Structure and Development of Oocyst and Sporocyst Walls

 描述(申请人提供)：球虫寄生虫是重要的人类病原体，可导致婴儿严重腹泻(微小隐孢子虫)和胎儿播散性感染(弓形虫)。隐孢子虫和弓形虫都会在免疫抑制的成年人中引起慢性感染。卵囊是球虫的感染期，通过粪便-口腔途径传播。虽然已经在卵囊壁中发现了许多蛋白质(例如隐孢子虫卵囊壁蛋白(COMP))，但还没有关于卵囊壁弓形虫和隐孢子虫如何组装的模型。在这里，我们发现弓形虫卵囊壁的内层是一种存在于真菌壁中的糖聚合物--1，3-葡聚糖的纤维构成的多孔支架。虽然葡聚糖合成酶抑制剂(棘球菌素)会杀死真菌，但这些药物会阻止艾美耳球虫卵囊壁的发育，艾美耳球虫是一种鸡寄生虫，与弓形虫卵囊非常相似。弓形虫卵囊壁的外层含有类似于覆盖在分枝杆菌表面的抗酸脂质。有机溶剂释放卵囊壁脂，这是带有多羟基脂肪酰基链的甘油三酯，类似于植物蜡质角质层的甘油三酯。 隐孢子虫的卵囊壁含有抗酸脂肪，但不含葡聚糖纤维。相反，隐孢子虫壁的内层含有一种新型糖聚合物的纤维。建议研究目的1测试隐孢子虫卵囊壁的两层模型。我们将在外壁坚硬的双层中鉴定糖蛋白，该双层也含有抗酸类脂。我们将确定卵囊壁内层的纤维的化学成分，并确定牛皮是否是结合这些糖纤维的凝集素。目的2中提出的研究重点是弓形虫卵囊壁的结构和发育。我们将鉴定卵囊壁蛋白，并确定外层抗酸的蛋白是否具有脂质结合特性，而内层的蛋白是与1，3-葡聚糖纤维结合的凝集素。我们将测试DEA，即1，3-葡聚糖、抗酸脂肪和壁蛋白在卵囊壁的发育过程中分别在不同的分泌室中产生，我们将确定对分泌和决定卵囊壁形状重要的细胞骨架元件。建议的具体目标研究3集中在弓形虫孢子囊壁的结构和发育。我们将检验这样一种假设，即弓形虫的孢囊壁结构类似于隐孢子虫卵囊壁，隐孢子虫卵囊壁也是抗酸的，缺少1，3-葡聚糖原纤维，并含有子孢子。我们认为，孢子形成(孢子囊壁和子孢子的发育)明显不同于速殖子的营养生长，这是很好的特征。尤其是核分裂 孢子母细胞的形成是通过二次分裂而不是萌发(内源作用)；每个子孢子都是同步但独立地发育的，使用与速殖子相似的IMC复合体。

 DESCRIPTION (provided by applicant): Coccidian parasites are important human pathogens that cause severe diarrhea in infants (Cryptosporidium parvum) and disseminated infections in fetuses (Toxoplasma gondii). Both Cryptosporidium and Toxoplasma cause chronic infections in immunosuppressed adults. Oocysts are infectious stages of coccidia, which are spread by the fecal-oral route. While numerous proteins have been identified in oocyst walls (e.g. Cryptosporidium oocyst wall proteins (COWPs)), there are no models for how oocyst walls Toxoplasma and Cryptosporidium are assembled. Here we show that the inner layer of the oocyst wall of Toxoplasma is a porous scaffold of fibrils of ß-1,3-glucan, a sugar polymer present in fungal walls. While glucan synthase inhibitors (echinocandins) kill fungi, these drugs arrest the development of oocyst walls of Eimeria, a chicken parasite that closely models Toxoplasma oocysts. The outer layer of the oocyst wall of Toxoplasma contains acid-fast lipids like those that cover the surface of mycobacteria. Organic solvents release oocyst wall lipids, which are triglycerides with polyhydroxy fatty acyl chain like those of the waxy cuticle of plants. The oocyst wall of Cryptosporidium contains acid-fast lipids but does not contain glucan fibrils. Instead the inner layer of the Cryptosporidium wall contains fibrils of a novel sugar polymer. Proposed studies in Aim 1 test a two-layer model of the Cryptosporidium oocyst wall. We will identify glycoproteins in the outer rigid bilayer of wall that also contains acid-fast lipids. We wll determine the chemical composition of fibrils in the inner layer of the oocyst wall and determine whether COWPS are lectins that bind these sugar fibrils. Proposed studies in Aim 2 focus on the structure and development of the oocyst wall of Toxoplasma. We will identify oocyst wall proteins and determine whether those in the outer, acid-fast layer have lipid-binding properties, while those in the inner layer are lectins that bind to fibrils of ß-1,3-glucan. We will test the dea that ß-1,3-glucan, acid-fast lipids, and wall proteins are made in separate secretory compartments during development of the oocyst wall, and we will identify cytoskeletal elements important for secretion and for determining oocyst wall shape. Proposed studies in Specific Aim 3 focus on the structure and development of the sporocyst wall of Toxoplasma. We will test the hypothesis that sporocyst wall of Toxoplasma is structural similar to the Cryptosporidium oocyst wall, which is also acid-fast, is missing fibrils of ß-1,3-glucan, and contains sporozoites. We tet the idea that sporulation (development of the sporocyst wall and sporozoites) differs markedly from vegetative growth by tachyzoites, which is well-characterized. In particular, nuclear division is uncoupled from cell division; sporoblast formation is by binary fission rather than budding (endodyogeny); and each sporozoite develops synchronously but independently from others, using an IMC complex similar to that of tachyzoites.