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

Entamoeba histolytica is the causative agent of amoebiasis. Pathogenesis is associated with profound damage to human tissues. We previously showed that amoebae kill human cells through trogocytosis. Trogocytosis is likely to underlie tissue damage during infection, although the mechanism is still unknown. Trogocytosis is difficult to assay quantitatively, which makes it difficult to study. Here, we developed two new, complementary assays to measure trogocytosis by quantifying human cell death. One assay uses CellTiterGlo, a luminescent readout for ATP, as a proxy for cell death. We found that the CellTiterGlo could be used to detect death of human cells after co-incubation with amoebae, and that it was sensitive to inhibition of actin or the amoeba surface Gal/GalNAc lectin, two conditions that are known to inhibit amoebic trogocytosis. The other assay uses two fluorescent nuclear stains to directly differentiate live and dead human cells by microscopy, and is also sensitive to inhibition of amoebic trogocytosis through interference with actin. 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 trogocytosis and amoebiasis pathogenesis.