Detection of the Endosomal Sorting Complex Required for Transport inEntamoeba histolyticaand Characterization of the EhVps4 Protein

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.

A Bioinformatical Approach to Study the Endosomal Sorting Complex Required for Transport (ESCRT) Machinery in Protozoan Parasites: The Entamoeba histolytica Case

“…In E. histolytica, we found a highly conserved ESCRT machinery with 19 putative components representing all complexes. These findings have been experimentally confirmed by determining the expression of most ESCRT gene transcripts (López-Reyes, et al, 2010). Furthermore, our current in silico results suggest that some E. histolytica ESCRT-0 to -III components contain putative FYVE or ubiquitin binding domains, both important to recruit cargo molecules to endosomal membranes.…”

“…Interestingly, this protein is located at MVB-like structures in E. histolytica trophozoites and is structurally related to Bro1 (Bañuelos et al, 2005), an accessory protein that interacts with the ESCRT-III complex in yeast. Recently, our research group reported the presence of a set of 19 putative ESCRT proteins in this parasite and characterized a yeast Vps4 homologue by analyzing its ATPase function and relationship to parasite virulence in wild type and mutant cells (López-Reyes et al, 2010). Results derived from these studies strongly suggest that E. histolytica possesses a well conserved ESCRT machinery.…”

“…Assays demonstrated that EhADH112 binds through its Nterminus to a recombinant protein of EhVps32 fused to GST (our unpublished data). Since yeast Vps4 and its orthologues have been previously described as key molecules for ESCRT dissociation and recycling, López-Reyes et al, (2010) characterized the EhVps4 protein in more detail. Protein domain predictions, as well as tertiary structure modeling and phylogenetic trees assayed for EhVps4 suggest, that it conserves a typical Vps4 architecture (Babst et al, 1998) and is more related to protozoan Vps4 homologues than to that of higher eukaryotes.…”

Intracellular traffic of the lysine and glutamic acid rich protein KERP1 reveals features of endomembrane organization inEntamoeba histolytica

“…We then focused on Alix (EHI_167710), which has two homologues (sharing the BRO domain) previously described in E. histolytica : the ADH112 adhesin (EHI_181220) (Lopez‐Reyes et al ., ) and a second protein sharing domain similarities (EHI_165220), (Perdomo et al ., ). We detected Alix with an antibody specifically recognizing EHI_167710, a confirmed protein present in isolated amoebic vesicles and involved in endosomal trafficking (Perdomo et al ., ).…”

Resolving the homology—function relationship through comparative genomics of membrane-trafficking machinery and parasite cell biology

“…An ATPase assay confirmed Vps4 ATPase activity, and overexpression of an enzymatically dead mutant impairs phagocytosis and the organism’s ability to cause hepatic abscesses in hamsters [100]. Three E. histolytica proteins contain a Bro1 domain, and thus may be homologues of Bro1/Vps31: ADH112, ADH112-like 1 and ADH112-like 2.…”