The Ca2+-sensing machinery operating on exocytosis in Toxoplasma

项目摘要 弓形虫病是由专性细胞内顶复体寄生虫弓形虫引起的。30%的 全球人口慢性感染弓形虫，通常没有症状。然而，在这方面， 免疫功能低下的患者体内潜伏感染的再次觉醒可导致危及生命的脑炎 在艾滋病患者中，分别有50%和10%观察到心肌炎。感染无法治愈， 通过预防性药物给药来控制疾病，这具有严重的毒性，特别是在长时间给药后。 用于艾滋病患者。因此，迫切需要新的药物。 弓形虫病的病理起源于细胞内重复的复制和寄生虫的出现 从宿主细胞中。因此，宿主细胞的侵入对于弓形虫病的进展是必不可少的。的 这一步骤的重要性使其成为新的抗弓形虫治疗剂的极好靶点， 这一提议的理由。我们已经证明，入侵是完全依赖于Ca2 +- 微丝和棒状体的依赖性分泌。有趣的是，蛋白质运输的最后一段 微丝体和棒状体的分泌途径似乎不依赖于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Ca 2+响应蛋白（TgDOC 2 和三种ferlin）在Ca 2+介导的胞吐中起作用。初步调查显示， 不同的分泌事件，代表不同的功能，因此突出了一种新的致病机制。 这一建议将建立一个机制的理解钙依赖性分泌，并将摆脱很多- 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，为未来的药物 设计为此，我们将结合联合收割机遗传，细胞生物学和生物化学的方法在三个具体目标：1。 检验以下假设：在弓形虫中，ferlin是不同胞吐途径中的不同Ca2+传感器， 而TgDOC 2作为一般的Ca2+依赖性分泌激活剂发挥作用。这将通过 在这些基因中产生特定突变并评估细胞器分泌动力学2.确定 通过SILAC研究不同微线体分泌物含量的功能，并确定不同突变体如何显示 Ca 2+胞吐作用的明显缺陷与沿流出-运动-侵入轨迹的明显步骤沿着相关。 3.确定可能的非正统的膜融合机制，其中这些Ca2 +-传感器和激活剂 功能使用TAP下拉和质谱法。在成功完成拟议的 我们希望这项工作已经解决了Ca2+依赖性分泌的分子基础，并了解了 在寄生虫的发病机制中，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， 这将是有效控制艾滋病患者机会性弓形虫病的一个重大进展。

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.