Reactivation of Chronic Toxoplasmosis

项目摘要/摘要 弓形虫是一种广泛存在的动物寄生虫，在猫之间循环，在猫之间传播传染性疾病。 它们粪便中的卵囊，以及各种携带长期慢性感染的动物。人类也是 通常因摄入受污染的水中的卵囊或因摄入受污染的水中的组织囊而感染 未煮熟的肉。感染后，寄生虫最初以快速生长的速殖子的形式增殖，这 在体内广泛传播。然后分化成驻留在组织内的半休眠缓虫体。 囊肿，最常见于肌肉和大脑。尽管健康个体的感染是由 免疫系统、组织囊肿面持续免疫，药物治疗不能消除。因此， 慢性感染弓形虫对艾滋病毒感染的艾滋病患者构成严重威胁，因为寄生虫从 半休眠组织囊肿和高度增殖的速殖子阶段的重新出现，这可能导致生命 危险的并发症。 尽管曾经被认为是潜伏的，但最新的研究表明，缓虫体可以复制，尽管是异步的。 而且很少见。此外，组织囊定期破裂，释放出感染新宿主细胞的缓殖子。 并产生子体组织囊肿。缓殖子出口的一个障碍是它们坚硬的囊壁，由 蛋白质和碳水化合物，包括优势抗原CST1，它由O-连接的高度糖基化 加糖。CST1的缺失或其粘蛋白结构域的缺失会导致脆弱的包囊，这意味着这种糖蛋白 为细胞壁提供刚性。这与碳水化合物构成囊壁的关键部分的观点一致， 我们的研究表明，外源添加的葡聚糖酶和几丁质酶消化囊壁和 导致缓殖子的释放。为了探索控制这一过程的内生途径，我们将研究 两种寄生虫糖基水解酶的作用，这两种酶在缓殖子中表达，由寄生虫分泌，以及 定位于囊壁。铜绿假单胞菌葡聚糖酶(GLN1)和几丁质酶样蛋白(CLP1)基因的遗传破坏 弓形虫致囊菌株导致子囊的形成减少，支持了这样的假设 这些酶有助于包囊的成熟和周转。我们将研究这些物质的底物特异性。 酶的体外实验，并评估它们在体内组织囊变形成和周转中的作用。我们还将检查 这些糖基水解酶在免疫低下小鼠模型慢性感染中的作用 模仿感染艾滋病毒的艾滋病患者的免疫力受损。拟议的研究将探索这一假说。 糖基水解酶有助于组织包囊的周转，从而重新激活 弓形虫病，这对感染艾滋病毒的艾滋病患者造成严重疾病的主要风险。

Project Summary/Abstract Toxoplasma gondii is a widespread parasite of animals that cycles between cats, which shed infectious oocysts in their feces, and a variety of animals that harbor long-lived chronic infections. Humans are also commonly infected either by ingestion of oocysts in contaminated water, or by ingestion of tissue cysts in undercooked meat. Following infection, the parasite initially proliferates as fast-growing tachyzoites, which disseminate widely in the body. It then differentiates into semi-dormant bradyzoites that reside within tissue cysts, most commonly in muscle and brain. Although infections in healthy individuals are controlled by the immune system, tissue cysts persist in the face of immunity and are not eliminated by drug treatment. As such, chronic infections with T gondii pose a serious risk to HIV-infected AIDS patients due release of parasites from semi-dormant tissue cysts and re-emergence of the highly proliferative tachyzoite stage, which can lead to life threatening complications. Although once considered latent, newer studies reveal that bradyzoites replicate, albeit asynchronously and infrequently. Moreover, tissue cysts periodically rupture to release bradyzoites that infect new host cells and give rise to daughter tissue cysts. One barrier to egress of bradyzoites is their rigid cyst wall, comprised of proteins and carbohydrates, including the dominant antigen CST1, which is heavily glycosylated by O-linked sugars. Loss of CST1, or deletion of its mucin domain, results in fragile cysts, implying that this glycoprotein provides rigidity to the cell wall. Consistent with the idea that carbohydrates form a critical part of the cyst wall, our studies demonstrate that exogenously added glucanase and chitinase enzymes digest the cyst wall and result in release of bradyzoites. To explore endogenous pathways that control this process, we will examine the roles of two parasite glycosyl hydrolases that are expressed in bradyzoites, secreted from the parasite, and localized to the cyst wall. Genetic disruption of glucanase (GLN1) and chitinase-like protein (CLP1) genes in a cystogenic strain of T. gondii results in reduced formation of daughter cysts, supporting the hypothesis that these enzymes contribute to cyst maturation and turnover. We will examine the substrate specificity of these enzymes in vitro and assess their roles in vivo in formation and turnover of tissue cysts. We will also examine the role of these glycosyl hydrolases in chronic infection of immunocompromised mouse models that closely mimic impaired immunity seen in HIV-infected AIDS patients. The proposed studies will explore the hypothesis that glycosyl hydrolase enzymes contribute to the turnover of tissue cysts resulting in reactivation of toxoplasmosis, which poses a major risk for severe disease in HIV-infected AIDS patients.