EhNPC1 and EhNPC2 Proteins Participate in Trafficking of Exogenous Cholesterol in Entamoeba histolytica Trophozoites: Relevance for Phagocytosis

Entamoeba histolytica, the highly phagocytic protozoan causative of human amoebiasis lacks the machinery to synthesize cholesterol. Here, we investigated the presence of NPC1 and NPC2 proteins in this parasite, which are involved in cholesterol trafficking in mammals. Bioinformatics analysis revealed one Ehnpc1 and two Ehnpc2 genes. EhNPC1 appeared as a transmembrane protein and both EhNPC2 as peripheral membrane proteins. Molecular docking predicted that EhNPC1 and EhNPC2 bind cholesterol and interact with each other. Genes and proteins were identified in trophozoites. Serum pulse-chase and confocal microscopy assays unveiled that after trophozoites sensed the cholesterol source, EhNPC1 and EhNPC2 were organized around the plasma membrane in a punctuated pattern. Vesicles emerged and increased in number and size and some appeared full of cholesterol with EhNPC1 or EhNPC2 facing the extracellular space. Both proteins, but mostly EhNPC2, were found out of the cell associated with cholesterol. EhNPC1 and cholesterol formed networks from the plasma membrane to the nucleus. EhNPC2 appeared in erythrocytes that were being ingested by trophozoites, co-localizing with cholesterol of erythrocytes, whereas EhNPC1 surrounded the phagocytic cup. EhNPC1 and EhNPC2 co-localized with EhSERCA in the endoplasmic reticulum and with lysobisphosphatidic acid and EhADH (an Alix protein) in phagolysosomes. Immunoprecipitation assays confirmed the EhNPC1 and EhNPC2 association with cholesterol, EhRab7A and EhADH. Serum starved and blockage of cholesterol trafficking caused a low rate of phagocytosis and incapability of trophozoites to produce damage in the mouse colon. Ehnpc1 and Ehnpc2 knockdown provoked in trophozoites a lower intracellular cholesterol concentration and a diminished rate of phagocytosis; and Ehnpc1 silencing also produced a decrease of trophozoites movement. Trafficking of EhNPC1 and EhNPC2 during cholesterol uptake and phagocytosis as well as their association with molecules involved in endocytosis strongly suggest that these proteins play a key role in cholesterol uptake.
Author Summary NPC1 and NPC2 proteins are involved in cholesterol trafficking in mammals. Using different approaches, we have detected the orthologues EhNPC1 and EhNPC2 proteins in Entamoeba histolytica. Trophozoites are particularly rich in membranes and vacuoles, but they do not possess the machinery to synthetize cholesterol. Thus, they are completely dependent on molecules able to “fish” cholesterol from the medium. The relevance of our findings lies in the fact that cholesterol is fundamental for endocytosis and motility; and, phagocytosis is an important nutritional and virulence factor for E. histolytica. In silico and experimental strategies, using U18666A to arrest cholesterol trafficking, as well as, knockdown mutants, showed that EhNPC1 and EhNPC2 participate in cholesterol uptake and trafficking in this parasite. They are secreted by trophozoites and directly involved in erythrophagocytosis and motility. Our findings revealed E. histolytica as one of the first protozoa in which these proteins are being characterized. Moreover, E. histolytica provides an excellent and less complicated model to elucidate the intricate event of cholesterol trafficking in eukaryotic cells. The relevance of cholesterol transport for the parasite virulence and the involvement of EhNPC1 and EhNPC2 in this process, make these proteins promising targets for therapy strategies development against the parasite.