Acylation in trypanosomatids: an essential process and potential drug target.

Fatty acylation--the addition of fatty acid moieties such as myristate and palmitate to proteins--is essential for the survival, growth, and infectivity of the trypanosomatids: Trypanosoma brucei, Trypanosoma cruzi, and Leishmania. Myristoylation and palmitoylation are critical for parasite growth, targeting and localization, and the intrinsic function of some proteins. The trypanosomatids possess a single N-myristoyltransferase (NMT) and multiple palmitoyl acyltransferases, and these enzymes and their protein targets are only now being characterized. Global inhibition of either process leads to cell death in trypanosomatids, and genetic ablation of NMT compromises virulence. Moreover, NMT inhibitors effectively cure T. brucei infection in rodents. Thus, protein acylation represents an attractive target for the development of new trypanocidal drugs.

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.

Exposure to host ligands correlates with colocalization of Gal/GalNAc lectin subunits in lipid rafts and phosphatidylinositol (4,5)-bisphosphate signaling in Entamoeba histolytica.

Entamoeba histolytica is an intestinal parasite that causes dysentery and liver abscess. Parasite cell surface receptors, such as the Gal/GalNAc lectin, facilitate attachment to host cells and extracellular matrix. The Gal/GalNAc lectin binds to galactose or N-acetylgalactosamine residues on host components and is composed of heavy (Hgl), intermediate (Igl), and light (Lgl) subunits. Although Igl is constitutively localized to lipid rafts (cholesterol-rich membrane domains), Hgl and Lgl transiently associate with this compartment in a cholesterol-dependent fashion. In this study, trophozoites were exposed to biologically relevant ligands to determine if ligand binding influences the submembrane distribution of the subunits. Exposure to human red blood cells (hRBCs) or collagen, which are bona fide Gal/GalNAc lectin ligands, was correlated with enrichment of Hgl and Lgl in rafts. This enrichment was abrogated in the presence of galactose, suggesting that direct lectin-ligand interactions are necessary to influence subunit location. Using a cell line that is able to attach to, but not phagocytose, hRBCs, it was shown that physical attachment to ligands was not sufficient to induce the enrichment of lectin subunits in rafts. Additionally, the mutant had lower levels of phosphatidylinositol (4,5)-bisphosphate (PIP(2)); PIP(2) loading restored the ability of this mutant to respond to ligands with enrichment of subunits in rafts. Finally, intracellular calcium levels increased upon attachment to collagen; this increase was essential for the enrichment of lectin subunits in rafts. Together, these data provide evidence that ligand-induced enrichment of lectin subunits in rafts may be the first step in a signaling pathway that involves both PIP(2) and calcium signaling.

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.