[PMC free article] [PubMed] [CrossRef] [Google Scholar] 36. variances from two animals. For the and groups, the data shown are the averages and SD. *, animals that died. (C) Survival curve of Ifngr1?/? mice infected with different transgenic parasites. Animals correspond to those shown in panels A and B. For the WT group, four Ifngr1?/? mice were gavaged with 2??104 oocysts. Download FIG?S1, DOCX file, 0.4 MB. Copyright ? 2021 Xu et al. This content is distributed under the terms of the Creative Commons Attribution 4.0 International license. TABLE?S1. Oligonucleotides used in this study. Download Table?S1, XLSX file, 0.01 MB. Copyright ? 2021 Xu et al. This content is distributed under the terms of the Creative Commons Attribution 4.0 International license. Data Availability StatementAll of the data are found in the manuscript or supplemental material. ABSTRACT The apicomplexan parasite contains an expanded family of 22 insulinase-like proteases (INS), a feature that contrasts with their otherwise streamlined genome. Here, we examined the function of INS1, which is most similar to the human insulinase protease that cleaves a variety of small peptide substrates. INS1 is an M16A clan member and contains a signal peptide, an N-terminal domain with the HXXEH active site, followed by three inactive domains. Unlike previously studied INS proteins that are expressed in sporozoites and during merogony, INS1 was expressed exclusively in macrogamonts, where it was localized in small cytoplasmic vesicles. Although INS1 did not colocalize with the oocyst wall protein recognized by the antibody OW50, immune-electron microscopy indicated that INS1 resides in small vesicles in the secretory system. Notably, these small INS1-positive vesicles were often in close proximity to large OW50-positive vacuoles resembling wall-forming bodies, which contain precursors for oocyst wall formation. Genetic deletion of INS1, or replacement with an active-site mutant, UR 1102 resulted in lower formation of macrogamonts and reduced oocyst shedding spp. are apicomplexan parasites that cause diarrheal disease in humans and animals. Human infection is primarily caused by two species, occurs in a single host, Akt1s1 leading to efficient fecal-oral transmission (5). Following the ingestion of oocysts, sporozoites emerge and invade intestinal epithelial cells, where they develop in a unique vacuole formed at the apex of the host cell (6). The parasite initially grows as a trophozoite before undergoing multiple rounds of asexual replication during merogony (7, 8). The parasite then differentiates to the sexual phase and develops as macrogamonts or microgamonts that begin appearing after 44 to 48?h postinfection (hpi) (7, 8). However, in transformed cell lines grown cultures in adenocarcinoma cell lines to the developmental process that occurs in the intestine of UR 1102 mice revealed that the block to complete development is due to a lack of fertilization despite the fact that both gametocyte forms develop normally (7). Biological investigations of have been hampered by limitations in experimental platforms for growth. Despite this limitation, propagation UR 1102 systems have been used to generate antibodies that identify different stages of (10), leading to a better understanding the life cycle (8). Recent developments have also provided systems that allow complete development of infectious oocysts in stem cell-derived cultures (11, 12). Finally, advances in CRISPR/Cas9 technology have allowed genetic modification in to tag genes for localization and disrupt them to study function (13). Despite these advances, we lack an understanding of the function of most genes in is highly streamlined, with short intergenic regions, limited introns, and the loss of many metabolic pathways (16). Greater than 98% of genes in are present as a single copy, and only a limited number of multigene families include insulinase-like proteases (INS), predicted secretory proteins containing the amino acid sequence MEDLE, and mucin-type glycoproteins (17). INS proteins belong to the M16 UR 1102 family of metallopeptidases that play diverse roles in cells, and they can be found in the cytosol, organelles, and even the cell surface (18). M16 metalloproteases typically bind UR 1102 zinc as part of their active site (HXXEH), and they cleave short polypeptides, the size of which is constrained by a conserved small barrel fold that forms the catalytic chamber (19). The apicomplexan parasite contains 50 metalloproteases, including 11 members of the M16 clan (20),.