Cytoskeletal control of Leishmania infection

描述（由申请人提供）：利什曼原虫是一种寄生虫，可导致生活在发展中国家的人严重的皮肤溃疡或内脏疾病。每年，它感染数百万人，并杀死超过50，000人。治疗利什曼原虫感染的药物毒性很大，寄生虫正在对它们产生抗药性。由于这些原因，需要新的药物来治疗这种寄生虫的感染。利什曼原虫必须生活在巨噬细胞内才能在宿主中生存并引起疾病。为了进入巨噬细胞，利什曼原虫与细胞表面的整合素受体结合。然后，寄生虫通过吞噬作用触发自身的摄取。利什曼原虫进入细胞的机制还不清楚。该项目旨在表征将细胞表面受体的结合翻译为细胞骨架重组的信号事件，这是利什曼原虫内化所必需的。了解利什曼原虫进入细胞的机制将有助于解释这种寄生虫如何引起疾病。此外，如果能够破坏导致利什曼原虫侵入巨噬细胞的途径，那么寄生虫就不应该能够在宿主体内存活并引起疾病。因此，这些研究也可能提出治疗这种寄生虫感染的新药。 Abl家族激酶是将信号从细胞外整联蛋白受体转移到细胞骨架以协调细胞运动的蛋白质。已知这些激酶可激活皮质蛋白，从而触发基于肌动蛋白的细胞边缘突起。HS 1（造血谱系细胞特异性蛋白1）是一种与巨噬细胞中发现的皮质激素相关的蛋白质。实验室的初步工作表明，来自细胞表面受体的信号激活Abl家族激酶并刺激吞噬作用。然而，需要Abl家族激酶或HS 1的信号传导途径与巨噬细胞对利什曼原虫的摄取无关。 该提议的中心假设是利什曼原虫使用需要整合素受体、Abl家族激酶和HS 1的信号通路进入细胞并引起疾病。具体目标1解决了Abl家族激酶是否介导利什曼原虫的细胞进入和感染，以及22个整联蛋白是否直接结合并激活Abl家族激酶。具体目标2解决了利什曼原虫是否需要HS 1信号传导用于细胞进入和感染;它还测试了Abl家族激酶是否激活HS 1。在这两个具体目标中，巨噬细胞将被利什曼原虫感染以了解这些信号传导组分中的每一种对于细胞进入的需求，小鼠将被利什曼原虫感染以确定这些蛋白质在疾病中的意义。用纯化蛋白质和分子生物学技术破坏蛋白质之间的相互作用的生化测定也将用于阐明该信号通路中关键组分之间的界面。通过鉴定整合素、Abl家族激酶和HS 1是否允许利什曼原虫进入细胞并在小鼠中引起疾病，我们对利什曼原虫感染过程的理解将得到改善。 公共卫生相关性：该项目旨在解释一种名为利什曼原虫的人类寄生虫如何进入细胞以生存。利什曼原虫每年在全世界感染数百万人，并导致其中5万多人死亡。我们希望，如果我们了解利什曼原虫如何进入细胞，就可以开发出新的药物来阻止细胞进入，从而阻止这种寄生虫引起的疾病。

DESCRIPTION (provided by applicant): Leishmania is a parasite that causes severe skin ulcers or visceral disease in people living in the developing world. Every year, it infects millions of people, and kills over 50,000. Drugs for treating infection by Leishmania are very toxic, and the parasite is developing resistance to them. For these reasons, new drugs are needed to treat infections by this parasite. Leishmania must live inside macrophages to survive in and cause disease within a host. To enter macrophages, Leishmania binds to integrin receptors on the cell surface. The parasite then triggers its own uptake through the process of phagocytosis. The mechanisms that permit cell entry by Leishmania are not well understood. This project aims to characterize the signaling events that translate the binding of cell surface receptors to the reorganization of the cytoskeleton, which is required for internalization of Leishmania. Understanding the mechanisms of cell entry by Leishmania will help explain how this parasite causes disease. Furthermore, if one could disrupt the pathways that lead to invasion of macrophages by Leishmania, the parasite should not be able to survive within a host to cause disease. Therefore, these studies may also suggest new drugs to treat this parasitic infection. Abl family kinases are proteins that transfer signals from extracellular integrin receptors to the cytoskeleton in order to orchestrate cell movement. These kinases are known to activate cortactin, which triggers actin-based cell edge protrusions. HS1 (hematopoietic lineage cell-specific protein 1) is a protein related to cortactin that is found in macrophages. Preliminary work in the laboratory demonstrates that signals from cell surface receptors activate Abl family kinases and stimulate phagocytosis. However, signaling pathways requiring Abl family kinases or HS1 have not been linked to uptake of Leishmania by macrophages. The central hypothesis in this proposal is that Leishmania uses a signaling pathway that requires integrin receptors, Abl family kinases, and HS1 to enter cells and cause disease. Specific Aim 1 addresses whether Abl family kinases mediate cell entry and infection by Leishmania, and if 22 integrins directly bind and activate Abl family kinases. Specific Aim 2 addresses whether Leishmania requires HS1 signaling for cell entry and infection; it also tests whether Abl family kinases activate HS1. In both specific aims, macrophages will be infected with Leishmania to understand the requirement for each of these signaling components for cell entry, and mice will be infected with Leishmania to determine the significance of these proteins in disease. Biochemical assays with purified proteins and molecular biology techniques to disrupt interactions between proteins will also be employed to elucidate the interfaces between key components in this signaling pathway. By identifying whether signaling though integrins, Abl family kinases, and HS1 allows Leishmania to enter cells and cause disease in mice, our understanding of the process of Leishmania infection will improve. PUBLIC HEALTH RELEVANCE: This project seeks to explain how a human parasite called Leishmania gains entry into cells in order to survive. Leishmania infects millions of people every year worldwide, and kills more than 50,000 of them. We hope that if we understand how Leishmania enters cells, new drugs can be developed that block cell entry, and therefore, disease caused by this parasite.