PDF(Pubmed)
Abstract：
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.
摘要：
隐孢子虫是人畜共患疾病的常见原因，也是新生儿腹泻的主要原因。仍然缺乏有效的药物或疫苗。卵囊是寄生虫的感染形式；摄入后，卵囊排出并释放四个子孢子进入宿主肠道，迅速攻击肠细胞。膜蛋白CpRom1是一种大的菱形蛋白酶，由子孢子表达并被宿主免疫系统识别为抗原。在这项研究中,我们观察到CpRom1与细胞外囊泡（EV）的释放，这是以前没有描述过的。为了研究这种现象，我们通过差别式超速离心从细胞外化培养基中分离和分离EV。荧光流式细胞术和透射电子显微镜（TEM）实验鉴定了两种子孢子衍生的囊泡：大的细胞外囊泡（LEV）和小的细胞外囊泡（SEV）。纳米粒子跟踪分析（NTA）显示，LEV的模式直径为181nm，SEV的模式直径为105nm，分别。免疫检测实验证明在LEV中存在CpRom1和高尔基体蛋白CpGRASP，而免疫电子显微镜试验证明了CpRom1在LEVs表面的定位。TEM和扫描电子显微镜（SEM）表明，LEV是通过子孢子外膜的出芽产生的；相反，SEV的起源仍然不确定。在LEV和SEV之间观察到不同的蛋白质组成,如它们相应的电泳图谱所证明的。的确,专门的蛋白质组学分析确定了LEV和SEV特有的5和16种蛋白质，分别。总的来说,在两种类型的囊泡的蛋白质组中鉴定了60种蛋白质，并且这些蛋白质中的大多数（数量为48种）已经在来自其他生物体的细胞外囊泡的分子货物中鉴定。值得注意的是,我们鉴定出12种隐孢子虫特有的蛋白质.最后一组包括免疫显性寄生虫抗原糖蛋白GP60，它是LEV和SEV中最丰富的蛋白质之一。
