopy (MoTissU-ExM) enables ultrastructural and anatomical analysis of malaria parasites and their mosquito

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