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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

mRNA expression profile of E. histolytica putative ESCRT machinery genes. (a) RT-PCR products obtained from 1 μg of total RNA from trophozoites growing in TYI-S-33 medium (0 minute) or after 5 minutes of erythrophagocytosis. (b) Densitometric analysis of RT-PCR products in (a). Pixels corresponding to Eh25S rRNA amplified product were taken as 100% in each lane. Data represent the mean of three independent experiments performed by duplicate for each gene. Asterisk, genes whose transcription is significantly changed according to T Student test.
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fig1: mRNA expression profile of E. histolytica putative ESCRT machinery genes. (a) RT-PCR products obtained from 1 μg of total RNA from trophozoites growing in TYI-S-33 medium (0 minute) or after 5 minutes of erythrophagocytosis. (b) Densitometric analysis of RT-PCR products in (a). Pixels corresponding to Eh25S rRNA amplified product were taken as 100% in each lane. Data represent the mean of three independent experiments performed by duplicate for each gene. Asterisk, genes whose transcription is significantly changed according to T Student test.

Mentions: To determine whether the putative E. histolytica ESCRT genes were expressed, we performed RT-PCR assays for the 16 most conserved genes. In basal culture conditions, 15 genes were transcribed, but the Ehvps22 transcript was not detected in these experiments (see Figures 1(a) and 1(b)). We also performed RT-PCR assays using RNA obtained from trophozoites incubated with RBC for 5 minutes to investigate whether erythrophagocytosis has an effect on ESCRT gene expression (see Figures 1(a) and 1(b)). In these assays, we found that all genes were expressed during erythrophagocytosis at similar levels as in basal conditions. Again, we did not detect the Ehvps22 gene expression. Interestingly, Ehvps23 and Ehadh112 were 2- and 3-fold up-regulated, respectively. By densitometric analysis, the band given by the amplified Eh25S rRNA product in both conditions was taken as 100% and used to normalize ESCRT genes data.


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)

mRNA expression profile of E. histolytica putative ESCRT machinery genes. (a) RT-PCR products obtained from 1 μg of total RNA from trophozoites growing in TYI-S-33 medium (0 minute) or after 5 minutes of erythrophagocytosis. (b) Densitometric analysis of RT-PCR products in (a). Pixels corresponding to Eh25S rRNA amplified product were taken as 100% in each lane. Data represent the mean of three independent experiments performed by duplicate for each gene. Asterisk, genes whose transcription is significantly changed according to T Student test.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2875786&req=5

fig1: mRNA expression profile of E. histolytica putative ESCRT machinery genes. (a) RT-PCR products obtained from 1 μg of total RNA from trophozoites growing in TYI-S-33 medium (0 minute) or after 5 minutes of erythrophagocytosis. (b) Densitometric analysis of RT-PCR products in (a). Pixels corresponding to Eh25S rRNA amplified product were taken as 100% in each lane. Data represent the mean of three independent experiments performed by duplicate for each gene. Asterisk, genes whose transcription is significantly changed according to T Student test.
Mentions: To determine whether the putative E. histolytica ESCRT genes were expressed, we performed RT-PCR assays for the 16 most conserved genes. In basal culture conditions, 15 genes were transcribed, but the Ehvps22 transcript was not detected in these experiments (see Figures 1(a) and 1(b)). We also performed RT-PCR assays using RNA obtained from trophozoites incubated with RBC for 5 minutes to investigate whether erythrophagocytosis has an effect on ESCRT gene expression (see Figures 1(a) and 1(b)). In these assays, we found that all genes were expressed during erythrophagocytosis at similar levels as in basal conditions. Again, we did not detect the Ehvps22 gene expression. Interestingly, Ehvps23 and Ehadh112 were 2- and 3-fold up-regulated, respectively. By densitometric analysis, the band given by the amplified Eh25S rRNA product in both conditions was taken as 100% and used to normalize ESCRT genes data.

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