Rapid polymerization of Entamoeba histolytica actin induced by interaction with target cells.

Abstract

The mechanism by which trophozoites of the protozoan parasite, Entamoeba histolytica destroy mammalian cells is not clear. A variety of amoeba-associated properties have been correlated with virulence (reviewed in 1 and 2). In vitro studies (3-6) have shown that contact of the trophozoite with a target cell is required. Contact killing has been described (6) as occurring in the sequence: adherence, cytolysis, then phagocytosis (possibly) of the target or its remnants. Dependence of in vivo virulence upon the phagocytic competence of the amoeba has also been reported (7). All steps of the sequence are inhibited by cytochalasins (6,8,9), indicating that dynamic participation of the amoeba actin cytoskeleton is required. However, the role of actin in the attack process has not been defined.By fluorescence microscopy of glutaraldehyde-fixed and rhodamine-phalloidin-stained (10) trophozoites, we have revealed the organization and distribution of polymerized actin in Entamoeba involved in a variety of motility-related activities (11, 12), including target cell interactions. Furthermore, by methanol extraction and spectrofluorometric measurement of the bound fluorescence, we have been able to quantitate polymerized actin in trophozoites. In this report, we describe this latter procedure and studies of the organization and quantity of polymerized actin in E. histolytica trophozoites before and immediately after challenge with human red blood cells (RBC) 1. Within 5 s after challenge, polymerized actin appeared at the contact interface with many adherent RBC. A net increase in amoeba polymerized actin content was detectable 1 rnin after the challenge, and reached a maximum of approximately twice the value in unchallenged cells within 4 min. Latex beads, which were phagocytized by E. histolytica trophozoites, neither stimulated an actin response nor were able to diminish the interaction with RBC. RBC, on the other hand, inhibited uptake of latex beads.These results indicate that the initial interaction of E. histolytica trophozoites with target cells is a recognition-specific process that triggers rapid polymerization of amoeba actin at the site of target contact. This actin appears to be involved in phagocytosis of the target cells. The procedure developed to quanThis work was supported in part by grants AI 19023 and RR 08006 from the National Institutes of Health, Bethesda, MD.i Abbreviations used in this paper: BSA, bovine serum albumin; PBS, phosphate-buffered saline; RBC, red blood cell. 546J. ExP. M~D.

Involvement of the actin cytoskeleton and p21rho-family GTPases in the pathogenesis of the human protozoan parasite Entamoeba histolytica

“…While latex beads are phagocytized by amebae, they do not stimulate an actin response, nor are they able to inhibit the interaction with red blood cells. Erythrocytes, however, diminish the phagocytosis of latex beads (30). These studies on amebic phagocytosis indicate that galactose-or GalNAc-containing lipids or proteins can instigate a full phagocytic response (including mobilization of the actin cytoskeleton) in the amebae, when they appear in the context of negatively charged phospolipids.…”

“…This correlates with the extension of an engulfing pseudopod. The polymerized actin content in a challenged ameba is double that of unchallenged cells, reaching this total within 4 min of adherence to target red blood cells (30). Actin polymerization can be inhibited by pre-incubating amebae with galactose or GalNAc before challenge (33), suggesting that the Gal/GalNAc lectin is involved in the initiation of actin polymerization.…”

“…Details are sparse, but localized actin polymerization around the site of contact is among the first events (30). Upon binding to either a red blood cell or a negatively charged liposome harboring galactosecontaining glycoproteins/glycosphingolipids, an increase in cortical actin polymerization can be seen in the ameba at the site of contact within five seconds (Figure 2) (30)(31)(32)(33). This correlates with the extension of an engulfing pseudopod.…”

“…Actin polymerization is essential for the cytolytic as well as the phagocytic activity of the ameba since treatment with cytochalasin D at 37 o C abolishes both activities (8,9,30), and induces the amebae to become spherical. Cytochalasins block actin polymerization by binding to the fast-growing end of actin, but do not cause actin depolymerization.…”

New insights into the role of the cytoskeleton in phagocytosis of Entamoeba histolytica

“…Multiple lines of evidence suggest that actin also plays a major role in the process of particle uptake: actin ®laments are concentrated in the pseudopod and cytoplasm surrounding phagosomes (Wang et al, 1984), cytochalasin blocks phagocytosis by neutrophils and macrophages (Zigmond and Hirsch, 1972;Axline and Reaven, 1974) and F-actin assembles around IgG-coated erythrocytes during phagocytosis by macrophages (Greenberg et al, 1988). In the case of E. histolytica, rapid actin polymerization is observed upon contact with target cells (Bailey et al, 1985;Bailey et al, 1990;Voigt, 1999), which, in turn, can lead to an upregulation of the actin mRNA transcription level (Manning-Cela and Meza, 1997). Latex beads of similar size to erythrocytes are phagocytosed more slowly than red blood cells and do not induce an evident actin polymerization (Bailey et al, 1985), suggesting that speci®c ligand interactions play a role in inducing actin polymerization.…”