Contribution of N-glycosylation to the Toxoplasma glycoproteome

N-糖基化对弓形虫糖蛋白质组的贡献

• 批准号: 8320531
• 负责人: ANTHONY P. SINAI
• 金额: $ 21.73万
• 依托单位: UNIVERSITY OF KENTUCKY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-03-01 至 2014-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8320531
• 关键词: Acquired Immunodeficiency Syndrome; Acute; Address; Affinity; Affinity Chromatography; Animals; Apicomplexa; Area; Asparagine; Bioinformatics; Biological Process; Biology; Blood; Brain; Cataloging; Catalogs; Cells; Chronic; Chronic Phase; Complex; Computer Simulation; Coupled; Cyst; Development; Disease; Elements; Enzymes; Exhibits; Foundations; Funding Mechanisms; Future; Gene Expression; Genes; Genetic; Genome; Glycoproteins; Golgi Apparatus; HIV; Homologous Gene; Immune; Immune response; Immune system; Incidence; Individual; Infection; Investigation; Knock-out; Knowledge; Lectin; Life; Light; Link; Maintenance; Mass Spectrum Analysis; Mediating; Methods; Muscle; Nature; Neospora; Opportunistic Infections; Organelles; Parasites; Pathogenesis; Pathology; Pathway interactions; Patients; Pattern; Phase; Play; Polysaccharides; Protein Glycosylation; Proteins; Proteome; Proteomics; Protozoa; Role; Seroprevalences; Site-Directed Mutagenesis; Sterility; Structure; System; Technology; Testing; Tetanus Helper Peptide; Tissues; Toxoplasma; Toxoplasma gondii; Vision; Work; asexual; base; genetic manipulation; glycosylation; high risk; infancy; knowledge base; member; microbial; rhoptry; sugar

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.

描述（由申请人提供）：原生动物寄生虫弓形虫是一种重要的机会性感染，会导致艾滋病毒/艾滋病和其他免疫受损疾病患者罹患危及生命的疾病。全球范围内弓形虫血清阳性率较高，但有症状疾病的发生率较低，这反映出免疫反应可以有效应对感染的急性期。不幸的是，没有实现无菌治疗。相反，寄生虫会通过通常在大脑和肌肉中形成的组织囊肿介导形成终生慢性感染。组织囊肿受到高度糖基化的囊肿壁的保护。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，我们解决了弓形虫中 N-连接糖基化的贡献。我们建议使用凝集素亲和力结合质谱鉴定来对组织囊肿中 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-糖基化中的一个关键基因 TgALG7，以评估其在寄生虫生物学中的作用，重点是针对其多种独特分泌细胞器的蛋白质。鉴于糖基化在组织囊肿中的核心作用，我们提出了一种实验策略来选择性敲除组织囊肿中的基因表达。该技术将应用于确定 TgALG7 的贡献以及 N-糖基化对组织囊肿形成和稳定性的影响。这项工作代表了首次使用最先进的方法来调节基因表达，对弓形虫蛋白糖基化的细胞生物学基础进行深入研究。 公共卫生相关性：弓形虫会导致免疫功能低下的个体（包括艾滋病毒/艾滋病患者）患上严重疾病。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，我们直接探索休眠组织囊肿形式中的 N-糖基化。我们希望为这一正在探索的调查领域的未来研究奠定坚实的基础。