IAI Accepts, published online ahead of print on 9 November 2009

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Infect. Immun. doi:10.1128/IAI.00719-09
Copyright (c) 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Localization of phosphatidylinositol (3,4,5)-trisphosphate to phagosomes in Entamoeba histolytica using GST- and GFP-tagged lipid biosensors

Yevgeniya A. Byekova, Rhonda R. Powell, Brenda H. Welter, and Lesly A. Temesvari^*

Department of Biological Sciences, Clemson University, Clemson, SC 29634, USA

^* To whom correspondence should be addressed. Email: LTEMESV{at}clemson.edu.

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 role of many of the proteins that regulate phagocytosis-related signaling events in E. histolytica has been characterized, the functions of lipids in this cellular process remain largely unknown in this parasite. In other systems, phosphatidylinositol (3, 4, 5)-trisphosphate (PIP₃), a major product of PI 3-kinase activity, is essential for phagocytosis. Pleckstrin homology (PH) domains are protein domains that specifically bind to PIP₃. In this study, we utilized GST- and GFP-labeled PH domains as lipid biosensors to characterize the spatiotemporal aspects of PIP₃ distribution during various endocytic processes in E. histolytica. PIP₃-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 PIP₃ is involved in the early stages of phagosome formation in E. histolytica. In addition, we demonstrated that PIP₃ exists in high steady-state levels in the plasma membrane of E. histolytica, and that these levels, unlike 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 PI 3-kinase signaling in these processes in E. histolytica.