Localization of phosphatidylinositol (3,4,5)-trisphosphate to phagosomes in entamoeba histolytica achieved using glutathione S-transferase- and green fluorescent protein-tagged lipid biosensors.
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.
Byekova
Yevgeniya A
YA
Department of Biological Sciences, Clemson University, Clemson, South Carolina 29634, USA.
Powell
Rhonda R
RR
Welter
Brenda H
BH
Temesvari
Lesly A
LA
D000077329:Agammaglobulinaemia Tyrosine Kinase; D000818:Animals; D015374:Biosensing Techniques; D004705:Endocytosis / Q000502:physiology; D004748:Entamoeba histolytica / Q000166:cytology / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000502:physiology; D005982:Glutathione Transferase / Q000737:chemistry / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000378:metabolism; D008055:Lipids / Q000737:chemistry*; D010588:Phagosomes / Q000378:metabolism*; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011505:Protein-Tyrosine Kinases / Q000737:chemistry
D008055:Lipids; D018129:Phosphatidylinositol Phosphates; C060974:phosphatidylinositol 3,4,5-triphosphate; D049452:Green Fluorescent Proteins; D005982:Glutathione Transferase; D011505:Protein-Tyrosine Kinases; D000077329:Agammaglobulinaemia Tyrosine Kinase