Identifying Genes Involved in Trogocytosis (cell‐nibbling) in Entamoeba histolytica

Abstract

Entamoeba histolytica (histo‐: tissue; lytic‐: dissolving) is a eukaryotic pathogen that causes amoebic dysentery in humans. Amoebic dysentery is a considerable global burden resulting in about 50 million diarrheal infections and 100,000 deaths per year. The ability of E. histolytica to cause profound tissue damage is likely to be driven by human cell killing. While many other cell‐killing organisms release cell‐killing effector toxins, this is not the case for E. histolytica, and the mechanism by which amoebae kill cells was previously unclear. We recently uncovered a new paradigm for cell killing by showing that amoebae kill human cells by performing trogocytosis (trogo‐: nibble), or taking ‘nibbles', of living human cells leading to cell death. In contrast, amoebae engulf dead (pre‐killed) human cells whole via phagocytosis (phago‐: devour). While there are some shared features between trogocytosis and phagocytosis, we hypothesize that amoebic trogocytosis requires signaling machinery distinct from phagocytosis. There is evidence that phagocytosis is under feed‐forward gene regulation in E. histolytica. We have shown that amoebae that are exposed to living human cells perform more trogocytosis after a second round of exposure to living human cells, suggesting that trogocytosis is likewise under feed‐forward gene regulation. To identify genes differentially involved in trogocytosis versus phagocytosis, we will feed E. histolytica live or dead human cells, respectively, to prompt genes that are involved in these processes to be up‐regulated. We will then perform RNA‐seq analysis to identify genes that have changed substantially in comparison to the reference RNA from amoebae that have not been fed human cells. From this, we expect to develop a list of candidate genes that are differentially important for either trogocytosis or phagocytosis, and genes that are important for both processes. To prepare for performing RNA‐seq analysis on amoebae that have undergone trogocytosis and phagocytosis, we have established amoebic ingestion conditions to maximize ingestion, quantified methods of digestion via imaging flow cytometry, and used qRT‐PCR to analyze gene expression before and after ingestion. After performing RNA‐seq analysis and generating a list of candidate genes that appear to be uniquely involved in trogocytosis, we will then perform functional studies in order to validate the roles of these genes in trogocytosis. Since trogocytosis appears to be a fundamental form of cell‐cell interaction that is present in many eukaryotes, and exaggerated and exploited for cell killing by eukaryotic microbes, this work will have broad impact.Support or Funding InformationNIH MCB T32 GM007377; NIH NIAID K22 Career Transition Award Al108814This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.