Involvement of Raft-Like Plasma Membrane Domains of Entamoeba histolytica in Pinocytosis and Adhesion

Abstract

Lipid rafts are highly ordered, cholesterol-rich, and detergent-resistant microdomains found in the plasma membrane of many eukaryotic cells. These domains play important roles in endocytosis, secretion, and adhesion in a variety of cell types. The parasitic protozoan Entamoeba histolytica, the causative agent of amoebic dysentery, was determined to have raft-like plasma membrane domains by use of fluorescent lipid analogs that specifically partition into raft and nonraft regions of the membrane. Disruption of raft-like membrane domains in Entamoeba with the cholesterol-binding agents filipin and methyl-␤-cyclodextrin resulted in the inhibition of several important virulence functions, fluid-phase pinocytosis, and adhesion to host cell monolayers. However, disruption of raft-like domains did not inhibit constitutive secretion of cysteine proteases, another important virulence function of Entamoeba. Flotation of the cold Triton X-100-insoluble portion of membranes on sucrose gradients revealed that the heavy, intermediate, and light subunits of the galactose-N-acetylgalactosamine-inhibitible lectin, an important cell surface adhesion molecule of Entamoeba, were enriched in cholesterol-rich (raft-like) fractions, whereas EhCP5, another cell surface molecule, was not enriched in these fractions. The subunits of the lectin were also observed in high-density, actin-rich fractions of the sucrose gradient. Together, these data suggest that pinocytosis and adhesion are raft-dependent functions in this pathogen. This is the first report describing the existence and physiological relevance of raft-like membrane domains in E. histolytica.Recent evidence suggests that plasma membrane lipids are nonhomogeneously distributed and that microdomains with specialized functions exist in the membrane. One such domain, a lipid raft, is a highly ordered, less-fluid, and tightly packaged membrane domain enriched in cholesterol (or other sterols), glycosphingolipids, and phospholipids with a higher degree of saturated fatty acyl chains than those of the rest of the membrane (reviewed in references 37 and 54). These domains are also detergent insoluble and are thus often referred to as detergent-resistant membranes (DRMs). The presence of microdomains in membranes allows for the inclusion or exclusion of membrane proteins based on their attachment to the membrane via lipid anchors or specific protein-lipid interactions. For example, proteins that are modified with a hydrophobic attachment, such as a glycosylphosphatidylinositol (GPI) anchor, or double acylation or transmembrane proteins with the capacity to interact with cholesterol are often found in lipid microdomains.The physiological role of lipid rafts has been the subject of numerous recent studies, and it has recently become clear that these membrane regions play an important role in a variety of cellular functions, including polarization, signal transduction, endocytosis, secretion, and cell-cell and cell-pathogen adhesion (17,18,21,34,36,44). A range of cell surface receptors m...

EhLimA, a Novel LIM Protein, Localizes to the Plasma Membrane inEntamoeba histolytica

“…In this case also, a significant portion of EhLimA was found in the Triton X-100-insoluble pellet fraction. Interestingly, a similar distribution of the amoebic Gal-lectin light subunit (Ehlgl), which was reported to be present in raft-like fractions (33), was observed in these fractions as well.…”

“…Association of EhLimA with lipid rafts was shown by the distribution of a portion of EhLimA to the lower-density regions of a sucrose gradient. Since raft-like microdomains have only recently been identified in E. histolytica (33), at present there is no well-accepted raft marker for this organism. We used the light subunit of the amoebic Gallectin (Ehlgl) that was shown to interact with lipid microdomains in E. histolytica (33) and found that it was indeed enriched in the lower-density regions of the sucrose gradient.…”

“…This was expected, since we have shown that EhLimA associates with the actin cytoskeleton that itself distributes to the high-density regions of the gradient, as seen here by the predominance of actin in these fractions. Ehlgl, which as already mentioned has been shown to interact with lipid microdomains in E. histolytica (33), was used as a marker for raft-like domains and was indeed found to be enriched in the lower-density regions of the gradient, though it also appeared in the higher-density regions. Results obtained in three independent experiments revealed some differences in the distribution of EhLimA, but in all experiments, a portion of EhLimA was found in association with the lower-density regions of the gradient.…”

“…Lipid rafts are membrane domains that have been found to play a role in various cellular functions, including membrane trafficking, endocytosis, secretion, and signal transduction (13,52,53). Recently, raft-like microdomains were identified in E. histolytica and were found to be involved in pinocytosis and adhesion (33). Insolubility in Triton X-100, which is a property common to cytoskeletonassociated proteins, can also serve as an indication that the protein associates with lipid rafts (25,49) that are insoluble in Triton X-100 due to their high cholesterol and sphingolipid content (14,52).…”

Localization of Phosphatidylinositol 4,5-Bisphosphate to Lipid Rafts and Uroids in the Human Protozoan Parasite Entamoeba histolytica

“…Rafts are specialized membrane microdomains that are cho-lesterol and sphingolipid rich and are known to organize signaling molecules that participate in a variety of cellular functions, including cell polarization (21,22), endocytosis (reviewed in reference 23), secretion (reviewed in reference 24), and adhesion (25). Previously, E. histolytica was shown to possess lipid rafts that regulate parasite-host interaction (26,27). Disruption of rafts with cholesterol-binding agents like methyl-␤-cyclodextrin (M␤CD) inhibits adhesion of E. histolytica to host cells (26) and host extracellular matrix components (27).…”