PTEN differentially regulates endocytosis, migration, and proliferation in the enteric protozoan parasite Entamoeba histolytica

Abstract

PTEN is a lipid phosphatase that is highly conserved and involved in a broad range of biological processes including cytoskeletal reorganization, endocytosis, signal transduction, and cell migration in all eukaryotes. Although regulation of phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P3] signaling via PTEN has been well established in model organisms and mammals, it remains elusive in the parasitic protist E. histolytica, which heavily relies on PtdIns phosphate(s)-dependent membrane traffic, migration, and phago- and trogocytosis for its pathogenesis. In this study, we characterized the major PTEN from E. histolytica, EhPTEN1, which shows the highest expression at the transcript level in the trophozoite stage among 6 possible PTENs, to understand the significance of PtdIns(3,4,5)P3 signaling in this parasite. Live imaging of GFP-EhPTEN1 expressing amebic trophozoites showed localization mainly in the cytosol with a higher concentration at pseudopods and the extending edge of the phago- and trogocytic cups. Furthermore, quantitative analysis of phago- and trogocytosis using a confocal image cytometer showed that overexpression of EhPTEN1 caused reduction in trogo- and phagocytosis while transcriptional gene silencing of EhPTEN1 gene caused opposite phenotypes. These data suggest that EhPTEN1 has an inhibitory role in these biological processes. Conversely, EhPTEN1 acts as a positive regulator for fluid-phase and receptor-mediated endocytosis in E. histolytica trophozoites. Moreover, we showed that EhPTEN1 was required for optimal growth and migration of this parasite. Finally, the phosphatase activity of EhPTEN1 towards PtdIns(3,4,5)P3 was demonstrated, suggesting that the biological roles of EhPTEN1 are likely linked to its catalytic function. Taken together, these results indicate that EhPTEN1 differentially regulates multiple cellular activities essential for proliferation and pathogenesis of the organism, via PtdIns(3,4,5)P3 signaling. Elucidation of biological roles of PTEN and PtdIns(3,4,5)P3 signaling at the molecular levels promotes our understanding of the pathogenesis of this parasite and potentially leads to the design of novel therapeutics against amebiasis. Author summaryEntamoeba histolytica is an intestinal protozoan parasite that causes amoebic dysentery and liver abscesses in humans. It has been well understood how the amoebas ability to ingests and destroy human cells and invade tissues contributes to disease symptoms such as bloody diarrhea. The underlying mechanisms for such activities, called pathogenicity, include trafficking (transport) and secretion of cytolytic proteins, migration (ameboid movement), and ingestion and destruction of human cells, heavily rely on the signal transduction system via metabolism (synthesis and decomposition) of phosphoinositides (phosphatidylinositols containing 0-5 phosphates), and downstream regulation of cytoskeleton (dynamic network of interlinking protein filaments, such as actin, in the cytoplasm). In this study, we characterized one enzyme called EhPTEN1, which degrades and inactivate PtdIns(3,4,5)P3. We have shown that EhPTEN1 is involved in migration, internalization of soluble and solid materials (endocytosis, trogo-, and phagocytosis). EhPTEN1 apparently regulates cell migration, endocytosis, trogo-, phagocytosis, and proliferation in a complex fashion. Our findings help in the elucidation of the physiological significance of PTEN and cellular events regulated via phosphoinositides in this enteric parasite and other pathogenic parasites, and potentially lead to the development of new control measures against parasitic diseases.