The Ca2+-sensing machinery operating on exocytosis in Toxoplasma

弓形虫胞吐作用中的 Ca2 感应机制

• 批准号: 9203658
• 负责人: Marc-Jan Gubbels
• 金额: $ 40.68万
• 依托单位: BOSTON COLLEGE
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-06-16 至 2021-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9203658
• 关键词: Acquired Immunodeficiency Syndrome; Activator Appliances; Address; Affect; Alleles; Binding; Biochemical; Biological; Biology; Boxing; Cardiac; Cell-Matrix Junction; Cells; Comparative Study; Complex; Data; Disease; Dominant-Negative Mutation; Drug Design; Drug Targeting; Encephalitis; Event; Exocytosis; Fluorescence; Future; Genes; Genetic; Goals; Human; Immunocompromised Host; Infection; Invaded; Investigation; Kinetics; Lead; Leg; Life; Life Style; Light; Mass Spectrum Analysis; Mediating; Membrane; Membrane Fusion; Microscopy; Molecular; Mutation; Myocarditis; Opportunistic Infections; Organelles; Parasites; Pathogenesis; Pathology; Pathway interactions; Patients; Pharmaceutical Preparations; Physiology; Point Mutation; Population; Process; Proteins; Receptor Signaling; Resolution; SNAP receptor; Signaling Protein; Specificity; Symptoms; System; Testing; Therapeutic; Tissues; Toxic effect; Toxoplasma; Toxoplasma gondii; Toxoplasmosis; Work; Yeasts; base; cell motility; design; drug development; insight; knock-down; mutant; novel; novel therapeutics; pathogen; prophylactic; protein function; protein transport; research study; rhoptry; sensor; seropositive; trafficking; transmission process; yeast two hybrid system

Project Summary Toxoplasmosis is caused by the obligate intracellular apicomplexan parasite Toxoplasma gondii. 30% of the global human population is chronically infected with Toxoplasma, typically without symptoms. However, reawakening of a dormant infection in immunocompromised patients can lead to life-threatening encephalitis and myocarditis observed in 50% and 10% of AIDS patients, respectively. Infection cannot be cured and the disease is managed by prophylactic drug administration, which has severe toxicity, particularly upon prolonged use as applied in AIDS patients. Thus, there is an urgent need for new drugs. Toxoplasmosis pathology originates in repeated rounds of intracellular replication and emergence of parasites from the host cell. Host cell invasion is therefore essential for the progression of toxoplasmosis. The essentiality of this step makes it an excellent target for new anti-Toxoplasma therapeutics, which is the rationale for this proposal. We have already demonstrated that invasion is completely reliant upon the Ca2+- dependent secretion of both micronemes and rhoptries. Interestingly, the last leg of protein trafficking through the secretory pathway to the micronemes and rhoptries does not appear to rely on Rab or SNARE proteins and therefore the Ca2+-dependent membrane fusion machinery involved in this process is likely unorthodox (i.e. a specific drug target). In support of this hypothesis, we identified unusual Ca2+-responsive proteins (TgDOC2 and three ferlins) acting in Ca2+-mediated exocytosis. Preliminary investigation revealed that they operate on distinct secretion events, which represent different functions and thus highlight a novel pathogenic mechanism. This proposal will establish a mechanistic understanding of Ca2+-dependent secretion and will shed much- needed light on the physiology and pathogenesis of Toxoplasma to effectively exploit this target for future drug design. Hereto we will combine genetic, cell biological and biochemical approaches in three specific aims: 1. Test the hypothesis that, in Toxoplasma, the ferlins are distinct Ca2+-sensors in different exocytic pathways, whereas TgDOC2 functions as a general Ca2+-dependent activator for secretion. This will be accomplished by generating specific mutations in these genes and assessing organelle secretion dynamics 2. Determine the function of different microneme secretion content by SILAC and establish how the different mutants displaying distinct deficiencies in Ca2+-exocytosis correlate with distinct steps along the egress-motility-invasion trajectory. 3. Identify the likely unorthodox membrane fusion machinery in which these Ca2+-sensors and activators function using a TAP pull-down and mass spectrometry approach. Upon successfully completing the proposed work we expect to have resolved the molecular basis of Ca2+-dependent secretion and to understand the complexity of differential microneme and rhoptry secretion events in the parasite's pathogenesis. These findings will provide the basis for rational new drug development, which would represent a novel mechanism of action and would be a major advance toward effective control of opportunistic toxoplasmosis in AIDS patients.

项目摘要 弓形虫病是由强制性细胞内的寄生虫弓形虫弓形虫引起的。 30％ 全球人口长期感染弓形虫，通常没有症状。然而， 免疫功能低下的患者中休眠感染的重新训练会导致威胁生命的脑炎 在50％和10％的艾滋病患者中观察到心肌炎。感染无法治愈， 疾病是由预防药物管理严重毒性的预防药物管理 使用在艾滋病患者中应用。因此，迫切需要新药。 弓形虫病病理学起源于反复的细胞内复制和寄生虫的出现 来自主机单元。因此，宿主细胞侵袭对于弓形虫病的进展至关重要。这 这一步骤的重要性使其成为新抗氧化质治疗剂的绝佳目标，这是 该提议的理由。我们已经证明，入侵完全依赖于Ca2+ - 微生物和rhoptries的依赖分泌。有趣的是，蛋白质贩运的最后一圈通过 微分和rhoptries的分泌途径似乎并不依赖于Rab或snare蛋白，并且 因此，参与此过程的Ca2+依赖性膜融合机械可能是非正统的（即 特定的药物靶标）。为了支持这一假设，我们确定了异常的Ca2+反应性蛋白（TGDOC2 和三个作用于Ca2+介导的胞吐作用的Ferlins。初步调查表明，他们在 不同的分泌事件，代表不同的功能，因此突出了一种新型的致病机制。 该建议将对Ca2+依赖性分泌建立机械理解，并将其抛弃 - 需要对弓形虫的生理和发病机理有效利用该靶标的未来药物 设计。迄今为止，我们将在三个特定目标中结合遗传，细胞生物学和生化方法：1。 检验以下假设：在毒品中，在不同的外生途径中，ferlins是不同的Ca2+传感器， 而TGDOC2作为分泌的一般Ca2+依赖性激活剂起作用。这将通过 在这些基因中产生特定的突变并评估细胞器分泌动力学2。 SILAC通过不同的MicroNeme分泌含量的功能，并确定不同突变体如何显示 Ca2+ - 细胞增多症的明显缺陷与沿着出口侵入性轨迹的不同步骤相关。 3。确定这些CA2+传感器和激活剂的可能非传统膜融合机制 使用TAP下拉和质谱法的功能。成功完成建议的 我们期望解决Ca2+依赖性分泌的分子基础的工作，并了解 寄生虫发病机理中差异微神经和变体分泌事件的复杂性。这些 调查结果将为理性新药开发提供基础，这将代表一种新的机制 行动，将是有效控制艾滋病患者机会性弓形虫病的重大进步。