Novel membrane defense systems utilized by the human pathogen Leishmania

项目摘要 抗利什曼病药物利什曼菌素B的治疗指数较低，这可能是由于保护了利什曼病的 利什曼原虫质膜中的麦角固醇。利什曼原虫隐藏麦角固醇的程度以及它如何 重新密封它的膜从未被研究过。因此，迫切需要确定 利什曼原虫通过这种方式保护它们的固醇以防止膜损伤， 膜。没有这些信息，抗麦角固醇药物如麦角碱B的全部潜力将无法发挥。 实现，并且改进的膜破坏策略将不会被识别。长期目标是确定 膜机制和脂质的利什曼原虫，将提供高选择性控制寄生虫。的 总体目标是确定利什曼原虫用于预防和重新密封膜损伤的机制。中央 假设利什曼原虫通过保护麦角固醇不与固醇结合来防止膜损伤 毒素和药物通过主要利什曼原虫鞘脂，肌醇磷酸神经酰胺（IPC），并有效地 通过Ca 2+非依赖性修复重新修复膜损伤。该项目的基本原理是，利什曼原虫是 遗传上易于驾驭的原生动物，在进化上与哺乳动物不同，可能为我们提供关键的见解， 抵抗和重新密封质膜损伤。确定膜防御和修复的差异 利什曼原虫和哺乳动物之间的关系将提供一个强有力的科学框架， 可以改进治疗方法，开发新的治疗方法。为了实现这些目标，将制定以下具体目标： 追求：1）确定减少利什曼原虫膜损伤的机制，和2）确定利什曼原虫膜损伤的机制。 促进利什曼原虫膜修复的机制。在目标1中，工作假设利什曼原虫 通过在IPC下保护脆弱的甾醇来防止膜损伤将通过挑战L.主要 前鞭毛体在遗传上或酶促上缺乏鞘脂、磷脂或含固醇的毒力因子- 结合毒素或去污剂并通过流式细胞术测量甾醇可及性、毒素结合和致死率。在 目的2，工作假设利什曼原虫使用Ca 2+非依赖性ESCRT- 将通过测量膜脱落、补片修复和 毒素攻击L.通过流式细胞术主要表达GFP标记的ESCRT蛋白， 超离心和活细胞成像。完成这项工作的预期成果是确定 利什曼原虫保护其固醇免受攻击的机制，以及 在进化上远离哺乳动物的人类病原体中恢复体内平衡。拟议的研究是 创新，因为它通过揭示甾醇可及性的新范式而脱离现状， 膜修复这些结果将产生积极的影响，因为更好地了解利什曼原虫如何 保护它们的细胞膜将提供新的药物靶点。利什曼原虫的靶向保护机制可能 增强药物如利什曼原虫素B，并为利什曼原虫和其他原生动物提供选择性靶点。

PROJECT ABSTRACT The anti-Leishmaniasis drug amphotericin B has a low therapeutic index, which may be due to protection of ergosterol in the Leishmania plasma membrane. The extent to which Leishmania hides its ergosterol and how it reseals its membrane have never been examined. Thus, there is a critical need to determine the mechanisms by which Leishmania protect their sterols to prevent membrane damage, and by which they reseal damaged membranes. Without this information, the full potential of anti-ergosterol drugs like amphotericin B will not be realized, and improved membrane disrupting strategies will not be identified. The long-term goal is to identify membrane mechanisms and lipids in Leishmania that will provide high selectivity for controlling the parasite. The overall goal is to identify the mechanisms Leishmania use to prevent and reseal membrane damage. The central hypothesis is that Leishmania prevent membrane damage by protecting ergosterol from access to sterol-binding toxins and drugs via the primary Leishmania sphingolipid, inositol phosphorylceramide (IPC), and potently reseals membrane damage via Ca2+-independent repair. The rationale for the project is that Leishmania are genetically tractable protozoa evolutionarily distinct from mammals that are likely to provide key insights into resisting and resealing plasma membrane damage. Determining the differences in membrane defense and repair between Leishmania and mammals will provide a strong scientific framework in which existing anti-Leishmania therapies can be improved, and new therapies developed. To attain the objectives, these specific aims will be pursued: 1) Determine the mechanisms that reduce membrane damage in Leishmania, and 2) Determine the mechanisms that promote membrane repair in Leishmania. In Aim 1, the working hypothesis that Leishmania prevent membrane damage by sheltering vulnerable sterols under IPC will be tested by challenging L. major promastigotes genetically or enzymatically lacking sphingolipids, phospholipids or virulence factors with sterol- binding toxins or detergents and measuring sterol accessibility, toxin binding, and lethality by flow cytometry. In Aim 2, the working hypothesis that Leishmania reseal their membrane using Ca2+ independent, ESCRT- dependent shedding of damaged membranes will be tested by measuring membrane shedding, patch repair and ESCRT trafficking in toxin-challenged L. major expressing GFP-tagged ESCRT proteins by flow cytometry, ultracentrifugation, and live cell imaging. The expected outcomes of completing this work are to have defined the mechanisms by which Leishmania protect their sterols from attack, and membrane repair pathways that restore homeostasis in human pathogens evolutionarily distant from mammals. The proposed research is innovative because it departs from the status quo by revealing new paradigms of sterol accessibility and membrane repair. These results will have a positive impact because a better understanding of how Leishmania protect their membrane will provide new drug targets. Targeting protective mechanisms in Leishmania may potentiate drugs like amphotericin B, and provide a selective target for Leishmania and other protozoa.