Clustering of adenosine A2B receptors with ectonucleotidases in caveolin-rich lipid rafts underlies immunomodulation by Leishmania amazonensis.

Extracellular adenosine plays important roles in modulating the immune responses. We have previously demonstrated that infection of dendritic cells (DC) by Leishmania amazonensis leads to increased expression of CD39 and CD73 and to the selective activation of the low affinity A2B receptors (A2B R), which contributes to DC inhibition, without involvement of the high affinity A2A R. To understand this apparent paradox, we now characterized the alterations of both adenosine receptors in infected cells. With this aim, bone marrow-derived DC from C57BL/6J mice were infected with metacyclic promastigotes of L. amazonensis. Fluorescence microscopy revealed that L. amazonensis infection stimulates the recruitment of A2B R, but not of A2A R, to the surface of infected DC, without altering the amount of mRNA or the total A2B R density, an effect dependent on lipophosphoglycan (LPG). Log-phase promastigotes or axenic amastigotes of L. amazonensis do not stimulate A2B R recruitment. A2B R clusters are localized in caveolin-rich lipid rafts and the disruption of these membrane domains impairs A2B R recruitment and activation. More importantly, our results show that A2B R co-localize with CD39 and CD73 forming a "purinergic cluster" that allows for the production of extracellular adenosine in close proximity with these receptors. We conclude that A2B R activation by locally produced adenosine constitutes an elegant and powerful evasion mechanism used by L. amazonensis to down-modulate the DC activation.

Proteomic analysis of detergent-resistant membrane microdomains in trophozoite blood stage of the human malaria parasite Plasmodium falciparum.

Intracellular pathogens contribute to a significant proportion of infectious diseases worldwide. The successful strategy of evading the immune system by hiding inside host cells is common to all the microorganism classes, which exploit membrane microdomains, enriched in cholesterol and sphingolipids, to invade and colonize the host cell. These assemblies, with distinct biochemical properties, can be isolated by means of flotation in sucrose density gradient centrifugation because they are insoluble in nonionic detergents at low temperature. We analyzed the protein and lipid contents of detergent-resistant membranes from erythrocytes infected by Plasmodium falciparum, the most deadly human malaria parasite. Proteins associated with membrane microdomains of trophic parasite blood stages (trophozoites) include an abundance of chaperones, molecules involved in vesicular trafficking, and enzymes implicated in host hemoglobin degradation. About 60% of the identified proteins contain a predicted localization signal suggesting a role of membrane microdomains in protein sorting/trafficking. To validate our proteomic data, we raised antibodies against six Plasmodium proteins not characterized previously. All the selected candidates were recovered in floating low-density fractions after density gradient centrifugation. The analyzed proteins localized either to internal organelles, such as the mitochondrion and the endoplasmic reticulum, or to exported membrane structures, the parasitophorous vacuole membrane and Maurer's clefts, implicated in targeting parasite proteins to the host erythrocyte cytosol or surface. The relative abundance of cholesterol and phospholipid species varies in gradient fractions containing detergent-resistant membranes, suggesting heterogeneity in the lipid composition of the isolated microdomain population. This study is the first report showing the presence of cholesterol-rich microdomains with distinct properties and subcellular localization in trophic stages of Plasmodium falciparum.

Sink or swim: lipid rafts in parasite pathogenesis.

Lipid rafts, sterol- and sphingolipid-rich membrane microdomains, have been extensively studied in mammalian cells. Recently, lipid rafts have been shown to control virulence in a variety of parasites including Entamoeba histolytica, Giardia intestinalis, Leishmania spp., Plasmodium spp., Toxoplasma gondii, and Trypanosoma spp. Parasite rafts regulate adhesion to host and invasion, and parasite adhesion molecules often localize to rafts. Parasite rafts also control vesicle trafficking, motility, and cell signaling. Parasites disrupt host cell rafts; the dysregulation of host membrane function facilitates the establishment of infection and evasion of the host immune system. Discerning the mechanism by which lipid rafts regulate parasite pathogenesis is essential to our understanding of virulence. Such insight may guide the development of new drugs for disease management.

Localisation to lipid rafts correlates with increased function of the Gal/GalNAc lectin in the human protozoan parasite, Entamoeba histolytica.

Entamoeba histolytica is the causative agent of dysentery and liver abscess and is prevalent in developing countries. Adhesion to the host is critical to infection and is mediated by amoebic surface receptors. One such receptor, the Gal/GalNAc lectin, binds to galactose or N-acetylgalactosamine residues on host components and consists of heavy (Hgl), light (Lgl) and intermediate (Igl) subunits. The mechanism by which the lectin assembles into a functional complex is not known. The parasite also relies on cholesterol-rich domains (lipid rafts) for adhesion. Therefore, it is conceivable that rafts regulate the assembly or function of the lectin. To test this, amoebae were loaded with cholesterol and lipid rafts were purified and characterised. Western blotting showed that cholesterol loading resulted in co-compartmentalisation of all three subunits in rafts. This co-compartmentalisation was accompanied by an increase in the ability of the amoebae to bind to host cells in a galactose-specific manner, suggesting that there is a correlation between location and function of the Gal/GalNAc lectin. Cholesterol loading did not increase the surface levels of the lectin subunits. Therefore, the cholesterol-induced increase in adhesion was not the result of externalisation of an internal pool of subunits. A mutant cell line that modestly responded to cholesterol with a slight increase in adhesion exhibited only a slight enrichment of Hgl and Lgl in rafts. This supports the connection between location and function of the Gal/GalNAc lectin. Actin can also influence the interaction of proteins with rafts. Therefore, the sub-membrane distribution of the lectin subunits was also assessed after treatment with an actin depolymerising agent, cytochalasin D. Cytochalasin D-treatment had no effect on the submembrane distribution of the subunits, suggesting that actin does not prevent the association of lectin subunits with rafts in this system. Together, these data provide insight into the molecular mechanisms regulating the location and function of this adhesin.

Stage-specific pathways of Leishmania infantum chagasi entry and phagosome maturation in macrophages.

The life stages of Leishmania spp. include the infectious promastigote and the replicative intracellular amastigote. Each stage is phagocytosed by macrophages during the parasite life cycle. We previously showed that caveolae, a subset of cholesterol-rich membrane lipid rafts, facilitate uptake and intracellular survival of virulent promastigotes by macrophages, at least in part, by delaying parasitophorous vacuole (PV)-lysosome fusion. We hypothesized that amastigotes and promastigotes would differ in their route of macrophage entry and mechanism of PV maturation. Indeed, transient disruption of macrophage lipid rafts decreased the entry of promastigotes, but not amastigotes, into macrophages (P<0.001). Promastigote-containing PVs were positive for caveolin-1, and co-localized transiently with EEA-1 and Rab5 at 5 minutes. Amastigote-generated PVs lacked caveolin-1 but retained Rab5 and EEA-1 for at least 30 minutes or 2 hours, respectively. Coinciding with their conversion into amastigotes, the number of promastigote PVs positive for LAMP-1 increased from 20% at 1 hour, to 46% by 24 hours, (P<0.001, Chi square). In contrast, more than 80% of amastigote-initiated PVs were LAMP-1+ at both 1 and 24 hours. Furthermore, lipid raft disruption increased LAMP-1 recruitment to promastigote, but not to amastigote-containing compartments. Overall, our data showed that promastigotes enter macrophages through cholesterol-rich domains like caveolae to delay fusion with lysosomes. In contrast, amastigotes enter through a non-caveolae pathway, and their PVs rapidly fuse with late endosomes but prolong their association with early endosome markers. These results suggest a model in which promastigotes and amastigotes use different mechanisms to enter macrophages, modulate the kinetics of phagosome maturation, and facilitate their intracellular survival.

Host-cell lipid rafts: a safe door for micro-organisms?

The lipid raft hypothesis proposed that these microdomains are small (10-200 nM), highly dynamic and enriched in cholesterol, glycosphingolipids and signalling phospholipids, which compartmentalize cellular processes. These membrane regions play crucial roles in signal transduction, phagocytosis and secretion, as well as pathogen adhesion/interaction. Throughout evolution, many pathogens have developed mechanisms to escape from the host immune system, some of which are based on the host membrane microdomain machinery. Thus lipid rafts might be exploited by pathogens as signalling and entry platforms. In this review, we summarize the role of lipid rafts as players in the overall invasion process used by different pathogens to escape from the host immune system.

Participation of macrophage membrane rafts in Trypanosoma cruzi invasion process.

Membrane rafts are small and dynamic regions enriched in sphingolipids, cholesterol, ganglioside GM1 and protein markers like flotillins, forming the flatter domains or caveolins, which are characterized as stable flask-shape invaginations. We explored whether membrane rafts participate in the entry of Trypanosoma cruzi's trypomastigotes into murine macrophages through transient depletion of macrophage membrane cholesterol with methyl-beta-cyclodextrin and treatment with filipin. These treatments led to a decrease in the trypomastigote invasion process. Macrophage pre incubated with increasing concentrations of cholera toxin B, that binds GM1, inhibited the adhesion and invasion of trypomastigote and amastigote forms. Immunofluorescence analysis demonstrated a colocalization of GM1, flotillin 1 and caveolin 1 in the T. cruzi parasitophorous vacuole. Taken together these data suggest that membrane rafts, including caveolae, are involved in the process of T. cruzi invasion of macrophages.

Cytoplasmic remodeling of erythrocyte raft lipids during infection by the human malaria parasite Plasmodium falciparum.

Studies of detergent-resistant membrane (DRM) rafts in mature erythrocytes have facilitated identification of proteins that regulate formation of endovacuolar structures such as the parasitophorous vacuolar membrane (PVM) induced by the malaria parasite Plasmodium falciparum. However, analyses of raft lipids have remained elusive because detergents interfere with lipid detection. Here, we use primaquine to perturb the erythrocyte membrane and induce detergent-free buoyant vesicles, which are enriched in cholesterol and major raft proteins flotillin and stomatin and contain low levels of cytoskeleton, all characteristics of raft microdomains. Lipid mass spectrometry revealed that phosphatidylethanolamine and phosphatidylglycerol are depleted in endovesicles while phosphoinositides are highly enriched, suggesting raft-based endovesiculation can be achieved by simple (non-receptor-mediated) mechanical perturbation of the erythrocyte plasma membrane and results in sorting of inner leaflet phospholipids. Live-cell imaging of lipid-specific protein probes showed that phosphatidylinositol (4,5) bisphosphate (PIP(2)) is highly concentrated in primaquine-induced vesicles, confirming that it is an erythrocyte raft lipid. However, the malarial PVM lacks PIP(2), although another raft lipid, phosphatidylserine, is readily detected. Thus, different remodeling/sorting of cytoplasmic raft phospholipids may occur in distinct endovacuoles. Importantly, erythrocyte raft lipids recruited to the invasion junction by mechanical stimulation may be remodeled by the malaria parasite to establish blood-stage infection.

Delivery of the malaria virulence protein PfEMP1 to the erythrocyte surface requires cholesterol-rich domains.

The particular virulence of the human malaria parasite Plasmodium falciparum derives from export of parasite-encoded proteins to the surface of the mature erythrocytes in which it resides. The mechanisms and machinery for the export of proteins to the erythrocyte membrane are largely unknown. In other eukaryotic cells, cholesterol-rich membrane microdomains or "rafts" have been shown to play an important role in the export of proteins to the cell surface. Our data suggest that depletion of cholesterol from the erythrocyte membrane with methyl-beta-cyclodextrin significantly inhibits the delivery of the major virulence factor P. falciparum erythrocyte membrane protein 1 (PfEMP1). The trafficking defect appears to lie at the level of transfer of PfEMP1 from parasite-derived membranous structures within the infected erythrocyte cytoplasm, known as the Maurer's clefts, to the erythrocyte membrane. Thus our data suggest that delivery of this key cytoadherence-mediating protein to the host erythrocyte membrane involves insertion of PfEMP1 at cholesterol-rich microdomains. GTP-dependent vesicle budding and fusion events are also involved in many trafficking processes. To determine whether GTP-dependent events are involved in PfEMP1 trafficking, we have incorporated non-membrane-permeating GTP analogs inside resealed erythrocytes. Although these nonhydrolyzable GTP analogs reduced erythrocyte invasion efficiency and partially retarded growth of the intracellular parasite, they appeared to have little direct effect on PfEMP1 trafficking.

Lipid rafts and malaria parasite infection of erythrocytes.

Infection of human erythrocytes by the malarial parasite, Plasmodium falciparum, results in complex membrane sorting and signaling events in the mature erythrocyte. These events appear to rely heavily on proteins resident in erythrocyte lipid rafts. Over the past five years, we and others have undertaken a comprehensive characterization of major proteins present in erythrocyte detergent-resistant membrane lipid rafts and determined which of these proteins traffic to the host-derived membrane that bounds the intraerythrocytic parasite. The data suggest that raft association is necessary but not sufficient for vacuolar recruitment, and that there is likely a mechanism of active uptake of a subset of erythrocyte detergent-resistant membrane proteins. Of the ten internalized proteins, few have been evaluated for a role in malarial entry. The beta(2)-adrenergic receptor and heterotrimeric G protein G(s) signaling pathway proteins regulate invasion. The implications of these differences are discussed. In addition, the latter finding indicates that erythrocytes possess important signaling pathways. These signaling cascades may have important influences on in vivo malarial infection, as well as on erythrocyte membrane flexibility and adhesiveness in sickle cell anemia. With respect to malarial infection, host signaling components alone are not sufficient to induce formation of the malarial vacuole. Parasite proteins are likely to have a major role in making the intraerythrocytic environment conducive for vacuole formation. Such interactions should be the focus of future efforts to understand malarial infection of erythrocytes since host- and parasite-targeted interventions are urgently needed to combat this terrible disease.

Molecular partitioning during host cell penetration by Toxoplasma gondii.

During invasion by Toxoplasma gondii, host cell transmembrane proteins are excluded from the forming parasitophorous vacuole membrane (PVM) by the tight apposition of host and parasite cellular membranes. Previous studies suggested that the basis for the selective partitioning of membrane constituents may be a preference for membrane microdomains, and this hypothesis was herein tested. The partitioning of a diverse group of molecular reporters for raft and nonraft membrane subdomains was monitored during parasite invasion by time-lapse video or confocal microscopy. Unexpectedly, both raft and nonraft lipid probes, as well as both raft and nonraft cytosolic leaflet proteins, flowed unhindered past the host-parasite junction into the PVM. Moreover, neither a raft-associated type 1 transmembrane protein nor its raft-dissociated counterpart accessed the PVM, while a multispanning membrane raft protein readily did so. Considered together with previous data, these studies demonstrate that selective partitioning at the host-parasite interface is a highly complex process, in which raft association favors, but is neither necessary nor sufficient for, inclusion into the T. gondii PVM.

Characterization of a membrane-associated rhoptry protein of Plasmodium falciparum.

Invasive forms of apicomplexan parasites contain secretory organelles called rhoptries that are essential for entry into host cells. We present a detailed characterization of an unusual rhoptry protein of the human malaria parasite Plasmodium falciparum, the rhoptry-associated membrane antigen (RAMA) that appears to have roles in both rhoptry biogenesis and host cell invasion. RAMA is synthesized as a 170-kDa protein in early trophozoites, several hours before rhoptry formation and is transiently localized within the endoplasmic reticulum and Golgi within lipid-rich microdomains. Regions of the Golgi membrane containing RAMA bud to form vesicles that later mature into rhoptries in a process that is inhibitable by brefeldin A. Other rhoptry proteins such as RhopH3 and RAP1 are found in close apposition with RAMA suggesting direct protein-protein interactions. We suggest that RAMA is involved in trafficking of these proteins into rhoptries. In rhoptries, RAMA is proteolytically processed to give a 60-kDa form that is anchored in the inner face of the rhoptry membrane by means of the glycosylphosphatidylinositol anchor. The p60 RAMA form is discharged from the rhoptries of free merozoites and binds to the red blood cell membrane by its most C-terminal region. In early ring stages RAMA is found in association with the parasitophorous vacuole.