Leishmania adleri, a lizard parasite, expresses structurally similar glycoinositolphospholipids to mammalian Leishmania

J.O.Previato 1 CJones 1 R-Wait 1 F.Routier 0 1 E.Saraiva 1 L.Mendonca-Previato 1 0 Laboratoire de Chimie Biologique , UMR 111 du CNRS, USTL, 59 655 Villeneuve d'Ascq, France 1 Instituto de Microbiologia, Universidade Federal do Rio de Janeiro , 21 944 970, Cidade Universiuiria, Rio de Janeiro-RJ, Brazil , 'Laboratory for Molecular Structure , NIBSC, Potters Bar, Herts EN6 3QG, UK 2 n t r e for Applied Microbiology and Research , Salisbury SP4 OJG, UK *To whom correspondence should be addressed - Glycoinositolphospholipids (GIPLs) were isolated from promastigotes of the lizard parasites Leishmania adleri by phenol/water extraction. Phosphoinositol oligosaccharides were liberated by mild alkaline hydrolysis, purified by gel filtration and high pH anion exchange chromatography, and characterized by methylation analysis, fast atom bombardment mass spectrometry, and nuclear magnetic resonance spectroscopy. The four major compounds (I-IV) from L.adUri were linked to alkylacyl glycerol, and their glycan moieties had the following structures: M a n a ( l 2)Mana(l-6)[ Mana(l-3)] Mana(l^t)GlcNa(l-6)Ins-lPO4 (I), G a l p a ( l - 6 ) G a l p a ( l - 3 ) G a ( / P ( l - 3 ) M a n a ( l 3 ) M a n a ( l - 4 ) G l c N a ( l - 6 ) I n s - l - P O 4 (II), G a l ^ a ( l 3)Gal/P(l-3)Mana(l-3)Mana(l-4)GlcNa(l-6)Ins-l-PO4 (III), M a n a ( l - 2 ) [ E t N P ( - 6 ) ] M a n a ( l - 6 ) [ M a n a ( l - 3 ) ] Mana(l-4)GIcNa(l-6)Ins-l-PO4 (IV). These compounds are analogous to the previously characterized GIPLs from New and Old World leishmania] parasites of mammals designated iM4 (identical to compound I), GIPLs 3 and 2 (identical to compounds II and III, respectively), and EPiM4 (identical to compound IV), which is consistent with a close phylogenetic relationship between lizard and mammalian Leishmania, However, in contrast to the mamma- Han parasites, the abundant surface glycoconjugate known as lipophosphoglycan was either absent or confined to the flagellar pocket region in L.adlert Introduction Parasites of the Trypanosomatidae family are responsible for many diseases of clinical and veterinary importance. Examples include Leishmania species, Trypanosoma cruzi, the etiological agent of Chagas's disease, and the members of the T.brucei complex which cause sleeping sickness in humans and nagana in cattle. The genus Leishmania contains a large number of pathogenic species and subspecies, which are responsible for an overlapping complex of visceral, cutaneous and mucocutaneous clinical syndromes. These range from relatively mild selflimiting cutaneous forms caused by (among other species) Lmajor and Ltropica in the Old World and Lmexicana and LbrazMensis in South America, to visceral leishmaniasis (kala azar) caused by Ldonovani and Linfantum (Old World) and Lchagasi (New World), which, if untreated, results in extremely high mortality (Grimaldi and Tesh, 1993). Leishmaniases are endemic in 82 countries on four continents, with an estimated 12 million individuals infected (Desjeux, 1992). Although cases are concentrated in third-world countries, the disease is not exclusive to developing nations, and occurs, for example, throughout die Mediterranean basin. The number of Linfantum and HTV coinfections in southern Europe is increasing rapidly (Alvar, 1994). Leishmania species which parasitize non-human hosts have also been identified, including several parasites of Old World lizards. The promastigote stages of these lizard parasites are morphologically similar to those of mammalian Leishmania, and both groups are transmitted by blood-sucking sandflies. However, as amastigote forms are rarely observed, and multiplication within macrophages has not been unequivocally demonstrated in vivo, the taxonomic status of these organisms is ambiguous, and a new genus, the Sauroleishmania has been proposed to accommodate them (Saf janova, 1986; Lainson and Shaw, 1987). Since these organisms have been extensively used as experimental systems, it is important to clarify their relationship to the leishmanial parasites of mammals. The cell surfaces of Leishmania species which parasitize mammals are coated with glycosylphosphatidylinositol (GPI)anchored proteins and two classes of protein-free GPIcontaining lipids: the lipophosphoglycans (LPGs), and the lowmolecular mass glycoinositolphospholipids (GIPLs) (McConville and Ferguson, 1993). The structures of LPGs from five species of Leishmania (L.major, L.mexicana, Ldonovani, Ltropica and Laethiopica) have now been elucidated (McConville et al., 1995), and indicate the existence of significant intra- and inter-specific polymorphism, though die variability is confined to the capping oligosaccharides and the side-chains, since the phosphosaccharide repeats and die GPI core are apparently conserved. The GIPLs of Leishmania have been classified into three lineages on the basis of the linkage between the two mannose residues proximal to inositol (McConville and Ferguson, 1993). In type-1 GIPLs, the second mannose is (1-6) linked to the first, as in the GPI-protein anchors. Type-2 GIPLs differ in that this linkage is (1-3), as in the LPG anchor. Hybrid type GEPLs are branched structures with both (1-3) and (1-6) mannose residues linked to the first mannose. Lmajor promastigotes synthesize predominantly galactose-terminating GIPLs belonging to type-2 lineage, whereas L.mcxicana, Ldonovani, Ltropica, and Laethiopica express mannoseterminating materials from the type-1 and hybrid series. The function of these molecules is still unclear. However, because their structures vary between species and genera they could provide useful phylogenetic markers in the trypanosomatidae family. In evolutionary terms, the GPI-anchored glycoproteins seem to precede the GIPLs. For example, the earliest-diverging trypanosomatid lineage is T.brucei, which is unable to synthesize GIPLs although its glycoproteins have typical GPI-anchors. T.cruzi is the first trypanosomatid lineage in which free GIPLs appear, and these compounds are closely related in structure to the GPI anchors of the surface glycoproteins of this organism (Previato et al, 1995; Carreira et al., 1996). However, the GIPLs of homoxenous and heteroxenous species, which have separated from the trypanosomatid lineage more recently, are increasingly divergent in structure from GPI-protein anchors (Previato et al., 1994; Routier et al., 1995). Comparative examination of GIPL and LPG structures may thus clarify the phylogenetic relationship between the leishmanial parasites of lizards and those of mammals. Ladleri is a lizard parasite that is intermediate in some respects between saurian and mammalian Leishmania. Like mammalian Leishmania (subgenus Leishmania), but in contrast to most other lizard Leishmania, it undergoes the sandfly vector stage of its developmental cycle in the anterior portion of the mid-gut (Heisch, 1958), and is able to establish transient cutaneous infections when experimentally inoculated into humans (...truncated)