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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-human C1q on the activation of classical complement pathway: hemolysis assay. 1: corresponding to NHS (positive control); 2: human C1q-depleted serum (NHSC1q−); 3: (NHSC1q−) + C1q; 4: (NHSC1q−) + C1q + EhCRT; 5: (NHSC1q−) + C1q + EhCRT + IgG anti-EhCRT. Assays were performed in triplicate; values are the mean of three different experiments ± SD. Differences between groups ∗, ●, and ▼ were compared through ANOVA test. Statistical significance (P = 0.012).
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fig3: EhCRT-human C1q on the activation of classical complement pathway: hemolysis assay. 1: corresponding to NHS (positive control); 2: human C1q-depleted serum (NHSC1q−); 3: (NHSC1q−) + C1q; 4: (NHSC1q−) + C1q + EhCRT; 5: (NHSC1q−) + C1q + EhCRT + IgG anti-EhCRT. Assays were performed in triplicate; values are the mean of three different experiments ± SD. Differences between groups ∗, ●, and ▼ were compared through ANOVA test. Statistical significance (P = 0.012).

Mentions: To confirm that this activity is the result of the interaction of human C1q with EhCRT or EdCRT, we used C1q-depleted human serum; when this serum was added to human C1q in the presence of EhCRT, haemolysis was inhibited. Moreover, when EhCRT was pretreated with anti-CRT antibodies, EhCRT could not bind to C1q, and the activation of the classical complement pathway was restored (Figure 3).


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-human C1q on the activation of classical complement pathway: hemolysis assay. 1: corresponding to NHS (positive control); 2: human C1q-depleted serum (NHSC1q−); 3: (NHSC1q−) + C1q; 4: (NHSC1q−) + C1q + EhCRT; 5: (NHSC1q−) + C1q + EhCRT + IgG anti-EhCRT. Assays were performed in triplicate; values are the mean of three different experiments ± SD. Differences between groups ∗, ●, and ▼ were compared through ANOVA test. Statistical significance (P = 0.012).
© Copyright Policy
Related In: Results  -  Collection

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

fig3: EhCRT-human C1q on the activation of classical complement pathway: hemolysis assay. 1: corresponding to NHS (positive control); 2: human C1q-depleted serum (NHSC1q−); 3: (NHSC1q−) + C1q; 4: (NHSC1q−) + C1q + EhCRT; 5: (NHSC1q−) + C1q + EhCRT + IgG anti-EhCRT. Assays were performed in triplicate; values are the mean of three different experiments ± SD. Differences between groups ∗, ●, and ▼ were compared through ANOVA test. Statistical significance (P = 0.012).
Mentions: To confirm that this activity is the result of the interaction of human C1q with EhCRT or EdCRT, we used C1q-depleted human serum; when this serum was added to human C1q in the presence of EhCRT, haemolysis was inhibited. Moreover, when EhCRT was pretreated with anti-CRT antibodies, EhCRT could not bind to C1q, and the activation of the classical complement pathway was restored (Figure 3).

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