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

López-Reyes I, García-Rivera G, Bañuelos C, Herranz S, Vincent O, López-Camarillo C, Marchat LA, Orozco E - J. Biomed. Biotechnol. (2010)

Bottom Line: The EhVps4-FLAG was located in cytosol and at plasma membrane, whereas the EhVps4-E211Q-FLAG was detected as abundant cytoplasmic dots in trophozoites.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.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Infectómica y Patogénesis Molecular, Centro de Investigación y de Estudios Avanzados del IPN, México, CP 07360, Mexico.

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.

No MeSH data available.


Related in: MedlinePlus

Cellular localization of EhVps4 protein during erythrophagocytosis. Trophozoites transfected with pEhVps4 were incubated with RBC, treated with diaminobenzidine, anti-rEhVps4 and FITC-labeled secondary antibodies, and analyzed through confocal laser microscopy. (a) Cells observed in phase contrast. (b) Trophozoites observed in the green (FITC) channel and phase contrast. (c) Magnification of diaminobenzidine stained RBC squared in (b). Arrowhead, EhVps4 signal around RBC. N, nuclei.
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fig8: Cellular localization of EhVps4 protein during erythrophagocytosis. Trophozoites transfected with pEhVps4 were incubated with RBC, treated with diaminobenzidine, anti-rEhVps4 and FITC-labeled secondary antibodies, and analyzed through confocal laser microscopy. (a) Cells observed in phase contrast. (b) Trophozoites observed in the green (FITC) channel and phase contrast. (c) Magnification of diaminobenzidine stained RBC squared in (b). Arrowhead, EhVps4 signal around RBC. N, nuclei.

Mentions: To determine the localization of EhVps4 in pEhVps4 transfected trophozoites during erythrophagocytosis, we performed immunofluorescence assays with anti-rEhVps4 serum. Antibodies detected EhVps4 as a signal surrounding ingested erythrocytes, suggesting that this protein may be involved in phagocytosis (see Figure 8).


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

López-Reyes I, García-Rivera G, Bañuelos C, Herranz S, Vincent O, López-Camarillo C, Marchat LA, Orozco E - J. Biomed. Biotechnol. (2010)

Cellular localization of EhVps4 protein during erythrophagocytosis. Trophozoites transfected with pEhVps4 were incubated with RBC, treated with diaminobenzidine, anti-rEhVps4 and FITC-labeled secondary antibodies, and analyzed through confocal laser microscopy. (a) Cells observed in phase contrast. (b) Trophozoites observed in the green (FITC) channel and phase contrast. (c) Magnification of diaminobenzidine stained RBC squared in (b). Arrowhead, EhVps4 signal around RBC. N, nuclei.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2875786&req=5

fig8: Cellular localization of EhVps4 protein during erythrophagocytosis. Trophozoites transfected with pEhVps4 were incubated with RBC, treated with diaminobenzidine, anti-rEhVps4 and FITC-labeled secondary antibodies, and analyzed through confocal laser microscopy. (a) Cells observed in phase contrast. (b) Trophozoites observed in the green (FITC) channel and phase contrast. (c) Magnification of diaminobenzidine stained RBC squared in (b). Arrowhead, EhVps4 signal around RBC. N, nuclei.
Mentions: To determine the localization of EhVps4 in pEhVps4 transfected trophozoites during erythrophagocytosis, we performed immunofluorescence assays with anti-rEhVps4 serum. Antibodies detected EhVps4 as a signal surrounding ingested erythrocytes, suggesting that this protein may be involved in phagocytosis (see Figure 8).

Bottom Line: The EhVps4-FLAG was located in cytosol and at plasma membrane, whereas the EhVps4-E211Q-FLAG was detected as abundant cytoplasmic dots in trophozoites.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.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Infectómica y Patogénesis Molecular, Centro de Investigación y de Estudios Avanzados del IPN, México, CP 07360, Mexico.

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.

No MeSH data available.


Related in: MedlinePlus