Source data.

Background
Cryptosporidium parvum is a common protozoan pathogen responsible for moderate to severe diarrhea in humans and animals. Parasite invasion and egress cause damage to intestinal epithelial cells, which is mediated by a variety of secretory proteins from several unique organelles, such as micronemes. Previous spatial proteomic analysis has identified insulinase-like protease 6 (INS6) as a putative microneme protein in C. parvum. However, the functional contribution of INS6 to Cryptosporidium pathogenicity remains poorly characterized. In this study, we used genetic manipulation techniques to investigate the expression and biological functions of INS6 in C. parvum.
Methodology/principal findings
The INS6 gene was tagged and deleted in C. parvum using CRISPR/Cas9 technology. The expression of INS6 was determined by immunofluorescence analysis, ultrastructure-expansion microscopy, and immunoelectron microscopy. Endogenous labelling showed low levels of INS6 expression, which is found in C. parvum micronemes and is absent during the male gamont stage. The effect of INS6 deletion on parasite growth and pathogenicity was assessed in vitro using HCT-8 cultures and in vivo by infection of interferon-γ knockout mice. Deletion of the INS6 gene impaired C. parvum proliferation in vitro and significantly reduced the parasite burden in infected mice. In addition, mice infected with the Δins6 strain showed a significant reduction in the intestinal villus-to-crypt ratio, attenuated body weight loss and increased survival rates, compared to those infected with the INS6–3HA strain.
Conclusions/significance
These results indicate that INS6 protein is involved in C. parvum proliferation and plays a critical role in modulating the pathogenicity of this zoonotic parasite. Deletion of this gene affects the invasion efficiency and pathogenicity of the parasite.