Limits...
Entamoeba histolytica and E. dispar Calreticulin: inhibition of classical complement pathway and differences in the level of expression in amoebic liver abscess.

Ximénez C, González E, Nieves ME, Silva-Olivares A, Shibayama M, Galindo-Gómez S, Escobar-Herrera J, García de León Mdel C, Morán P, Valadez A, Rojas L, Hernández EG, Partida O, Cerritos R - Biomed Res Int (2014)

Bottom Line: In the presence of peripheral mononuclear blood cells, the distribution of EhCRT and C1q is clearly over the surface membrane of trophozoites.Nevertheless, the level of expression of CRT in situ in lesions of amoebic liver abscess (ALA) in the hamster model is different in both Entamoeba species; this molecule is expressed in higher levels in E. histolytica than in E. dispar.This result suggests that EhCRT may modulate some functions during the early moments of the host-parasite relationship.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Medicina Experimental, Facultad de Medicina, UNAM, Dr. Balmis 148, Colonia Doctores, 06726 México, DF, Mexico.

ABSTRACT
The role of calreticulin (CRT) in host-parasite interactions has recently become an important area of research. Information about the functions of calreticulin and its relevance to the physiology of Entamoeba parasites is limited. The present work demonstrates that CRT of both pathogenic E. histolytica and nonpathogenic E. dispar species specifically interacted with human C1q inhibiting the activation of the classical complement pathway. Using recombinant EhCRT protein, we demonstrate that CRT interaction site and human C1q is located at the N-terminal region of EhCRT. The immunofluorescence and confocal microscopy experiments show that CRT and human C1q colocalize in the cytoplasmic vesicles and near to the surface membrane of previously permeabilized trophozoites or are incubated with normal human serum which is known to destroy trophozoites. In the presence of peripheral mononuclear blood cells, the distribution of EhCRT and C1q is clearly over the surface membrane of trophozoites. Nevertheless, the level of expression of CRT in situ in lesions of amoebic liver abscess (ALA) in the hamster model is different in both Entamoeba species; this molecule is expressed in higher levels in E. histolytica than in E. dispar. This result suggests that EhCRT may modulate some functions during the early moments of the host-parasite relationship.

Show MeSH

Related in: MedlinePlus

EhCRT/EdCRT interaction with human C1q (ELISA). Microtiter wells were coated with 2.5 to 200 μM of the EhCRT or EdCRT and incubated with 1 : 10 diluted normal human serum supplemented with 4 μg of human C1q; the interaction of EhCRT-C1q was revealed using an anti-human C1q monoclonal antibody produced in mice and then an anti-mouse IgG produced in goat conjugated to peroxidase. Values are the mean of three different assays performed in triplicate ± SD.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4016843&req=5

fig1: EhCRT/EdCRT interaction with human C1q (ELISA). Microtiter wells were coated with 2.5 to 200 μM of the EhCRT or EdCRT and incubated with 1 : 10 diluted normal human serum supplemented with 4 μg of human C1q; the interaction of EhCRT-C1q was revealed using an anti-human C1q monoclonal antibody produced in mice and then an anti-mouse IgG produced in goat conjugated to peroxidase. Values are the mean of three different assays performed in triplicate ± SD.

Mentions: To assess the interaction between EhCRT and human C1q, a direct binding ELISA was conducted. Figure 1 shows data of the interaction assay between rEhCRT (full-length molecule, N-terminal binding domain, or C-terminal binding domain), nEhCRT or nEdCRT, and human C1q. Differences observed in binding between the native EhCRT or EdCRT and the full-length rEhCRT or rEhCRT-N were not statistically significant. The interaction was dose dependent and saturable; the maximum absorbance was obtained when 100 μM of CRT was used; the OD remained constant in the presence of larger quantities of CRT. In contrast, the rEhCRT-C-terminal protein did not bind to human C1q.


Entamoeba histolytica and E. dispar Calreticulin: inhibition of classical complement pathway and differences in the level of expression in amoebic liver abscess.

Ximénez C, González E, Nieves ME, Silva-Olivares A, Shibayama M, Galindo-Gómez S, Escobar-Herrera J, García de León Mdel C, Morán P, Valadez A, Rojas L, Hernández EG, Partida O, Cerritos R - Biomed Res Int (2014)

EhCRT/EdCRT interaction with human C1q (ELISA). Microtiter wells were coated with 2.5 to 200 μM of the EhCRT or EdCRT and incubated with 1 : 10 diluted normal human serum supplemented with 4 μg of human C1q; the interaction of EhCRT-C1q was revealed using an anti-human C1q monoclonal antibody produced in mice and then an anti-mouse IgG produced in goat conjugated to peroxidase. Values are the mean of three different assays performed in triplicate ± SD.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4016843&req=5

fig1: EhCRT/EdCRT interaction with human C1q (ELISA). Microtiter wells were coated with 2.5 to 200 μM of the EhCRT or EdCRT and incubated with 1 : 10 diluted normal human serum supplemented with 4 μg of human C1q; the interaction of EhCRT-C1q was revealed using an anti-human C1q monoclonal antibody produced in mice and then an anti-mouse IgG produced in goat conjugated to peroxidase. Values are the mean of three different assays performed in triplicate ± SD.
Mentions: To assess the interaction between EhCRT and human C1q, a direct binding ELISA was conducted. Figure 1 shows data of the interaction assay between rEhCRT (full-length molecule, N-terminal binding domain, or C-terminal binding domain), nEhCRT or nEdCRT, and human C1q. Differences observed in binding between the native EhCRT or EdCRT and the full-length rEhCRT or rEhCRT-N were not statistically significant. The interaction was dose dependent and saturable; the maximum absorbance was obtained when 100 μM of CRT was used; the OD remained constant in the presence of larger quantities of CRT. In contrast, the rEhCRT-C-terminal protein did not bind to human C1q.

Bottom Line: In the presence of peripheral mononuclear blood cells, the distribution of EhCRT and C1q is clearly over the surface membrane of trophozoites.Nevertheless, the level of expression of CRT in situ in lesions of amoebic liver abscess (ALA) in the hamster model is different in both Entamoeba species; this molecule is expressed in higher levels in E. histolytica than in E. dispar.This result suggests that EhCRT may modulate some functions during the early moments of the host-parasite relationship.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Medicina Experimental, Facultad de Medicina, UNAM, Dr. Balmis 148, Colonia Doctores, 06726 México, DF, Mexico.

ABSTRACT
The role of calreticulin (CRT) in host-parasite interactions has recently become an important area of research. Information about the functions of calreticulin and its relevance to the physiology of Entamoeba parasites is limited. The present work demonstrates that CRT of both pathogenic E. histolytica and nonpathogenic E. dispar species specifically interacted with human C1q inhibiting the activation of the classical complement pathway. Using recombinant EhCRT protein, we demonstrate that CRT interaction site and human C1q is located at the N-terminal region of EhCRT. The immunofluorescence and confocal microscopy experiments show that CRT and human C1q colocalize in the cytoplasmic vesicles and near to the surface membrane of previously permeabilized trophozoites or are incubated with normal human serum which is known to destroy trophozoites. In the presence of peripheral mononuclear blood cells, the distribution of EhCRT and C1q is clearly over the surface membrane of trophozoites. Nevertheless, the level of expression of CRT in situ in lesions of amoebic liver abscess (ALA) in the hamster model is different in both Entamoeba species; this molecule is expressed in higher levels in E. histolytica than in E. dispar. This result suggests that EhCRT may modulate some functions during the early moments of the host-parasite relationship.

Show MeSH
Related in: MedlinePlus