Erythrophagocytosis in Entamoeba histolytica and Entamoeba dispar: a comparative study

Abstract

Entamoeba histolytica is the causative agent of human intestinal and liver amebiasis. The extraordinary phagocytic activity of E. histolytica trophozoites has been accepted as one of the virulence mechanisms responsible for their invasive capacity. The recognition of the noninvasive Entamoeba dispar as a different species has raised the question as to whether the lack of pathogenic potential of this ameba correlates with a limited phagocytic capacity. We have therefore compared the process of erythrophagocytosis in both species by means of light and video microscopy, hemoglobin measurement, and the estimation of reactive oxygen species (ROS). In the present study, we confirmed that E. dispar has lower erythrophagocytic capacity. We also observed by video microscopy a new event of erythrocyte opsonization-like in both species, being more characteristic in E. histolytica. Moreover, E. dispar showed a lower capacity to produce ROS compared with the invasive species and also showed a large population of amoebae that did not engulf any erythrocyte over time. Our results demonstrate that E. histolytica has a higher phagocytic capacity than E. dispar, including a higher rate of production of ROS in the course of ingesting red blood cells.

Figure 1

Erythrocyte uptake performed with E. dispar (blue) and E. histolytica (red) by light microscopy. The experiment was repeated three times independently in triplicates. The statistical comparisons showed significant differences in the erythrophagocytic capacity between species and among times (P < 0.001; different letters on top). Moreover, species compared with their reciprocal times also showed a significant difference (P < 0.001; bottom letters: a with b, c with d, and e with f) (*). Outliers.

Figure 2

Hemoglobin content in E. dispar (blue) and E. histolytica (red) after erythrophagocytosis, determined by spectrophotometry. The experiment was repeated three times independently in triplicates. Quantification of erythrophagocytosis measured by ingested hemoglobin indicated no significant differences among times (letters on top, connected by the same line segment; a and b). A significant difference between species compared with their reciprocal times (P < 0.001; bottom letters connected by the same line segment) was observed.

Figure 3

Scatter diagrams showing correlations between area and the number of ingested erythrocytes with respect to interaction time. There is a direct and significant relation (P < 0.001) for both strains, meaning that the larger is the area of the amoeba, these engulf more erythrocytes. This ratio increases as time passes. The same trend for both strains was observed; however, correlations were higher for E. histolytica (“r” value).

Figure 4

Real time video microscopy showing the opsonization-like event. (a) E. dispar: Figure 4(a)(A) shows a trophozoite (green) with a group of bound erythrocytes (red); then it can be seen how these erythrocytes completely detach from this amoeba (Figure 4(a)(B–D)), probably due to a low affinity binding. A new trophozoite (blue) appears in the scene (Figure 4(a)(D)) ready to bind the erythrocytes that had been bound to another amoeba (Figure 4(a)(E and F)). Once again, these erythrocytes will detach from this blue amoeba (Figure 4(a)(G–I)). (b) E. histolytica: Figure 4(b)(A) shows a trophozoite (green) which binds erythrocytes (red) sending them to the caudal pole until it encounters another trophozoite (blue) (Figure 4(b)(B–D)); after both amoebas bound to the same group of erythrocytes, it is appreciated how they “fight” to keep the erythrocytes clump (Figure 4(b)(E–H)). Finally, Figure 4(b)(I) shows how every amoeba keeps a portion of the erythrocytes. Numbers shown in the upper right corner of each image correspond to time in seconds.

Figure 5

Major nonphagocytic subpopulation of E. dispar versus a major phagocytic subpopulation of E. histolytica. Images taken from a video microscopy followup of the erythrocytes-trophozoites interaction showing the existence of a nonphagocytic subpopulation of E. dispar (most of the cells are nonphagocytic). Only a few cells contain erythrocytes (red dots) inside their cytoplasm even after 20 min interaction ((a)(A–I)). On the contrary, images taken from the video microscopy followup of E. histolytica trophozoites show that nearly 90% or higher of the population have ingested erythrocytes (red dots), after only 5 min of interaction ((b)(A–I)). Numbers shown in the upper right corner of each image correspond to time in seconds starting after 20 min incubation for E. dispar and 5 min incubation for E. histolytica.

Figure 6

Quantitation and confocal analysis of the nonphagocytic and phagocytic subpopulations of E. dispar and E. histolytica. Graph showing the percentage of nonphagocytic amoebas after different times of interaction for E. dispar (blue) and E. histolytica (red) (a). Experiments were done in triplicate for each of the analyzed times. (b) shows representative images of phagocytic and nonphagocytic subpopulations for E. dispar (left side) and E. histolytica (right side). Upper panels, light microscopy; lower panels, confocal images. Arrow head: a nonphagocytic trophozoite.

Figure 7

Evaluation of the difference in the oxide-reduction ability between E. dispar and E. histolytica using the NBT reduction assay. The experiment was repeated three times independently in triplicates. Results describing the oxide-reduction ability of both strains, measured by reduction of NBT, indicate that after 60 min incubation, there is not a significant change in the amount of formazan produced (letters on top connected by the same line segment); however, between 30 and 60 min there is a significant difference appreciated in both species (letters on top). Moreover, species compared with their reciprocal times also showed a significant difference (P < 0.001) (different letters on bottom connected by the same line segment).