Insulinase-like Protease 1 Contributes to Macrogamont Formation in Cryptosporidium parvum.
Insulinase-like Protease 1 Contributes to Macrogamont Formation in Cryptosporidium parvum.
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DOI：
10.1128/mbio.03405-20
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发表时间：
2021-03-09
期刊：
影响因子：
6.4
通讯作者：
Sibley LD
中科院分区：
文献类型：
--
作者：
Xu R;Feng Y;Xiao L;Sibley LD
隐孢子虫病是发展中国家幼儿的一种使人衰弱的肠道疾病。由于缺乏有效的治疗或疫苗，这种感染在易感人群中很难控制。隐孢子虫（Cryptosporidium parvum）的顶复形寄生虫含有一个由22个胰岛素酶样蛋白酶（INS）组成的扩展家族，这一特征与它们的流线型基因组形成鲜明对比。在这里，我们研究了INS 1的功能，它与切割各种小肽底物的人胰岛素酶蛋白酶最相似。INS 1是M16 A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，随后是三个非活性结构域。与以前研究的C.小孢子INS蛋白在子孢子中和在部分生殖期间表达，INS 1仅在大配子中表达，在那里它定位于小的细胞质囊泡中。虽然INS 1没有与抗体OW 50识别的卵囊壁蛋白共定位，但免疫电子显微镜表明INS 1存在于分泌系统的小泡中。值得注意的是，这些小的INS 1阳性囊泡往往在接近大的OW 50阳性空泡类似的壁形成机构，其中包含卵囊壁形成的前体。基因缺失的INS 1，或替换与活性位点突变体，导致在体外形成的巨配子和减少体内卵囊脱落。我们的研究结果表明，INS 1功能的形成或成熟的巨配子，其损失的结果在免疫功能低下的小鼠毒力减弱。
Cryptosporidiosis is a debilitating diarrheal disease in young children in developing countries. The absence of effective treatments or vaccines makes this infection very difficult to manage in susceptible populations. 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 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 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 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, resulted in lower formation of macrogamonts in vitro and reduced oocyst shedding in vivo. Our findings reveal that INS1 functions in the formation or maturation of macrogamonts and that its loss results in attenuated virulence in immunocompromised mice.