Erythrophagocytosis is pathognomonic of amebiasis (49
), and deficiencies in this process have been associated with reduced virulence (31
). In this work we attempted to determine what ligands were recognized on erythrocytes for ingestion by E. histolytica
and whether these were also recognized by Entamoeba dispar
. Our conclusions from this study are as follows: (i) E. histolytica
causes physical changes on the surfaces of erythrocytes prior to ingestion, including the exposure of PS; (ii) PS is specifically recognized by an amebic coreceptor during the ingestion of erythrocytes; and (iii) the commensal parasite E. dispar
is relatively deficient in each step of erythrophagocytosis.
caused apparent surface changes on erythrocytes, including a change in the cell shape from concave to spherical, which were followed by membrane blebbing and PS exposure. These changes are consistent with both the aging of erythrocytes (9
) and calcium treatment (7
). The physical modifications of the erythrocyte membranes led to changes in their properties of adherence to amebae. Galactose was able to only partially block the recognition and consequent ingestion of calcium-treated erythrocytes; this was consistent with the existence of an additional receptor that recognized a ligand specific to the altered red-blood-cell surface. This theory was corroborated by the reduced galactose inhibitions of adherence and ingestion of calcium-treated erythrocytes by amebae compared to those of healthy erythrocytes. We surmise from these data that surface changes rendered by contact with amebae expose a new ligand on the erythrocytes, which can be recognized by an amebic receptor apart from the Gal/GalNAc lectin.
Previous experiments had implicated negatively charged lipids in recognition and phagocytosis by E. histolytica
. Bailey et al. demonstrated that liposomes comprised of the negatively charged lipids PS and dicetyl phosphate triggered actin polymerization in E. histolytica
trophozoites, whereas phosphatidylcholine, phosphatidylethanolamine, and phosphatidic acid did not (1
). Entamoeba histolytica
also showed increased ingestion of Jurkat leukemia T cells with added PS but not with added phosphatidylcholine, phosphatidylethanolamine, or phosphatidic acid on their surfaces (26
). These experiments showed that PS promoted ingestion of host cells, but there was no direct evidence suggesting the utilization of host cell PS exposure for ingestion by E. histolytica
Phosphatidylserine involvement in erythrocyte recognition and uptake was tested by the addition of annexin V to bind and mask exposed PS on the erythrocyte surface. Annexin V has been shown to bind to PS on apoptotic cells (51
) and interfere with the interactions between apoptotic cells and macrophages (19
). Annexin V inhibition of both adherence and phagocytosis and its additive nature with galactose suggest a coordinated role of the Gal/GalNAc lectin and a PS coreceptor in erythrophagocytosis.
Phosphatidylserine exposure has been well characterized on the surfaces of apoptotic cells (18
) as a signal for clearance in metazoans. There are many examples of receptors that recognize PS in mammals, including the stereospecific macrophage PS receptor (17
) and multiple scavenger receptors, which include CD36 (20
). Contrary to evidence pertaining to macrophages, the amebic receptor does not appear to recognize PS in a stereospecific manner; both phospho-l
- and phospho-d
-serine inhibited amebic phagocytosis, suggesting the amebic receptor functions as a scavenger receptor. There are no homologues of scavenger receptors or the mammalian PS receptor in either E. histolytica
genome database (available from The Institute for Genomic Research and the Sanger Centre). In addition, recent evidence suggests that the mammalian PS receptor does not participate in the endocytosis of apoptotic cells (4
). Therefore, the nature of the PS receptor that mediates recognition and endocytosis remains obscure for both amebae and mammals. The use of a PS receptor to ingest apoptotic cells by amebae is an interesting example of convergent evolution and illustrates the pressures on a parasite to emulate its host.
was initially distinguished from E. histolytica
by biochemical differences (21
), but much effort is being made to determine the genetic differences between these two species (54
). E. dispar
is noninvasive and rarely ingests host cells in vivo. The nature of the differences separating E. dispar
and E. histolytica
remains unknown, but the genome sequences of the two species are very closely related. One important finding was that the Gal/GalNAc adherence lectin was less prevalent on the surface of E. dispar
than on that of E. histolytica
). Additional information has been published by Pimenta et al. describing smaller vesicle formation by E. dispar
than by E. histolytica
during the ingestion of bacteria (35
). Otherwise, little is known about the capacity for E. dispar
to perform phagocytosis.
Given the noninvasive nature of Entamoeba dispar, it was important to address whether deficiencies existed in the processes of adherence, cytolysis, or phagocytosis. E. dispar exhibited reduced adherence to erythrocytes and little ability to cause externalization of PS to the outer leaflet of the membrane of erythrocytes. However, E. dispar was able to ingest PS-exposing erythrocytes, albeit more slowly than E. histolytica. The discrepancy between the ingestion of erythrocytes by E. dispar and that by E. histolytica may be explained by multiple factors, such as reduced Gal/GalNAc lectin expression, cell size, and long-term xenic growth conditions. Regardless of these differences, E. dispar can ingest erythrocytes exposing PS in a fashion similar to E. histolytica.
Future studies will focus on identifying the amebic receptors for PS as well as other receptors for apoptotic cells. A few candidates have already been uncovered through the work of other laboratories. Identification of a receptor would allow us to directly address the contribution of phagocytosis to virulence. We propose a stepwise model for amebic pathogenicity. First, the Gal/GalNAc lectin is used to adhere amebae to terminal galactose residues on host cells (33
). Once the membranes are within close proximity, secretion of amebapore (6
) and cysteine proteinases (25
) can damage the host cells, allowing calcium to flow into the cells and activate host cell apoptotic factors. As a direct result of this damage, PS is exposed on the surfaces of the erythrocytes, where amebic coreceptors can recognize and bind to them. This recognition then stimulates the phagocytic machinery, yielding ingestion of the damaged host cells. We hypothesize that amebic induction of host cell apoptosis leads to the rapid removal of dying tissue prior to the release of toxic cellular content, allowing the amebae to cause chronic infection by limiting inflammation.