Contribution of N-glycosylation to the Toxoplasma glycoproteome

描述（由申请方提供）：原生动物寄生虫弓形虫是一种重要的机会性感染，可导致HIV-AIDS患者和其他免疫受损疾病的危及生命的疾病。T.弓形虫病在世界范围内流行，但有症状的发病率较低，这反映出免疫反应可有效地处理感染的急性期。不幸的是，没有实现无菌治疗。相反，寄生虫建立了一个终身的慢性感染介导的组织囊肿通常形成在大脑和肌肉。组织囊肿由高度糖基化的囊壁保护。尽管糖基化的重要性，很少有人知道这些途径的寄生虫。通过这项工作，我们解决了弓形虫N-连接糖基化的贡献。我们建议使用凝集素的亲和力加上质谱鉴定目录的N-糖基化的糖蛋白在组织囊肿的多样性。我们进一步计划有条件地敲除N-糖基化中的关键基因TgALG7，以评估其在寄生虫生物学中的作用，重点是蛋白质靶向其多样化的独特分泌细胞器。鉴于糖基化在组织囊肿中的核心作用，我们提出了一种选择性敲除组织囊肿中基因表达的实验策略。该技术将被应用于确定TgALG7和通过扩展N-糖基化对组织囊肿形成和稳定性的贡献。这项工作代表了第一次深入研究的细胞生物学基础的蛋白质糖基化的弓形虫使用最先进的方法来调节基因表达。

DESCRIPTION (provided by applicant): The protozoan parasite Toxoplasma gondii is an important opportunistic infection causing life threatening disease in patients with HIV-AIDS and other immune compromising conditions. The high seroprevalence of the T. gondii worldwide but low incidence of symptomatic disease reflects the fact that the acute phase of infection is effectively handled by the immune response. Unfortunately a sterile cure is not achieved. Rather the parasite establishes a life-long chronic infections mediated by tissue cysts typically formed in the brain and muscle. The tissue cyst is protected by a heavily glycosylated cyst wall. Despite the importance of glycosylation, little is known about these pathways in the parasite. With this work we address the contribution of N-linked glycosylation in Toxoplasma. We propose to use lectin affinity coupled with mass spectrometric identification to catalogue the diversity of N-glycosylated glycoproteins in tissue cysts. We further plan to conditionally knock out a key gene in the N-glycosylation, TgALG7, in order to assess its role in parasite biology with a focus on protein targeting to its diverse unique secretory organelles. Given the central role for glycosylation in the tissue cyst we propose an experimental strategy to selectively knock out gene expression in the tissue cyst. This technology will be applied to establishing the contribution of TgALG7 and by extension N-glycosylation to tissue cyst formation and stability. This work represents the first in depth study of the cell biologic basis of protein glycosylation i Toxoplasma using state of the art approaches to modulate gene expression.