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Cryptosporidium parvum rhomboid1 has an activity in microneme protein CpGP900 cleavage
Mingying Li1, Xichen Zhang1, Pengtao Gong1
1Key Laboratory of Zoonosis, Ministry of Education, College of Veterinary Medicine, Jilin University, Changchun, 130062, China.
Background:
Apicomplexan parasites actively release transmembrane (TM) adhesive proteins involved in host cell attachment and invasion. Rhomboids, a family of intramembrane serine proteases, cleave these secreted adhesive proteins within their TM domains as an essential step in completing the invasion process. In Cryptosporidium parvum, the activity of rhomboids in cleaving microneme proteins (MICs) has not been reported. In the present study, the interaction between C. parvum rhomboids (CpROM1 and CpROM4) and C. parvum microneme proteins (CpGP900 and CpTRAP-C1) was investigated using yeast two-hybrid assay and co-immunoprecipitation assays.
Results:
Our study demonstrated that CpROM1 protein could interact with CpGP900 protein in co-transformed AH109 yeasts. Analysis of these proteins in co-transfected mammalian cells showed that the cleavage product of the CpGP900 protein was detected in the co-transfected cells. As control, CpGP900 only was transfected into cells and no cleavage was observed. The results suggested that CpGP900 protein was the substrate of CpROM1. Moreover, CpROM1 and CpROM4 could not cleave CpTRAP-C1 protein, which is the substrate of T. gondii rhomboid 2.
Conclusions:
Our results showed that CpROM1 is an active protease that is involved in microneme protein CpGP900 cleavage, which lay the foundation for further research on the mechanisms of C. parvum invasion.
Insights
Cryptosporidium parvum rhomboid 1 (CpROM1) cleaves the microneme protein CpGP900, aiding parasite invasion. This study identifies CpROM1 as an active protease essential for C. parvum host cell attachment and invasion mechanisms.
Area of Science:
- Parasitology
- Molecular Biology
- Biochemistry
Background:
- Apicomplexan parasites utilize transmembrane adhesive proteins for host cell invasion.
- Rhomboid proteases are crucial for cleaving these proteins within their transmembrane domains.
- The role of rhomboids in cleaving microneme proteins in Cryptosporidium parvum was previously uncharacterized.
Purpose of the Study:
- To investigate the interaction between Cryptosporidium parvum rhomboids (CpROM1, CpROM4) and microneme proteins (CpGP900, CpTRAP-C1).
- To determine if C. parvum rhomboids are involved in the cleavage of C. parvum microneme proteins.
Main Methods:
- Yeast two-hybrid assays were employed to assess protein-protein interactions.
- Co-immunoprecipitation assays were used to validate interactions in a cellular context.
- Analysis of protein cleavage products in co-transfected mammalian cells.
Main Results:
- CpROM1 was found to interact with CpGP900 in yeast and mammalian cells.
- Cleavage of CpGP900 was observed in cells co-expressing CpROM1, indicating CpGP900 is a substrate.
- CpROM1 and CpROM4 did not cleave CpTRAP-C1, suggesting substrate specificity.
Conclusions:
- CpROM1 functions as an active protease that cleaves the microneme protein CpGP900.
- This cleavage is a key step in the invasion process of Cryptosporidium parvum.
- The findings provide a basis for further research into C. parvum invasion mechanisms.
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