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Background: Cryptosporidium parvum is a common protozoan pathogen responsible for moderate to severe diarrhea in humans and animals. The C. parvum genome contains 22 genes encoding insulinase-like M16 proteases (INS) with diverse structures and sequences, suggesting that members of the protein family may have distinct biological functions in the life cycle of parasites. Here, we investigated the role of INS15 and INS16, two proteases encoded by neighboring genes with high sequence identity, in the growth and development of C. parvum in vivo and in vitro. Methodology/Principal findings: INS15 and INS16 genes were tagged and knocked out using CRISPR/Cas9 technology in C. parvum IIdA20G1-HLJ isolate. The expression of INS15 and INS16 was determined by immunofluorescence analysis and immunoelectron microscopy. The effect of depletion of INS15 and INS16 on parasite growth and pathogenicity were assessed on HCT-8 cells and in interferon-γ knockout mice. Endogenous tagging showed that INS15 and INS16 expressed in the oocyst, trophozoite, meront and female gametes. INS15 also expressed in male gamonts, while INS16 was not detected in the male gamonts. Although depletion of the INS15 or INS16 gene affected late development of C. parvum in vitro, only depletion of INS15 significantly reduced parasite burden in infected mice. Mice infected with the INS15-depleted strain had reduced clinical signs, body weight, intestinal villus length to crypt height ratio, and survival time compared to infected with the tagging mutant. Conclusions/Significance: The results of this study indicate that INS15 is mainly involved in the late development of C. parvum. Depletion of this gene attenuates the pathogenicity of this important zoonotic parasite. Author summary: Cryptosporidium parvum is a common protozoan pathogen responsible for moderate to severe diarrhea in young children and animals and there is currently no effective treatment to prevent the many deaths it causes each year. Previous studies showed that C. parvum genome encodes 22 insulinase-like M16 proteases (INSs) with diverse structures and sequences, suggesting that members of this protein family may have distinct biological functions in the life cycle of parasites. Our work characterizes two INSs, INS15 and INS16, which are encoded by neighboring genes with high sequence identity. We used CRISPR/Cas9 genome editing to endogenous tagged INS15 and INS16 and showed that expression patterns of INS15 and INS16 were different, with INS16 was not detected in the male gamonts. Genetic ablation of INS15 or INS16 affected late development of C. parvum in vitro, while only depletion of INS15 significantly reduced parasite burden in infected interferon-γ knockout mice. The INS15-depleted strain resulted on a reduction of clinical signs and an extension of survival time in infected mice compared to the INS15-tagged strain. These findings demonstrate that the depletion of INS15 attenuates the pathogenicity of this important zoonotic parasite. [ABSTRACT FROM AUTHOR]

Eimeria stiedae is one of the few eukaryotic pathogens that exclusively infect the liver and serves as a good model to study the host–pathogen interactions in this vital organ. In this study, we show that rabbits infected with E. stiedae develop severe but self‐healing cholangitis. RNA‐seq analysis of the liver gene expression landscapes over the long course of E. stiedae infection identified 912 differentially expressed genes (DEGs) in the prepatent period (794 up‐ and 118 downregulated genes), 2889 DEGs in the early oocyst shedding period (1870 up‐ and 1019 downregulated genes), 2859 DEGs in the peak oocyst shedding period (1923 up‐ and 936 downregulated genes), and 327 DEGs in the recovery period (164 up‐ and 163 downregulated genes). Combined with pathological observations, we identified dynamic changes in host–parasite interactions involving multiple pathways. They showed that E. stiedae infection induced full‐blown inflammatory, Th1 and Th17 immune responses at all time points. This was associated with the strong innate immune responses during the prepatent period, including increased Toll‐like and NOD‐like receptor signaling. Despite mounting several damage control and repair responses, such as PI3K‐Akt signaling, Ras signaling, and extracellular matrix‐receptor interactions, the liver underwent severe metabolic dysfunction, oxidative damage, and coagulopathy after patency and at peak infection, possibly as a result of suppressed peroxisome activities and downregulated PPAR signaling. These responses largely disappeared during late infection, suggesting that the liver self‐heals after severe cholangitis. These data provide new insights into host–pathogen interactions during Eimeria infection and improve our understanding of the pathogenesis of parasitic cholangitis. [ABSTRACT FROM AUTHOR]