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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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Colocalization of EhCRT or EdCRT and human C1q after interaction with PMBC. C1q and EhCRT or EdCRT colocalization by confocal microscopy of (a) E. histolytica; (b) E. dispar trophozoites. Trophozoites were grown under axenic conditions and incubated during 30 min with PMBC; thereafter, C1q was added and incubated for 30 min. Then, the mixture was added with specific primary antibodies, anti-rabbit EhCRT IgG and mouse anti-human C1q IgG, respectively. The reaction was revealed with Alexa Fluor 555 goat anti-rabbit IgG and Alexa Fluor 488 goat anti-mouse IgG. The micrographs show the maximal projection of the z-series. Scale bar represents 20 μm. A: phase contrast microscopy; B: EhCRT (red); C: C1q (green); D: merge.
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fig6: Colocalization of EhCRT or EdCRT and human C1q after interaction with PMBC. C1q and EhCRT or EdCRT colocalization by confocal microscopy of (a) E. histolytica; (b) E. dispar trophozoites. Trophozoites were grown under axenic conditions and incubated during 30 min with PMBC; thereafter, C1q was added and incubated for 30 min. Then, the mixture was added with specific primary antibodies, anti-rabbit EhCRT IgG and mouse anti-human C1q IgG, respectively. The reaction was revealed with Alexa Fluor 555 goat anti-rabbit IgG and Alexa Fluor 488 goat anti-mouse IgG. The micrographs show the maximal projection of the z-series. Scale bar represents 20 μm. A: phase contrast microscopy; B: EhCRT (red); C: C1q (green); D: merge.

Mentions: To evaluate the interaction of EhCRT/EdCRT in trophozoites with human C1q, a colocalization assay was performed directly on trophozoites of both E. histolytica and E. dispar species by confocal microscopy. Both proteins clearly colocalized in previously permeabilized trophozoites; the fluorescent signal was detected in the cytoplasmic vesicles but was more concentrated near the cytoplasmic membrane (Figure 5); apparently, there are no differences in distribution of fluorescence in trophozoites between the two species. However, in nonpermeabilized trophozoites activated with peripheral mononuclear blood cells, the immunolocalization of CRT/C1q was detected on the surface membrane of trophozoites (Figure 6). Furthermore, when the trophozoites were incubated with NHS, the colocalization of both proteins was detected mainly in the cytoplasmic vesicles (Figure 7); this could be due to the destruction of trophozoites membranes induced by NHS.


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)

Colocalization of EhCRT or EdCRT and human C1q after interaction with PMBC. C1q and EhCRT or EdCRT colocalization by confocal microscopy of (a) E. histolytica; (b) E. dispar trophozoites. Trophozoites were grown under axenic conditions and incubated during 30 min with PMBC; thereafter, C1q was added and incubated for 30 min. Then, the mixture was added with specific primary antibodies, anti-rabbit EhCRT IgG and mouse anti-human C1q IgG, respectively. The reaction was revealed with Alexa Fluor 555 goat anti-rabbit IgG and Alexa Fluor 488 goat anti-mouse IgG. The micrographs show the maximal projection of the z-series. Scale bar represents 20 μm. A: phase contrast microscopy; B: EhCRT (red); C: C1q (green); D: merge.
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Related In: Results  -  Collection

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

fig6: Colocalization of EhCRT or EdCRT and human C1q after interaction with PMBC. C1q and EhCRT or EdCRT colocalization by confocal microscopy of (a) E. histolytica; (b) E. dispar trophozoites. Trophozoites were grown under axenic conditions and incubated during 30 min with PMBC; thereafter, C1q was added and incubated for 30 min. Then, the mixture was added with specific primary antibodies, anti-rabbit EhCRT IgG and mouse anti-human C1q IgG, respectively. The reaction was revealed with Alexa Fluor 555 goat anti-rabbit IgG and Alexa Fluor 488 goat anti-mouse IgG. The micrographs show the maximal projection of the z-series. Scale bar represents 20 μm. A: phase contrast microscopy; B: EhCRT (red); C: C1q (green); D: merge.
Mentions: To evaluate the interaction of EhCRT/EdCRT in trophozoites with human C1q, a colocalization assay was performed directly on trophozoites of both E. histolytica and E. dispar species by confocal microscopy. Both proteins clearly colocalized in previously permeabilized trophozoites; the fluorescent signal was detected in the cytoplasmic vesicles but was more concentrated near the cytoplasmic membrane (Figure 5); apparently, there are no differences in distribution of fluorescence in trophozoites between the two species. However, in nonpermeabilized trophozoites activated with peripheral mononuclear blood cells, the immunolocalization of CRT/C1q was detected on the surface membrane of trophozoites (Figure 6). Furthermore, when the trophozoites were incubated with NHS, the colocalization of both proteins was detected mainly in the cytoplasmic vesicles (Figure 7); this could be due to the destruction of trophozoites membranes induced by NHS.

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