Characterization of INS-15, A Metalloprotease Potentially Involved in the Invasion of Cryptosporidium parvum

Abstract

<i>Cryptosporidium parvum</i> is a protozoan parasite that can cause moderate-to-severe diarrhea. Insulinase-like proteases (INS) are one of the largest protein families within the small proteome of the pathogen. However, their roles in <i>C. parvum</i> biology remain un-elucidated. In this study, a member of the protein family, INS-15 of <i>C. parvum</i> encoded by <i>cgd3_4260</i>, was cloned, expressed and characterized to understand its function. INS-15 and its domain I were expressed in <i>Escherichia coli</i> and polyclonal antibodies against the domain I and one specific polypeptide were prepared in rabbits. The role of INS-15 protein in the <i>C. parvum</i> invasion was preliminarily studied. Recombinant INS-15 protein and its domain I were successfully expressed in <i>E. coli</i>, together with various degraded products. The cgd3<i>_4260</i> gene had a peak expression at 2 h of <i>in vitro</i> <i>C. parvum</i> culture, while the INS-15 protein was expressed in the mid-anterior region of sporozoites and the area of merozoites opposite to the nucleus. Anti-INS-15 domain I antibodies reduced the invasion of <i>C. parvum</i> sporozoites by over 40%. The anterior location of INS-15 in invasion stages and partial reduction of <i>in vitro</i> growth indicate that INS-15 plays some roles in the invasion or early development of <i>C. parvum</i>.