Structure and Development of Oocyst and Sporocyst Walls

 描述（由申请方提供）：球虫寄生虫是重要的人类病原体，可引起婴儿严重腹泻（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引起免疫抑制的成人慢性感染。卵囊是球虫的感染阶段，通过粪口途径传播。虽然已经在卵囊壁中鉴定出许多蛋白质（例如隐孢子虫卵囊壁蛋白（COWP）），但没有弓形虫和隐孢子虫卵囊壁如何组装的模型。 在这里，我们表明，弓形虫卵囊壁的内层是一个多孔的支架纤维的β-1，3-葡聚糖，糖聚合物存在于真菌壁。虽然葡聚糖合成酶抑制剂（棘白菌素）杀死真菌，但这些药物阻止了艾美球虫卵囊壁的发育，艾美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抗酸脂质，就像那些覆盖在分枝杆菌表面的脂质一样。有机溶剂释放卵囊壁脂质，其是具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质，但不含葡聚糖纤维。相反，隐孢子虫的内层壁含有一种新型糖聚合物的纤维。 目标1中提出的研究测试了隐孢子虫卵囊壁的双层模型。我们将确定糖蛋白的外部刚性双层的壁，也包含抗酸脂质。我们将确定卵囊壁内层纤维的化学组成，并确定COWPS是否是结合这些糖纤维的凝集素。 目标2中拟议的研究重点是弓形虫卵囊壁的结构和发育。我们将确定卵囊壁蛋白，并确定是否在外层，抗酸层具有脂质结合特性，而那些在内层是凝集素，结合到纤维的β-1，3-葡聚糖。我们将测试的dea，β-1，3-葡聚糖，抗酸脂质和壁蛋白是在单独的分泌室在卵囊壁的发展过程中，我们将确定细胞骨架的分泌和决定卵囊壁的形状重要的元素。 具体目标3中拟议的研究重点是弓形虫孢子囊壁的结构和发育。我们将检验弓形虫的孢子囊壁与隐孢子虫卵囊壁结构相似的假设，隐孢子虫卵囊壁也是抗酸的，缺少β-1，3-葡聚糖的原纤维，并含有子孢子。我们泰特孢子形成（孢子囊壁和子孢子的发育）与速殖子的营养生长明显不同。尤其是核分裂 从细胞分裂中分离;成孢子细胞的形成是通过二分裂而不是出芽（endodytic）;每个子孢子同步发育，但独立于其他子孢子，使用类似于速殖子的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.