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.
Highlights
Cryptosporidium spp. are intracellular protozoan parasites that cause moderate-tosevere diarrhea in humans and various animals
CpINS-4 has all four domains seen in functional M16 proteases, including a M16 peptidase domain, two M16 inactive domains, and a middle or third domain of peptidase
The predicted tertiary structures of CpINS-4 and CpINS-6 were modeled based on the crystal structure (PDB inactive domains (ID): 2jbu_B) of human insulin degrading enzyme and the structure of hIDE complex with its copurified peptides [20]
Summary
Cryptosporidium spp. are intracellular protozoan parasites that cause moderate-tosevere diarrhea in humans and various animals. C. hominis, are the major causative agents of human cryptosporidiosis [1]. Neonatal animals, and immunocompromised individuals are especially susceptible to infection [2]. In low-income countries, cryptosporidiosis is a common cause of diarrhea-associated mortality in young children [3]. It was estimated that in 2016 alone, acute Cryptosporidium infections caused more than 48,000 deaths and over 4.2 million disability-adjusted life-years in low- and middle-income countries [4]. No effective vaccines are in clinical use, and nitazoxanide, the sole drug approved by the U.S Food and Drug Administration for treating cryptosporidiosis, has poor efficacy in immunocompromised individuals [5]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
