Contribution of N-glycosylation to the Toxoplasma glycoproteome

描述(申请人提供)：原虫寄生虫弓形虫是一种重要的机会性感染，在艾滋病毒/艾滋病患者和其他免疫损害情况下会导致危及生命的疾病。全世界弓形虫的血清阳性率高，但症状性疾病的发病率低，反映了这样一个事实，即感染的急性阶段通过免疫反应得到了有效的处理。不幸的是，无菌疗法并未实现。相反，这种寄生虫建立了一种终生的慢性感染，这种感染是由通常在大脑和肌肉中形成的组织囊肿介导的。组织囊被高度糖化的囊壁保护。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-连接糖基化在弓形虫中的作用。我们建议使用凝集素亲和力结合质谱学鉴定来分类组织包囊中N-糖基化糖蛋白的多样性。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，重点是针对其各种独特的分泌细胞器的蛋白质。鉴于糖基化在组织囊中的中心作用，我们提出了一种实验策略，选择性地敲除组织囊中的基因表达。这项技术将被应用于确定TgALG7以及延伸的N-糖基化对组织囊形成和稳定性的贡献。这项工作代表了第一次深入研究弓形虫蛋白糖基化的细胞生物学基础，使用最先进的方法来调节基因表达。 公共卫生相关性：弓形虫会导致免疫受损的人患上严重疾病，包括艾滋病毒/艾滋病患者。我们对糖基化在寄生虫生物学中的作用的了解非常有限。通过这项工作，我们直接探索了N-糖基化在有焦点的休眠组织囊状结构中的作用。我们希望为今后在这一正在探索的调查领域的研究奠定坚实的基础。

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. PUBLIC HEALTH RELEVANCE: Toxoplasma gondii causes serious illness in immune compromised individuals including patients with HIV- AIDS. Our understanding of the role of glycosylation in parasite biology is very limited. With this work we directly explore N-glycosylation in the with a focus the dormant tissue cyst form. We hope to establish a strong foundation for future studies in this under explored area of investigation.