All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement Not applicable. Informed Consent Statement Not applicable. Data Availability Statement The original contributions Tyrphostin AG 183 presented in the study are included in the article/supplementary material, and further inquiries can be directed to the corresponding authors. Conflicts of Interest The authors declare no conflict of interest. Footnotes Publishers Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.. results of this study suggest that INS-16 may have important roles in the development of and could be a Tyrphostin AG 183 valid target for the development of effective treatments. spp. are gastrointestinal pathogens that can cause severe diarrhea in humans and various animals . Young children in developing countries infected with spp. can develop malnutrition and cognitive impairments in Tyrphostin AG 183 addition to clinical illness . In industrialized Tyrphostin AG 183 countries, waterborne outbreaks of cryptosporidiosis are common . Among the more than 40 named species, is the main species for cryptosporidiosis in farm animals and one of the two dominant species in humans . Currently, there is a lack of effective drugs against spp. Although significant progress has been made in the development of novel drugs against cryptosporidiosis in recent years, we still have poor understanding of the biology of the pathogens . The invasion of host cells by apicomplexan parasites is a complex process mediated by receptors and ligands that involve many proteins on both sides . Secreted proteases and protein kinases in secretory organelles of apicomplexans can modify invasion-related proteins or host cell activities, thus playing important roles in invasion . Comparative genomics analysis of multiple species has revealed the presence of numerous genes encoding secreted proteases in the compact genome. Among them, insulinase-like metalloproteinases (INS) are one of the largest protease families with 22 members in [8,9]. As the genome is only 9 Mb in size and most of the ~4000 genes are single copied, INS probably play important roles in the invasion and development of spp. INS are zinc metalloproteinases of the M16 family, which are widely distributed in nature and divided into the M16A, M16B, and M16C subfamilies. The M16 metalloproteinases MMP7 are characterized by the presence of a functional domain containing an inversion of the thermolysin zinc-binding motif, HXXEH . They have a wide range of substrates and cleave many proteins and small peptides including insulin, -amyloid, and glucagon [11,12]. In apicomplexans, falcilysin is an M16C insulinase involved in hemoglobin catabolism and may function as two different proteases in two subcellular organelles of after proteolytic processing [13,14]. An M16A protease, toxolysin-1 (TLN1) of indicate that some INS genes are highly expressed in the invasion stages of the pathogen . In a recent study, one INS protein of INS protein, INS-15, appeared to be post-translationally processed as several fragments and have biological activities similar to toxolysins . Another INS protein, INS-1, is expressed in secretory vesicles within the pathogen and contributes to the formation of macrogamonts . Nevertheless, the role, processing, and trafficking of other INS remain unclear. In this study, we characterized INS-16 encoded by the cgd3_4270 gene and examined its expression patterns in developmental stages of oocysts as the template, we successfully cloned the full-length cgd3_4270 gene and its domain I fragment (Figure S1A,D), producing recombinant proteins in (Figure S1B,E). The recombinant proteins were purified using Ni-NTA affinity chromatography, with the purity being confirmed using SDS-PAGE analysis (Figure S1C,F). Open in a separate window Figure 1 Domain structure and specific amino acid sequence of INS-16 of sporozoites. Lane M: protein marker; Lane 1: purified recombinant full-length INS-16. Lane 2: sporozoite lysate. The picture on the right shows the result of proteins reacting with pre-immune serum. 2.3. Proteolytical Processing of Native INS-16 To assess the expression of the native INS-16 protein in in HCT-8 cells as indicated with immunofluorescence microscopy. (A) The localization of INS-16 with antibodies against INS-16 domain I. (B) The localization of INS-16 with antibodies against INS-16-specific peptide. The reactivity of the antibodies with oocysts, free sporozoites, and merozoites in infected HCT-8 cells is shown (red). Nuclei were.