A Calcium/Cation Exchanger Participates in the Programmed Cell Death and in vitro Virulence of Entamoeba histolytica

Abstract

Entamoeba histolytica is the etiologic agent of human amoebiasis, disease that causes 40,000 to 100,000 deaths annually worldwide. The cytopathic activity as well as the growth and differentiation of this microorganism is dependent on both, extracellular and free cytoplasmic calcium. However, few is known about the proteins that regulate the calcium flux in this parasite. In many cells, the calcium extrusion from the cytosol is performed by plasma membrane Ca2+-ATPases and calcium/cation exchangers. The aim of this work was to identify a calcium/cation exchanger of E. histolytica and to analyze its possible role in some cellular processes triggered by calcium flux, such as the programmed cell death and in vitro virulence. By searching putative calcium/cation exchangers in the genome database of E. histolyica we identified a protein belonging to the CCX family (EhCCX). We generated a specific antibody against EhCCX, which showed that this protein was expressed in higher levels in E. histolytica than its orthologous in the non-pathogenic amoeba E. dispar. In addition, the expression of EhCCX was increased in trophozoites incubated with hydrogen peroxide. This E. histolytica exchanger was localized in the plasma membrane and in the membrane of some cytoplasmic vesicles. However, after 10 min of erythrophagocytosis, EhCCX was found predominantly in the plasma membrane of the trophozoites. On the other hand, the parasites that overexpress this exchanger contained higher cytosolic calcium levels than control, but the extrusion of calcium after the addition of hydrogen peroxide was more efficient in EhCCX-overexpressing trophozoites; consequently, the programmed cell death was retarded in these parasites. Interestingly, the overexpression of EhCCX increased the in vitro virulence of trophozoites. These results suggest that EhCCX plays important roles in the programmed cell death and in the in vitro virulence of E. histolytica.

Lysine Methyltransferase EhPKMT2 Is Involved in the In Vitro Virulence of Entamoeba histolytica

“…Transfection was carried out as described [ 18 ] using 5 μg/mL of G418 to select the transfected trophozoites. For higher knockdown, parasites were grown in the presence of 40 μg/mL of G418.…”

Entamoeba histolytica and pathogenesis: A calcium connection

“…More recently, 2 Ca 2 + -ATPases from E. histolytica (Eh), namely EhSPCA (secretory pathway calcium ATPase) and EhCCX (Ca 2+ /cation exchanger), have been identified. These are present on the membrane of some cytoplasmic vesicles [9,10]. Interestingly, overexpression of EhCCX enhanced the virulence and reduced the cell death of trophozoites [9].…”

“…These are present on the membrane of some cytoplasmic vesicles [9,10]. Interestingly, overexpression of EhCCX enhanced the virulence and reduced the cell death of trophozoites [9]. E. histolytica has ionophore-releasable Ca 2+ , comprising around 70% of the total Ca 2+ pool that can be divided into 2 parts.…”

Signals and signal transduction pathways in Entamoeba histolytica during the life cycle and when interacting with bacteria or human cells

“…The role of Ca 2+ ‐binding proteins (EhCaBPs) in erythrophagocytosis deserves special recognition according to recent evidence. First, a Ca 2+ ‐exchanger protein of E. histolytica accumulates at the amebic plasma membrane during the early stages of erythrophagocytosis and the increase in protein levels enhance virulence and delay amebic death during stress (Valle‐Solis et al, 2018). Furthermore, 27 EF‐hand‐containing proteins have been identified in E. histolytica (Babuta et al, 2020), which act as Ca 2+ buffers that control Ca 2+ levels in different cellular compartments.…”