Inhibition of Amebic Lysosomal Acidification Blocks Amebic Trogocytosis and Cell Killing

Abstract

Entamoeba histolytica ingests fragments of live host cells in a nibbling-like process termed amebic trogocytosis. Amebic trogocytosis is required for cell killing and contributes to tissue invasion, which is a hallmark of invasive amebic colitis. Work done prior to the discovery of amebic trogocytosis showed that acid vesicles are required for amebic cytotoxicity. In the present study, we show that acidified lysosomes are required for amebic trogocytosis and cell killing. Interference with lysosome acidification using ammonium chloride, a weak base, or concanamycin A, a vacuolar H+ ATPase inhibitor, decreased amebic trogocytosis and amebic cytotoxicity. Our data suggest that the inhibitors do not impair the ingestion of an initial fragment but rather block continued trogocytosis and the ingestion of multiple fragments. The acidification inhibitors also decreased phagocytosis, but not fluid-phase endocytosis. These data suggest that amebic lysosomes play a crucial role in amebic trogocytosis, phagocytosis, and cell killing.

Exploring the mechanism of amebic trogocytosis: the role of amebic lysosomes

“…Thus, it is likely that the amebae internalize plasma membrane and surface receptors as they ingest fragments. Our data show that interference with acidification blocks receptor-dependent processes - both amebic trogocytosis and phagocytosis - but does not impair a receptor-independent process, fluid-phase endocytosis 12 . Together these data suggest that rapid recycling of membrane and receptors, facilitated by the amebic lysosomes, may be required for continued amebic trogocytosis and, thus, cell killing.…”

“…Interestingly, studies comparing E. histolytica with the less-pathogenic species Entamoeba dispar have noted that acidification of the phagosomes takes significantly longer in E. dispar and does not reach the same level of acidification, suggesting a role for lysosomes in the pathogenesis of amebiasis 11 . In our recent work, we found that inhibition of lysosome acidification by either a weak base or a vacuolar -ATPase inhibitor significantly decreased amebic trogocytosis and cell killing 12 . We observed that interference with acidification does not impact the initiation of amebic trogocytosis, but rather impairs continued ingestion and cell killing.…”

“…Treatment with acidification inhibitors also decreased phagocytosis, suggesting that lysosomes play a crucial role in this process as well. Importantly, fluid-phase endocytosis was not affected, indicating that these drugs do not simply block all endocytic processes 12 .…”

The Ethanolamine Permease EutH Promotes Vacuole Adaptation of Salmonella enterica and Listeria monocytogenes during Macrophage Infection

“…For vacuole neutralization, BMDMs were incubated for 1 h prior to infection with the indicated concentration of concanamycin A (Sigma), dimethyl sulfoxide (DMSO) vehicle, or ammonium chloride (79). Following a 1-h pretreatment, cells were washed twice to remove concanamycin A or DMSO vehicle before continuing with a standard gentamicin protection assay as described above.…”

Functional Characterization of an Interferon Gamma Receptor-Like Protein on Entamoeba histolytica

“…histolytica trophozoites overcome innate host defenses with molecules such as cysteine proteases to degrade mucus (8) and prostaglandin E 2 (PGE 2 ) to stimulate epithelial cells to produce interleukin-8 (IL-8), a chemoattractant for neutrophils (9). Various virulence factors act on different stages of the invasion process, such as Gal/GalNAc lectin for adhesion to host cells (10), amebapores that cause cytolysis of immune cells (11), and cysteine proteases that degrade mucins (12) and immunoglobulins like IgA (13) and cause tissue destruction (14).…”