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Journal of Biomedicine and Biotechnology
Volume 2010 (2010), Article ID 890674, 15 pages
http://dx.doi.org/10.1155/2010/890674
Research Article

Detection of the Endosomal Sorting Complex Required for Transport in Entamoeba histolytica and Characterization of the EhVps4 Protein

1Departamento de Infectómica y Patogénesis Molecular, Centro de Investigación y de Estudios Avanzados del IPN, México, CP 07360, Mexico
2Departamento de Modelos Experimentales de Enfermedades Humanas, Instituto de Investigaciones Biomédicas CSIC-UAM, Madrid, CP 28029, Spain
3Universidad Autónoma de la Ciudad de México, Posgrado en Ciencias Genómicas, México, CP 03100, Mexico
4Escuela Nacional de Medicina y Homeopatía del IPN, Programa Institucional de Biomedicina Molecular, México, CP 07320, Mexico

Received 23 September 2009; Revised 1 March 2010; Accepted 1 March 2010

Academic Editor: Abhay R. Satoskar

Copyright © 2010 Israel López-Reyes et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Eukaryotic endocytosis involves multivesicular bodies formation, which is driven by endosomal sorting complexes required for transport (ESCRT). Here, we showed the presence and expression of homologous ESCRT genes in Entamoeba histolytica. We cloned and expressed the Ehvps4 gene, an ESCRT member, to obtain the recombinant EhVps4 and generate specific antibodies, which immunodetected EhVps4 in cytoplasm of trophozoites. Bioinformatics and biochemical studies evidenced that rEhVps4 is an ATPase, whose activity depends on the conserved E211 residue. Next, we generated trophozoites overexpressing EhVps4 and mutant EhVps4-E211Q FLAG-tagged proteins. The EhVps4-FLAG was located in cytosol and at plasma membrane, whereas the EhVps4-E211Q-FLAG was detected as abundant cytoplasmic dots in trophozoites. Erythrophagocytosis, cytopathic activity, and hepatic damage in hamsters were not improved in trophozoites overexpressing EhVps4-FLAG. In contrast, EhVps4-E211Q-FLAG protein overexpression impaired these properties. The localization of EhVps4-FLAG around ingested erythrocytes, together with our previous results, strengthens the role for EhVps4 in E. histolytica phagocytosis and virulence.