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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.

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EhCRT inhibits classical pathway-mediated hemolysis. Different proteins of (CRT+) EhCRT or EdCRT; (CRT−) BSA or EhCRT-C was added to 1 : 10 dilution of NHS (as source of C1q), incubated 30 min at 37°C, and then added to 108 cell/mL of EA; the mixtures were incubated for 60 min at 37° C. After centrifugation, the OD (550 nm) of the supernatants was measured. The percentage of lyses was calculated using as reference the 100% lyses of erythrocytes in water. Values are the mean of three independent experiments ± SD. Differences between groups ∗ were compared through ANOVA test detecting statistical significance (P = 0.05).
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fig2: EhCRT inhibits classical pathway-mediated hemolysis. Different proteins of (CRT+) EhCRT or EdCRT; (CRT−) BSA or EhCRT-C was added to 1 : 10 dilution of NHS (as source of C1q), incubated 30 min at 37°C, and then added to 108 cell/mL of EA; the mixtures were incubated for 60 min at 37° C. After centrifugation, the OD (550 nm) of the supernatants was measured. The percentage of lyses was calculated using as reference the 100% lyses of erythrocytes in water. Values are the mean of three independent experiments ± SD. Differences between groups ∗ were compared through ANOVA test detecting statistical significance (P = 0.05).

Mentions: The ability of EhCRT to inhibit the activation of the classical complement pathway by binding to C1q was tested in a simple assay of inhibition of the haemolysis of SRBCs previously sensitized with antibody (EAs). Human serum was used as the source of C1q. The optimal dilution of the human serum was determined previously by a complement titration curve (data not shown); the optimal dilution of serum was 1 : 10. Figure 2 shows the values of inhibition of the activation of the classical complement pathway assays, in the presence of different concentrations of nEhCRT or nEdCRT. Both proteins inhibited the lysis of EAs in a dose-dependent manner as shown by the decrease in haemolytic activity (34–22% of baseline). By contrast, in the control assay (without CRT−) or in case we use the recombinant protein EhCRT-C, there was no significant decrease in haemolytic activity (95–86% of baseline) due to the absence of C1q binding site.


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 inhibits classical pathway-mediated hemolysis. Different proteins of (CRT+) EhCRT or EdCRT; (CRT−) BSA or EhCRT-C was added to 1 : 10 dilution of NHS (as source of C1q), incubated 30 min at 37°C, and then added to 108 cell/mL of EA; the mixtures were incubated for 60 min at 37° C. After centrifugation, the OD (550 nm) of the supernatants was measured. The percentage of lyses was calculated using as reference the 100% lyses of erythrocytes in water. Values are the mean of three independent experiments ± SD. Differences between groups ∗ were compared through ANOVA test detecting statistical significance (P = 0.05).
© Copyright Policy
Related In: Results  -  Collection

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

fig2: EhCRT inhibits classical pathway-mediated hemolysis. Different proteins of (CRT+) EhCRT or EdCRT; (CRT−) BSA or EhCRT-C was added to 1 : 10 dilution of NHS (as source of C1q), incubated 30 min at 37°C, and then added to 108 cell/mL of EA; the mixtures were incubated for 60 min at 37° C. After centrifugation, the OD (550 nm) of the supernatants was measured. The percentage of lyses was calculated using as reference the 100% lyses of erythrocytes in water. Values are the mean of three independent experiments ± SD. Differences between groups ∗ were compared through ANOVA test detecting statistical significance (P = 0.05).
Mentions: The ability of EhCRT to inhibit the activation of the classical complement pathway by binding to C1q was tested in a simple assay of inhibition of the haemolysis of SRBCs previously sensitized with antibody (EAs). Human serum was used as the source of C1q. The optimal dilution of the human serum was determined previously by a complement titration curve (data not shown); the optimal dilution of serum was 1 : 10. Figure 2 shows the values of inhibition of the activation of the classical complement pathway assays, in the presence of different concentrations of nEhCRT or nEdCRT. Both proteins inhibited the lysis of EAs in a dose-dependent manner as shown by the decrease in haemolytic activity (34–22% of baseline). By contrast, in the control assay (without CRT−) or in case we use the recombinant protein EhCRT-C, there was no significant decrease in haemolytic activity (95–86% of baseline) due to the absence of C1q binding site.

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