Comparative Study of Two Insulinlike Proteases in Cryptosporidium parvum

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.