Leishmania major promastigote entry of the of the autophagic compartment and amastigote escape from the parasitophorous vacuole

在最初的三年中，我们将（i）使用最先进的高压冷冻和电子断层扫描技术观察人巨噬细胞中含有寄生虫空泡的大型利什曼原虫，重点是前鞭毛体与自噬的相互作用和无鞭毛体从寄生虫空泡中逃逸（ii）我们将尝试下调巨噬细胞系和原代巨噬细胞中的自噬和溶酶体成熟（iii）我们将建立一种新的相关电镜分析方法。在第二个3年资助期内，我们的目标是剖析导致前鞭毛体进入自噬途径和无鞭毛体逃逸的分子基础。(ii)我们将应用相关的电子显微镜技术对质谱筛选出的新靶蛋白进行分析。此外，我们将测试L。donovani已知阻断吞噬溶酶体成熟和L.已知amazonensis诱导大的寄生虫空泡。(iii)此外，我们将集中在人类嗜中性粒细胞中的寄生虫空泡，只能感染前鞭毛体和树突状细胞，只能感染无鞭毛体。

During the first three years we will (i) visualize the leishmania major containing parasitophorous vacuoles in human macrophages using state of the art high pressure freezing and electron tomography focusing on promastigote interactions with autophagy and amastigote escape from the parasitophorous vacuoles (ii) we will try to down regulate autophagy and lysosomal maturation in macrophage cell lines and primary macrophages (iii) we will establish a new assay for correlative electron microscopy. During the second 3-year funding period (i) we aim to dissect the molecular basis leading to promastigote entry of the autophagic pathway and amastigote escape. (ii) We will apply the correlative electron microcopy technique on new target proteins coming from our mass spectrometry screen. In addition we will test L. donovani known to block phagolysosomal maturation and L. amazonensis known to induce large parasitophorous vacuoles. (iii) Moreover we will focus on the parasitophorous vacuoles in human neutrophils that can only be infected with promastigotes and dendritic cells that can only be infected with amastigotes.