title: EhNPC1 and EhNPC2 Proteins Participate in Trafficking of Exogenous Cholesterol in Entamoeba histolytica Trophozoites: Relevance for Phagocytosis.
abstract: 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.
authors.affiliation: Departamento de Infectómica y Patogénesis Molecular, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, D.F., México.
authors.forename: Jeni
authors.initials: J
authors.lastname: Bolaños
authors.affiliation: Departamento de Infectómica y Patogénesis Molecular, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, D.F., México.|Cátedras, Consejo Nacional de Ciencia y Tecnología, D.F., México.
authors.forename: Abigail
authors.initials: A
authors.lastname: Betanzos
authors.affiliation: Departamento de Infectómica y Patogénesis Molecular, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, D.F., México.
authors.forename: Rosario
authors.initials: R
authors.lastname: Javier-Reyna
authors.affiliation: Departamento de Infectómica y Patogénesis Molecular, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, D.F., México.
authors.forename: Guillermina
authors.initials: G
authors.lastname: García-Rivera
authors.affiliation: Departamento de Infectómica y Patogénesis Molecular, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, D.F., México.
authors.forename: Miriam
authors.initials: M
authors.lastname: Huerta
authors.affiliation: Departamento de Infectómica y Patogénesis Molecular, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, D.F., México.
authors.forename: Jonnatan
authors.initials: J
authors.lastname: Pais-Morales
authors.affiliation: Departamento de Infectómica y Patogénesis Molecular, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, D.F., México.
authors.forename: Arturo
authors.initials: A
authors.lastname: González-Robles
authors.affiliation: Departamento de Infectómica y Patogénesis Molecular, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, D.F., México.
authors.forename: Mario A
authors.initials: MA
authors.lastname: Rodríguez
authors.affiliation: Departamento de Biomedicina Molecular, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, D.F., México.
authors.forename: Michael
authors.identifier: http://orcid.org/0000-0002-0269-5884
authors.initials: M
authors.lastname: Schnoor
authors.affiliation: Departamento de Infectómica y Patogénesis Molecular, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, D.F., México.
authors.forename: Esther
authors.identifier: http://orcid.org/0000-0002-8360-2968
authors.initials: E
authors.lastname: Orozco
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002784:Cholesterol / Q000378:metabolism*; D004195:Disease Models, Animal; D004748:Entamoeba histolytica / Q000378:metabolism*; D004749:Entamoebiasis / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation; D051379:Mice; D008810:Mice, Inbred C57BL; D018613:Microscopy, Confocal; D046529:Microscopy, Electron, Transmission; D008958:Models, Molecular; D062105:Molecular Docking Simulation; D010587:Phagocytosis / Q000502:physiology; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D021381:Protein Transport / Q000502:physiology; D015800:Protozoan Proteins / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D053058:Trophozoites / Q000378:metabolism*; D014774:Virulence / Q000502:physiology
chemical_list: D015800:Protozoan Proteins; D002784:Cholesterol