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.

Public Health Relevance

I propose to study the mechanism by which the parasite Entamoeba histolytica causes amebiasis. E. histolytica has the potent ability to kill human cells by ingesting fragments of live cells in a process called amoebic trogocytosis and I aim to understand this process. Since there is no vaccine for amebiasis and current treatments are inadequate, these studies have the potential to lead to new treatment and prevention strategies for this serious disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Individual Predoctoral NRSA for M.D./Ph.D. Fellowships (ADAMHA) (F30)
Project #
5F30AI114136-02
Application #
9302232
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Wali, Tonu M
Project Start
2016-06-01
Project End
2020-05-31
Budget Start
2017-06-01
Budget End
2018-05-31
Support Year
2
Fiscal Year
2017
Total Cost
Indirect Cost
Name
University of Virginia
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
065391526
City
Charlottesville
State
VA
Country
United States
Zip Code
22904
Gilmartin, Allissia A; Ralston, Katherine S; Petri Jr, William A (2017) Inhibition of Amebic Lysosomal Acidification Blocks Amebic Trogocytosis and Cell Killing. MBio 8: