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Published on: September 14, 2019
Comparative Study of Two Insulinlike Proteases in Cryptosporidium parvum
Wei He1, Cong Lai2, Fuxian Yang1
1Center for Emerging and Zoonotic Diseases, College of Veterinary Medicine, South China Agricultural University, Guangzhou, Guangdong 510642, China.
Abstract:
Cryptosporidiumparvum is a common protozoan pathogen responsible for moderate-to-severe diarrhea in humans and animals. The small genome of C. parvum has 22 genes encoding insulinlike proteases (INS) with diverse sequences, suggesting that members of the protein family may have different biological functions in the life cycle. In this study, two members of the INS family, CpINS-4 and CpINS-6 with the Zn2+-binding motif "HXXEH" but different numbers of function domains, were expressed in Escherichia coli and used in the generation of polyclonal antibodies. In both recombinant and native proteins, CpINS-4 and CpINS-6 were spliced into multiple fragments. The antibodies generated recognized their respective recombinant and native proteins and the spliced products, but had minimum cross-reactivity with each other. Anti-CpINS-4 antibodies reacted with the middle region of sporozoites and merozoites, while CpINS-6 had the highest reactivity to the apical region. Polyclonal anti-CpINS-4 antibodies produced 36% reduction in parasite load in HCT-8 cultures at 24 h, while those against CpINS-6, which has one of the function domains missing, failed in doing so. The genes encoding both CpINS-4 and CpINS-6 had the highest expression in the invasion phase of in vitro C. parvum culture. These data suggest that CpINS-4 and CpINS-6 might be expressed in different organelles and play different biological functions in the life cycle of C. parvum.
Insights
Two Cryptosporidium parvum insulin-like proteases, CpINS-4 and CpINS-6, show distinct functions. Anti-CpINS-4 antibodies reduced parasite load, suggesting therapeutic potential against cryptosporidiosis.
Area of Science:
- Parasitology
- Molecular Biology
- Immunology
Background:
- Cryptosporidium parvum is a significant protozoan pathogen causing diarrhea in humans and animals.
- The C. parvum genome contains 22 insulin-like protease (INS) genes with diverse sequences, hinting at varied biological roles.
- CpINS-4 and CpINS-6, two INS family members, possess a Zn2+-binding motif but differ in functional domains.
Purpose of the Study:
- To investigate the distinct biological functions of CpINS-4 and CpINS-6 in the C. parvum life cycle.
- To generate and characterize polyclonal antibodies against CpINS-4 and CpINS-6.
- To assess the potential of these antibodies in controlling C. parvum infection.
Main Methods:
- Recombinant expression of CpINS-4 and CpINS-6 in Escherichia coli.
- Generation of polyclonal antibodies against the recombinant proteins.
- Western blot analysis to detect native and spliced protein forms.
- Immunofluorescence assays to determine antibody reactivity with parasite stages.
- In vitro culture experiments to evaluate the efficacy of antibodies in reducing parasite load.
Main Results:
- Both CpINS-4 and CpINS-6 were found to be spliced into multiple fragments in native and recombinant forms.
- Generated antibodies showed specific recognition of their target proteins with minimal cross-reactivity.
- Anti-CpINS-4 antibodies localized to the middle region of sporozoites and merozoites, while anti-CpINS-6 targeted the apical region.
- Anti-CpINS-4 antibodies significantly reduced C. parvum load by 36% in HCT-8 cultures within 24 hours.
- CpINS-4 and CpINS-6 genes exhibited peak expression during the invasion phase of in vitro C. parvum culture.
Conclusions:
- CpINS-4 and CpINS-6 likely reside in different organelles and perform distinct functions in the C. parvum lifecycle.
- CpINS-4 plays a crucial role in parasite invasion and proliferation, as evidenced by antibody-mediated parasite load reduction.
- CpINS-6's function remains less clear, potentially due to the absence of a key domain in the studied form, but it localizes to the apical region during invasion.
