DataSheet_2_Unveiling Cryptosporidium parvum sporozoite-derived extracellular vesicles: profiling, origin, and protein composition.pdf

Cryptosporidium parvum is a common cause of a zoonotic disease and a main cause of diarrhea in newborns. Effective drugs or vaccines are still lacking. Oocyst is the infective form of the parasite; after its ingestion, the oocyst excysts and releases four sporozoites into the host intestine that rapidly attack the enterocytes. The membrane protein CpRom1 is a large rhomboid protease that is expressed by sporozoites and recognized as antigen by the host immune system. In this study, we observed the release of CpRom1 with extracellular vesicles (EVs) that was not previously described. To investigate this phenomenon, we isolated and resolved EVs from the excystation medium by differential ultracentrifugation. Fluorescence flow cytometry and transmission electron microscopy (TEM) experiments identified two types of sporozoite-derived vesicles: large extracellular vesicles (LEVs) and small extracellular vesicles (SEVs). Nanoparticle tracking analysis (NTA) revealed mode diameter of 181 nm for LEVs and 105 nm for SEVs, respectively. Immunodetection experiments proved the presence of CpRom1 and the Golgi protein CpGRASP in LEVs, while immune-electron microscopy trials demonstrated the localization of CpRom1 on the LEVs surface. TEM and scanning electron microscopy (SEM) showed that LEVs were generated by means of the budding of the outer membrane of sporozoites; conversely, the origin of SEVs remained uncertain. Distinct protein compositions were observed between LEVs and SEVs as evidenced by their corresponding electrophoretic profiles. Indeed, a dedicated proteomic analysis identified 5 and 16 proteins unique for LEVs and SEVs, respectively. Overall, 60 proteins were identified in the proteome of both types of vesicles and most of these proteins (48 in number) were already identified in the molecular cargo of extracellular vesicles from other organisms. Noteworthy, we identified 12 proteins unique to Cryptosporidium spp. and this last group included the immunodominant parasite antigen glycoprotein GP60, which is one of the most abundant proteins in both LEVs and SEVs.

微小隐孢子虫（Cryptosporidium parvum）是一种常见的人畜共患病病原体，也是新生儿腹泻的主要致病原。目前仍缺乏有效的治疗药物或疫苗。卵囊（oocyst）是该寄生虫的感染形式：宿主摄入卵囊后，卵囊会脱囊并释放4个子孢子侵入宿主肠道，快速攻击肠上皮细胞。膜蛋白CpRom1是一类大型菱形蛋白酶（rhomboid protease），由子孢子表达，可被宿主免疫系统识别为抗原。本研究首次观察到子孢子通过细胞外囊泡（extracellular vesicles, EVs）释放CpRom1，此前尚无相关报道。为探究该现象，我们通过差速超速离心法从脱囊培养基中分离并解析了细胞外囊泡。荧光流式细胞术与透射电子显微镜（transmission electron microscopy, TEM）实验证实存在两种源自子孢子的囊泡：大型细胞外囊泡（large extracellular vesicles, LEVs）与小型细胞外囊泡（small extracellular vesicles, SEVs）。纳米颗粒追踪分析（nanoparticle tracking analysis, NTA）结果显示，LEVs的模态直径为181 nm，SEVs则为105 nm。免疫检测实验证实LEVs中存在CpRom1与高尔基体蛋白CpGRASP；而免疫电子显微镜试验则证明CpRom1定位于LEVs的表面。透射电子显微镜与扫描电子显微镜（scanning electron microscopy, SEM）结果显示，LEVs由子孢子外膜出芽生成；与之相反，SEVs的起源仍不明确。LEVs与SEVs的蛋白质组成存在显著差异，电泳图谱可佐证这一点。具体而言，靶向蛋白质组学分析分别鉴定出5种LEVs特有蛋白与16种SEVs特有蛋白。两类囊泡的蛋白质组中共鉴定出60种蛋白，其中大多数（共48种）已在其他生物的细胞外囊泡分子货物中被报道。值得注意的是，我们鉴定出12种隐孢子虫属（Cryptosporidium spp.）特有蛋白，其中包括免疫优势寄生虫抗原糖蛋白GP60，该蛋白是LEVs与SEVs中丰度最高的蛋白之一。