Localization of Phosphatidylinositol (3,4,5)-Trisphosphate to Phagosomes in Entamoeba histolytica Achieved Using Glutathione S -Transferase- and Green Fluorescent Protein-Tagged Lipid Biosensors

Abstract

ABSTRACT Entamoeba histolytica is an intestinal protozoan parasite that causes amoebic dysentery and liver abscess. Phagocytosis by the parasite is a critical virulence process, since it is a prerequisite for tissue invasion and establishment of chronic infection. While the roles of many of the proteins that regulate phagocytosis-related signaling events in E. histolytica have been characterized, the functions of lipids in this cellular process remain largely unknown in this parasite. In other systems, phosphatidylinositol (3,4,5)-trisphosphate (PIP 3 ), a major product of phosphoinositide 3 kinase (PI3-kinase) activity, is essential for phagocytosis. Pleckstrin homology (PH) domains are protein domains that specifically bind to PIP 3 . In this study, we utilized glutathione S -transferase (GST)- and green fluorescent protein (GFP)-labeled PH domains as lipid biosensors to characterize the spatiotemporal aspects of PIP 3 distribution during various endocytic processes in E. histolytica . PIP 3 -specific biosensors accumulated at extending pseudopodia and in phagosomal cups in trophozoites exposed to erythrocytes but did not localize to pinocytic compartments during the uptake of a fluid-phase marker, dextran. Our results suggest that PIP 3 is involved in the early stages of phagosome formation in E. histolytica . In addition, we demonstrated that PIP 3 exists at high steady-state levels in the plasma membrane of E. histolytica and that these levels, unlike those in mammalian cells, are not abolished by serum withdrawal. Finally, expression of a PH domain in trophozoites inhibited erythrophagocytosis and enhanced motility, providing genetic evidence supporting the role of PI3-kinase signaling in these processes in E. histolytica .