The Ca2+-sensing machinery operating on exocytosis in Toxoplasma

弓形虫胞吐作用中的 Ca2 感应机制

基本信息

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
项目类别：
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

项目摘要

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.

项目概要弓形虫病是由专性细胞内顶复门寄生虫弓形虫引起的。 30%的全球人口长期感染弓形虫，通常没有症状。然而，免疫功能低下患者中休眠感染的重新唤醒可能导致危及生命的脑炎艾滋病患者中分别有 50% 和 10% 患有心肌炎。感染无法治愈，并且疾病是通过预防性给药来控制的，这种给药具有严重的毒性，特别是在长期给药后应用于艾滋病患者。因此，迫切需要新药。弓形虫病病理起源于反复的细胞内复制和寄生虫的出现来自宿主细胞。因此，宿主细胞入侵对于弓形体病的进展至关重要。这这一步骤的重要性使其成为新的抗弓形虫疗法的绝佳靶标，这是该提案的理由。我们已经证明入侵完全依赖于Ca2+- 微线体和菱形体的依赖性分泌。有趣的是，蛋白质贩运的最后一站是通过微线体和棒状体的分泌途径似乎并不依赖于 Rab 或 SNARE 蛋白因此，参与该过程的 Ca2+ 依赖性膜融合机制可能是非正统的（即特定药物靶点）。为了支持这一假设，我们鉴定了不寻常的 Ca2+ 反应蛋白 (TgDOC2 和三个 ferlins）在 Ca2+ 介导的胞吐作用中发挥作用。初步调查显示，他们的经营目的是不同的分泌事件代表不同的功能，从而突出了一种新的致病机制。该提案将建立对 Ca2+ 依赖性分泌的机制理解，并将产生很多- 需要了解弓形虫的生理学和发病机制，以有效地利用这一目标来开发未来的药物设计。在此，我们将结合遗传、细胞生物学和生化方法来实现三个具体目标：1. 检验以下假设：在弓形虫中，ferlins 是不同胞吐途径中不同的 Ca2+ 传感器，而 TgDOC2 则充当一般 Ca2+ 依赖性分泌激活剂。这将通过以下方式完成在这些基因中产生特定突变并评估细胞器分泌动态 2. 确定通过 SILAC 分析不同微线体分泌内容的功能，并确定不同突变体如何表现 Ca2+胞吐作用的明显缺陷与沿出口-运动-入侵轨迹的不同步骤相关。 3. 确定这些 Ca2+ 传感器和激活剂可能存在的非正统膜融合机制使用 TAP 下拉和质谱方法进行功能。成功完成提议后我们希望通过这项工作解决 Ca2+ 依赖性分泌的分子基础，并了解寄生虫发病机制中差异微线体和棒状体分泌事件的复杂性。这些研究结果将为合理的新药开发提供基础，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展。