Protozoan parasites of the phylum Apicomplexa contain three genetic elements: the nuclear and mitochondrial genomes characteristic of virtually all eukaryotic cells and a 35-kilobase circular extrachromosomal DNA. In situ hybridization techniques were used to localize the 35-kilobase DNA of Toxoplasma gondii to a discrete organelle surrounded by four membranes. Phylogenetic analysis of the tufA gene encoded by the 35-kilobase genomes of coccidians T. gondii and Eimeria tenella and the malaria parasite Plasmodium falciparum grouped this organellar genome with cyanobacteria and plastids, showing consistent clustering with green algal plastids. Taken together, these observations indicate that the Apicomplexa acquired a plastid by secondary endosymbiosis, probably from a green alga.

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The surface of the protozoan parasite Leishmania is unusual in that it consists predominantly of glycosylphosphatidylinositol-anchored glycoconjugates and proteins. Additionally, a family of hydrophilic acylated surface proteins (HASPs) has been localized to the extracellular face of the plasma membrane in infective parasite stages. These surface polypeptides lack a recognizable endoplasmic reticulum secretory signal sequence, transmembrane spanning domain, or glycosylphosphatidylinositol-anchor consensus sequence, indicating that novel mechanisms are involved in their transport and localization. Here, we show that the Nterminal domain of HASPB contains primary structural information that directs both N-myristoylation and palmitoylation and is essential for correct localization of the protein to the plasma membrane. Furthermore, the N-terminal 18 amino acids of HASPB, encoding the dual acylation site, are sufficient to target the heterologous Aequorea victoria green fluorescent protein to the cell surface of Leishmania. Mutagenesis of the predicted acylated residues confirms that modification by both myristate and palmitate is required for correct trafficking. These data suggest that HASPB is a representative of a novel class of proteins whose translocation onto the surface of eukaryotic cells is dependent upon a "nonclassical" pathway involving N-myristoylation/palmitoylation. Significantly, HASPB is also translocated on to the extracellular face of the plasma membrane of transfected mammalian cells, indicating that the export signal for HASPB is recognized by a higher eukaryotic export mechanism.Protozoan parasites of the genus Leishmania cause a spectrum of tropical and sub-tropical diseases termed the leishmaniases. Leishmania live as either extracellular, flagellated promastigotes in the digestive tract of their sandfly vector or as aflagellated amastigotes within the phagolysosomes of mammalian macrophages (1). Much research has focused on the unusually high levels of glycosylphosphatidylinositol (GPI) 1 -anchored surface molecules present in these organisms (2), particularly the unique glycoconjugate, lipophosphoglycan (LPG), abundant in promastigotes (3), and the metalloprotease GP63 (or leishmanolysin) (4). The hydrophilic acylated surface proteins (HASPs; originally named GBP and GA/CP in Leishmania major) are a family of related surface molecules expressed only in infective parasite stages (5, 6). These proteins are unusual in that they lack a "classical" endoplasmic reticulum (ER) secretory signal sequence, a GPI-anchor consensus, or membrane-spanning domains but are surface-localized (6), partition into the hydrophobic phase on Triton X-114 separation, and fractionate with lipid species (5). The mechanism of transport and attachment to the cell surface is unclear, although it has been suggested that this may occur by virtue of a close association with LPG via an HASP repeat region with homology to the peptidoglycan binding domain of Staphylococcus aureus protein A (6). More recent studies have not s...

Ether phospholipids are major components of the membranes of humans and Leishmania. In protozoan parasites they occur separately or as part of the glycosylphosphatidylinositol (GPI) anchor of molecules implicated in virulence, such as lipophosphoglycan (LPG), smaller glycosylinositolphospholipids (GIPLs), and GPIanchored proteins. We generated null mutants of the Leishmania major alkyldihydroxyacetonephosphate synthase (ADS), the first committed step of ether lipid synthesis. Enzymatic analysis and comprehensive mass spectrometric analysis showed that ads1 ؊ knock-outs lacked all ether phospholipids, including plasmalogens, LPG, and GIPLs. Leishmania ads1 ؊ thus represents the first ether lipid-synthesizing eukaryote for which a completely null mutant could be obtained. Remarkably ads1 ؊ grew well and maintained lipid rafts (detergentresistant membranes). In virulence tests it closely resembled LPG-deficient L. major, including sensitivity to complement and an inability to survive the initial phase of macrophage infection. Likewise it retained the ability to inhibit host cell signaling and to form infectious amastigotes from the few parasites surviving the establishment defect. These findings counter current proposals that GIPLs are required for amastigote survival in the mammalian host or that parasite lyso-alkyl or alkylacyl-GPI anchors are solely responsible for inhibition of macrophage activation.