The Ca2+-sensing machinery operating on exocytosis in Toxoplasma

项目摘要 弓形虫病是由弓形虫专性胞内寄生虫引起的。30%的 全球人口长期感染弓形虫，通常没有症状。然而， 免疫功能低下患者的潜伏感染重新苏醒可导致危及生命的脑炎 分别有50%和10%的艾滋病患者出现心肌炎。感染无法治愈，而 疾病是通过预防性给药来管理的，这种药物有严重的毒性，特别是在长期服用的情况下。 适用于艾滋病患者。因此，迫切需要新药。 弓形虫病的病理起源于反复的细胞内复制和寄生虫的出现 从宿主细胞。因此，宿主细胞的入侵对于弓形虫病的发展是必不可少的。这个 这一步骤的重要性使其成为新的抗弓形虫疗法的极佳靶点，这是 这项建议的理由。我们已经证明，侵袭完全依赖于钙离子- 微线粒和棒状染色体的依赖分泌。有趣的是，蛋白质运输的最后一段通过 微丝和棒状体的分泌途径似乎不依赖于Rab或SNARE蛋白和 因此，参与这一过程的依赖于钙的膜融合机制可能是非正统的(即 特定的药物靶点)。为了支持这一假设，我们发现了异常的钙反应蛋白(TgDOC2 和三种)在钙离子介导的胞吐作用中起作用。初步调查显示，他们在 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。 这一建议将建立对钙离子依赖的分泌的机械理解，并将在很大程度上减少 需要了解弓形虫的生理和发病机制，以便有效地开发这一靶点用于未来的药物 设计。在这里，我们将结合遗传学、细胞生物学和生物化学的三个具体目标：1. 测试假设，在弓形虫中，费林在不同的胞外途径中是不同的钙感受器， 而TgDOC2则是一种普通的钙依赖的分泌激活剂。这将通过以下方式实现 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.确定 SILAC对不同微线体分泌含量的作用及不同突变体的表现 钙离子排出功能的明显缺陷与出口-运动-侵袭轨迹上的不同步骤相关。 3.确定可能是非正统的膜融合机制，其中这些钙传感器和激活剂 功能使用分接下拉和质谱分析方法。在成功完成建议的 我们的工作有望解决钙依赖分泌的分子基础，并理解 在寄生虫发病机制中差异微线体和棒状细胞分泌事件的复杂性。这些 这些发现将为合理的新药开发提供依据，这将代表一种新的治疗机制 行动，将是朝着有效控制艾滋病患者机会性弓形虫病的方向取得的重大进展。

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.