LYOSOME--MEDIATED CELL INVASION BY TRYPANOSOMA CRUZI

我们实验室最近的研究揭示了细胞的一种新机制 原生动物克氏锥虫入侵。在进入非政府组织之前 吞噬哺乳动物细胞、宿主溶酶体被招募到寄生虫 附着部位并逐渐与质膜融合，起源于 含有寄生虫的液泡。在我们寻找信号转导的过程中 在这一过程中，我们发现具有感染性的锥体鞭毛虫 从宿主细胞内的存储中动员钙离子，诱导 细胞内游离钙离子浓度一过性升高 哺乳动物细胞类型的多样性。宿主细胞的快速重排 皮质肌动蛋白细胞骨架，这种情况已知会导致T。 CRUZI的侵袭，观察到是由锥虫诱导的结果 CA2+-瞬变。显著地防止这些瞬变 抑制寄生虫对宿主细胞的入侵。我们现在计划直接 克氏锥虫诱导的细胞内游离钙离子与细胞内钙离子的关系 加上瞬变和溶酶体的动员和融合 哺乳动物细胞的质膜。具体来说，我们将：1)使用 视频显微镜分析，检测宿主细胞胞浆游离钙的作用 再加上募集和/或质膜融合的瞬变 克鲁兹毛滴虫诱导的溶酶体；2)研究主要的PKC的作用 底物、钙调素和肌动蛋白结合蛋白标记 导致溶酶体融合和克氏锥虫入侵的宿主细胞事件；3) 使用通透性哺乳动物细胞系统来研究分子 钙离子调节溶酶体胞吐的机制， 特别是NSF/SNAP/SNARE融合复合体的作用， 突触素、膜联蛋白、单体和三聚体GTP结合蛋白； 4)将溶酶体胞吐试验获得的数据与T. 克鲁兹的侵袭，使用特定的抑制剂和细胞显微注射。 这些研究将促进我们对不寻常的细胞入侵的了解 人类重要病原体克氏锥虫利用的机制，以及 哺乳动物涉及溶酶体的基本膜交通事件 细胞。

Recent studies from our laboratory uncovered a novel mechanism for cell invasion by the protozoan Trypanosoma cruzi. Prior to entry into non- phagocytic mammalian cells, host lysosomes are recruited to the parasite attachment site and gradually fuse with the plasma membrane, originating the parasite-containing vacuole. In our search for signal transduction events involved in this process, we found that infective trypomastigotes mobilize Ca2 plus from host cell intracellular stores, inducing transient increases in the cytosolic free Ca2 plus concentration of a variety of mammalian cell types. Rapid rearrangements in the host cell cortical actin cytoskeleton, a condition known to result in enhanced T. cruzi invasion, are observed as a consequence of trypomastigote-induced Ca2 plus-transients. Prevention of these transients significantly inhibits host cell invasion by the parasites. We now plan to directly investigate the link between the T. cruzi-induced intracellular free Ca2 plus transients and the mobilization and fusion of lysosomes with the plasma membrane in mammalian cells. Specifically, we will: 1) using a video-microscopy assay, examine the role of host cell cytosolic free Ca2 plus transients on the recruitment and/or plasma membrane fusion of lysosomes induced by T. cruzi; 2) investigate the role of the major PKC substrate, Ca2 plus-calmodulin and actin-binding protein MARCKS in the host cell events leading to lysosome fusion and T. cruzi invasion; 3) use a permeabilized mammalian cell system to investigate the molecular mechanisms involved in Ca2 plus-regulated lysosome exocytosis, specifically the role of the NSF/SNAP/SNARE fusion complex, synaptotagmin, annexins and monomeric and trimeric GTP-binding proteins; 4) correlate the data obtained in the lysosome exocytosis assay with T. cruzi invasion, using specific inhibitors and cellular microinjection. These studies will advance our knowledge of the unusual cell invasion mechanism utilized by the important human pathogen T. cruzi, as well as of fundamental membrane traffic events involving lysosomes in mammalian cells.