The apicomplexan parasite Cryptosporidium parvum 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 a 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 C. parvum 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 often subtend large vacuoles resembling wall forming bodies, which contain precursors for oocyst wall formation. Genetic deletion of INS1, or replacement with an active site mutant, resulted in lower formation of macrogamonts in vitro and reduced oocyst shedding in vivo. Our findings reveal that INS1 functions in formation or maturation of macrogamonts and that its loss results in attenuated virulence in immunocompromised mice.

中文翻译：

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胰岛素酶（如蛋白酶1）有助于小隐孢子虫的大ga形成

apicomplexan寄生虫小球隐孢子虫包含22个胰岛素酶样蛋白酶（INS）的扩展家族，此特征与其精简的基因组形成鲜明对比。在这里，我们检查了INS1的功能，该功能与切割各种小肽底物的人胰岛素酶蛋白酶最相似。INS1是M16A家族成员，包含一个信号肽，一个具有HxxEH活性位点的N末端结构域，后跟三个非活性结构域。与以前研究的小孢梭菌INS蛋白在子孢子和子代期间表达的不同，INS1仅在巨分子中表达，而INS1则位于小细胞质囊泡中。尽管INS1不能与抗体OW50识别的卵囊壁蛋白共定位，免疫电子显微镜检查表明，INS1位于分泌系统的小囊泡中。值得注意的是，这些小的INS1阳性小囊泡通常对着类似于壁形成体的大液泡，其中包含形成卵囊壁的前体。INS1的基因缺失，或被一个活性位点突变体替代，导致体外巨球形成的降低和体内卵囊脱落的减少。我们的研究结果表明，INS1在巨猴的形成或成熟中起作用，并且它的丧失导致免疫受损小鼠的毒力减弱。导致体外巨球形成的减少，并降低了体内卵囊的脱落。我们的研究结果表明，INS1在巨猴的形成或成熟中起作用，并且它的丧失导致免疫受损小鼠的毒力减弱。导致体外巨球形成的减少，并降低了体内卵囊的脱落。我们的研究结果表明，INS1在巨猴的形成或成熟中起作用，并且它的丧失导致免疫受损小鼠的毒力减弱。