Contribution of N-glycosylation to the Toxoplasma glycoproteome

描述（由申请人提供）：弓形虫是一种重要的机会性感染，可导致HIV-AIDS和其他免疫损害患者的生命威胁疾病。弓形虫在世界范围内的高血清阳性率，但有症状疾病的低发病率反映了感染的急性期由免疫反应有效处理的事实。不幸的是，无法实现无菌治疗。相反，寄生虫通过通常在大脑和肌肉中形成的组织囊肿来介导终身慢性感染。组织囊肿被糖基化的囊壁保护。尽管糖基化很重要，但人们对寄生虫中的这些途径知之甚少。通过这项工作，我们解决了n -链糖基化在弓形虫中的贡献。我们建议利用凝集素亲和力结合质谱鉴定方法对组织囊肿中n -糖基化糖蛋白的多样性进行分类。我们进一步计划有条件地敲除n -糖基化的关键基因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种独特的分泌细胞器。鉴于糖基化在组织囊肿中的中心作用，我们提出了一种选择性敲除组织囊肿中的基因表达的实验策略。该技术将用于确定TgALG7及其延伸的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在细胞生物学基础的深入研究蛋白糖基化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.