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

 描述(申请人提供)：溶组织内阿米巴是一种引起阿米巴病的原生动物寄生虫，是全球腹泻疾病问题的重要贡献者。溶组织埃希氏菌具有很强的杀灭多种宿主细胞的能力，组织破坏是其侵袭性感染的标志，但对这两个过程的机制知之甚少。皮氏杆菌实验室最近发现，溶组织埃希氏菌通过摄取活宿主细胞的碎片来杀戮，我们称之为阿米巴巨噬细胞增多症。然而，人们对阿米巴巨噬细胞增多症的机制以及它如何导致细胞杀伤知之甚少。我的目标是了解溶组织肠杆菌导致宿主细胞死亡的机制。我们的数据表明，阿米巴巨噬细胞增多是细胞杀伤的主要机制，摄取单一片段不足以导致宿主细胞死亡，相反，阿米巴鼠在宿主细胞死亡之前继续摄取多个宿主细胞片段。阿米巴溶酶体pH升高可降低细胞毒性，我的初步数据表明，抑制溶酶体酸化可减少宿主物质的摄取和细胞杀伤。因此，我推测阿米巴溶酶体在持续的阿米巴巨噬细胞增多症和宿主细胞杀伤中起着关键作用。在真核生物中，溶酶体对摄取物质的周转至关重要。我认为阿米巴溶酶体通过有效地消化在巨噬细胞增多症中获得的宿主细胞片段来促进细胞杀伤。溶酶体酸化的药理抑制剂和溶酶体基因突变体将分别用于扰乱溶酶体的功能和运输到溶酶体。用成像流式细胞术测定巨噬细胞增多率和宿主细胞杀伤率，以及摄取的宿主物质的酸化率和周转率。此外，我还将研究阿米巴蛋白家族在阿米巴巨噬细胞增多症中的作用。阿米巴蛋白是一种成孔蛋白家族，长期以来被认为是分泌的毒力因子。基于阿米巴胺定位于溶酶体和需要pH~5.2才能形成孔道的发现，我还提出阿米巴不是分泌的细胞毒效应因子，而是在巨噬细胞增多症后阿米巴溶酶体内摄取的宿主片段的处理中发挥重要作用。我将在巨噬细胞增多症期间标记和跟踪变形虫，以确定它们是否定位于含有吞噬片段的吞噬溶菌体。我还将敲除阿米巴基因的表达，并评估阿米巴基因突变体中巨噬细胞增多症和宿主细胞杀伤率。这项工作将有助于更好地理解巨噬细胞增多症作为一种基本生物学过程的本质，将彻底改变目前对溶酶体和变形虫在细胞杀伤中作用的看法，并将使新的治疗靶点的确定成为可能。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内科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.