Direct and high-throughput assays for human cell killing through trogocytosis by Entamoeba histolytica

Abstract

Entamoeba histolytica is a microbial eukaryote and causative agent of the diarrheal disease amoebiasis. Pathogenesis is associated with profound damage to human tissues, and treatment options are limited. We discovered that amoebae attack and kill human cells through a cell-nibbling process that we named trogocytosis (trogo-: nibble). Trogocytosis is likely to underlie tissue damage during infection and it represents a potential target for therapeutic intervention, although the mechanism is still unknown. Assays in current use to analyze trogocytosis by amoebae have not been amenable to studying different types of human cells, or to high-throughput analysis. Here, we developed two complementary assays to measure trogocytosis by quantifying human cell viability, both of which can be used for suspension and adherent cells. The first assay uses CellTiterGlo, a luminescent readout for cellular ATP levels, as a proxy for cell viability. We found that the CellTiterGlo signal is proportional to the quantity of viable cells, and can be used to detect death of human cells after co-incubation with amoebae.We established a second assay that is microscopy-based and uses two fluorescent stains to directly differentiate live and dead human cells. Both assays are simple and inexpensive, can be used with suspension and adherent human cell types, and are amenable to high-throughput approaches. These new assays are tools to improve understanding of amoebiasis pathogenesis.

Molecular interplays of the Entamoeba histolytica endosomal sorting complexes required for transport during phagocytosis

“…To perform the capturing of the prey, in general, trophozoites require to adhere to the target. The virulent strains of E. histolytica display a high rate of pinocytosis (micropinocytosis and macropinocytosis) and endocytosis (trogocytosis and phagocytosis) (Rodrıǵuez and Orozco, 1986;Laughlin et al, 2004;Bettadapur and Ralston, 2020). Pinocytosis is the process by which the trophozoites absorb extracellular fluids and compounds (Laughlin et al, 2004), while in endocytosis, the cells capture macromolecules, cell surface components and even whole cells that include red blood cells (RBCs), live and apoptotic mammalian cells, and bacteria (Labruyère and Guilleń, 2006).…”

“…Endocytic processes include phagocytosis, that refers to live whole, damaged or dead cells engulfment; and trogocytosis, where trophozoites ingest part of the living cells ( Ralston et al., 2014 ; Bettadapur and Ralston, 2020 ; Nakada-Tsukui and Nozaki, 2021 ). The PM invagination and the formation of vesicles and vacuoles required for these processes, imply the renewal of the PM every 30 min ( Doherty and McMahon, 2009 ).…”

Trogocytosis between Non-Immune Cells for Cell Clearance, and among Immune-Related Cells for Modulating Immune Responses and Autoimmunity

“…Furthermore, Somlata et al [ 25 ] documented that AGC family kinase 1 was specifically involved in trogocytosis of living human cells but not participate in phagocytosis of dead cells by Eh. Recently, Bettadapur et al [ 26 ] by using direct and high-throughput assay demonstrated that inhibition of human cell actin or amoeba surface Gal/GalNAc lectin could inhibit amoebic trogocytosis.…”

Conserved actin machinery drives microtubule-independent motility and phagocytosis in Naegleria

“…0.5 ml of cells were seeded (at ∼5 × 10 5 cells/ml) into a 24-well plate preloaded with 0.5 ml fresh M7 medium with inhibitors or controls for final (1×) concentrations as follows: 5 µM LatB, 20 µM cytochalasin D, 140 nM phalloidin, 2 µM jasplakinolide, 50 µM CK-666, 25 µM SMIFH2, 50 µM CK-689, 0.1% DMSO, and 0.09% ethanol. Concentrations were selected based on work in other systems (Bettadapur and Ralston, 2020;Fritz-Laylin et al, 2017b;Jahan and Yumura, 2017;Jasnin et al, 2016;Manich et al, 2018;Olins and Olins, 2004;Ralston et al, 2014;Spector et al, 1983;Velle and Campellone, 2018;Wang et al, 2014;Williams and Kay, 2018;Wilson et al, 2013) and/ or were tested at 10-fold dilutions within the range recommended in the company-supplied data sheets in preliminary experiments. From preliminary experiments, the lowest concentration that had an observable phenotype in live cells was chosen, or in the case of cytochalasin D, jasplakinolide, and phalloidin, the highest concentrations we tested were chosen, which still did not produce any obvious phenotypes.…”