The Role of Lysosomes and Amoebapores in Amoebic Trogocytosis and Cell Killing

 描述（由申请方提供）：溶组织内阿米巴是一种引起阿米巴病的原生动物寄生虫，是全球性阿米巴病问题的重要贡献者。E.溶组织大肠杆菌具有杀死多种宿主细胞的能力，组织破坏是其侵袭性的标志。histolytica感染，但很少有人知道负责这两个过程的机制。Petri实验室最近发现，E.溶组织菌通过摄入活宿主细胞的碎片来杀死宿主，我们称之为阿米巴胞啃作用。然而，很少有人了解阿米巴胞刺的机制或它如何导致细胞杀伤。我的目标是了解E.溶组织菌引起宿主细胞死亡。我们的数据表明，阿米巴胞刺是细胞杀伤的主要机制，并且摄入单个片段不足以引起宿主细胞死亡，而是阿米巴在宿主细胞死亡之前继续摄入多个宿主细胞片段。阿米巴溶酶体pH值升高已被证明可降低细胞毒性，我的初步数据表明，抑制溶酶体酸化可减少宿主物质的摄入和细胞杀伤。因此，我推测阿米巴溶酶体在持续的阿米巴胞饮和宿主细胞杀伤中起着至关重要的作用。在真核生物中，溶酶体对于摄入物质的周转至关重要。我认为，阿米巴溶酶体有助于有效地消化在胞啃过程中获得的宿主细胞碎片杀死细胞。溶酶体酸化的药理学抑制剂和溶酶体遗传突变体将分别用于干扰溶酶体功能和向溶酶体的运输。将使用成像流式细胞术测定胞啃率和宿主细胞杀伤率以及摄入宿主材料的酸化率和周转率。此外，我将调查变形虫的作用，一个家庭的孔形成蛋白质长期被认为是分泌的毒力因子，在变形虫trogocytsis。基于发现，阿米巴定位于溶酶体，并需要pH ~5.2的孔形成活性，我还建议，阿米巴分泌的细胞毒性效应，而是在加工中发挥重要作用的摄入宿主片段内的阿米巴溶酶体胞饮。我将标记和跟踪的变形虫在trogocytsis，以确定他们是否本地化的吞噬溶酶体含有摄入的片段。我还将敲低阿米巴表达，并评估阿米巴突变体中的胞刺率和宿主细胞杀伤率。 这项工作将有助于更好地理解作为基本生物学过程的胞洞作用的性质，将完全改变目前对溶酶体和阿米巴在细胞杀伤中作用的看法，并将能够识别新的治疗靶点。重要的是，通过这些研究获得的技能和知识将对我未来作为一名医生科学家的职业生涯至关重要。

 DESCRIPTION (provided by applicant): Entamoeba histolytica is a protozoan parasite that causes amebiasis, and is an important contributor to the global problem of diarrheal disease. E. histolytica has the potent ability to kill a variety of host cell types, and tissue destruction is he hallmark of invasive E. histolytica infection, but little is known about the mechanisms responsible for these two processes. The Petri lab has recently discovered that E. histolytica kills by ingesting fragments of live host cells, which we have termed amoebic trogocytosis. However, very little is understood about the mechanism of amoebic trogocytosis or how it leads to cell killing. My goal is to understand the mechanism by which E. histolytica causes host cell death. Our data suggest that amoebic trogocytosis is the primary mechanism of cell killing and that ingestion of a single fragment is not sufficient to elicit host cell death, rather amoebae mus continue to ingest multiple host cell fragments before the host cell dies. Elevated amoebic lysosomal pH has been shown to decrease cytotoxicity and my preliminary data suggests that inhibiting lysosomal acidification decreases ingestion of host material and cell killing. Thus, I hypothesize that amoebic lysosomes play a crucial role in continued amoebic trogocytosis and host cell killing. In eukaryotes, lysosomes are crucial for the turnover of ingested material. I propose that amoebic lysosomes contribute to cell killing by efficiently digesting the host cell fragments acquired during trogocytosis. Pharmacological inhibitors of lysosomal acidification and a lysosome genetic mutant will be used to perturb lysosomal function and trafficking to the lysosome, respectively. The rate of trogocytosis and host cell killing, as well as the rate of acidification and turnover of ingested host material will be determined using imaging flow cytometry. In addition, I will investigate the role of amoebapores, a family of pore-forming proteins long considered secreted virulence factors, in amoebic trogocytosis. Based on findings that amoebapores localize to lysosomes and require pH ~5.2 for pore-forming activity, I also propose that amoebapores are not secreted cytotoxic effectors, but rather play an important role in the processing of ingested host fragments within the amoebic lysosome following trogocytosis. I will tag and track the amoebapores during trogocytosis to determine whether they localize to phagolysosomes containing ingested fragments. I will also knockdown amoebapore expression and assess the rate of trogocytosis and host cell killing in amoebapore mutants. This work will contribute to a better understanding of the nature of trogocytosis as a fundamental biological process, will completely change the current thinking on the role of lysosomes and amoebapores in cell killing, and will enable the identification of novel therapeutic targets. Importantly, the skills and knowledge gained through these studies will be crucial to my future career as a physician scientist.