Comparisons of mutants lacking the Golgi UDP-galactose or GDP-mannose transporters establish that phosphoglycans are important for promastigote but not amastigote virulence in Leishmania major.

Abundant surface Leishmania phosphoglycans (PGs) containing [Gal(beta1,4)Man(alpha1-PO(4))]-derived repeating units are important at several points in the infectious cycle of this protozoan parasite. PG synthesis requires transport of activated nucleotide-sugar precursors from the cytoplasm to the Golgi apparatus. Correspondingly, null mutants of the L. major GDP-mannose transporter LPG2 lack PGs and are severely compromised in macrophage survival and induction of acute pathology in susceptible mice, yet they are able to persist indefinitely and induce protective immunity. However, lpg2(-) L. mexicana amastigotes similarly lacking PGs but otherwise normal in known glycoconjugates remain able to induce acute pathology. To explore this further, we tested the infectivity of a new PG-null L. major mutant, which is inactivated in the two UDP-galactose transporter genes LPG5A and LPG5B. Surprisingly this mutant did not recapitulate the phenotype of L. major lpg2(-), instead resembling the L. major lipophosphoglycan-deficient lpg1(-) mutant. Metacyclic lpg5A(-)/lpg5B(-) promastigotes showed strong defects in the initial steps of macrophage infection and survival. However, after a modest delay, the lpg5A(-)/lpg5B(-) mutant induced lesion pathology in infected mice, which thereafter progressed normally. Amastigotes recovered from these lesions were fully infective in mice and in macrophages despite the continued absence of PGs. This suggests that another LPG2-dependent metabolite is responsible for the L. major amastigote virulence defect, although further studies ruled out cytoplasmic mannans. These data thus resolve the distinct phenotypes seen among lpg2(-) Leishmania species by emphasizing the role of glycoconjugates other than PGs in amastigote virulence, while providing further support for the role of PGs in metacyclic promastigote virulence.

Two functionally divergent UDP-Gal nucleotide sugar transporters participate in phosphoglycan synthesis in Leishmania major.

In the protozoan parasite Leishmania, abundant surface and secreted molecules, such as lipophosphoglycan (LPG) and proteophosphoglycans (PPGs), contain extensive galactose in the form of phosphoglycans (PGs) based on (Gal-Man-PO(4)) repeating units. PGs are synthesized in the parasite Golgi apparatus and require transport of cytoplasmic nucleotide sugar precursors to the Golgi lumen by nucleotide sugar transporters (NSTs). GDP-Man transport is mediated by the LPG2 gene product, and here we focused on transporters for UDP-Gal. Data base mining revealed 12 candidate NST genes in the L. major genome, including LPG2 as well as a candidate endoplasmic reticulum UDP-glucose transporter (HUT1L) and several pseudogenes. Gene knock-out studies established that two genes (LPG5A and LPG5B) encoded UDP-Gal NSTs. Although the single lpg5A(-) and lpg5B(-) mutants produced PGs, an lpg5A(-)/5B(-) double mutant was completely deficient. PG synthesis was restored in the lpg5A(-)/5B(-) mutant by heterologous expression of the human UDP-Gal transporter, and heterologous expression of LPG5A and LPG5B rescued the glycosylation defects of the mammalian Lec8 mutant, which is deficient in UDP-Gal uptake. Interestingly, the LPG5A and LPG5B functions overlap but are not equivalent, since the lpg5A(-) mutant showed a partial defect in LPG but not PPG phosphoglycosylation, whereas the lpg5B(-) mutant showed a partial defect in PPG but not LPG phosphoglycosylation. Identification of these key NSTs in Leishmania will facilitate the dissection of glycoconjugate synthesis and its role(s) in the parasite life cycle and further our understanding of NSTs generally.

Quantitation of Leishmania lipophosphoglycan repeat units by capillary electrophoresis.

The glycosylphosphatidylinositol (GPI)-anchored lipophosphoglycan (LPG) of Leishmania is the dominant cell surface glycoconjugate of these pathogenic parasites. LPG is structurally characterized by a series of phosphoglycan repeat units. Determining the number of repeat units per LPG molecule has proven difficult using current technologies, such as mass spectrometry. As an alternative method to quantitate the number of repeat units in LPG, a procedure based on capillary electrophoretic analysis of the proportion of mannose to 2,5-anhydromannose (derived from the nonacetylated glucosamine of the GPI anchor of LPG) was developed. The CE-based technique is sensitive and relatively rapid compared to GC-MS-based protocols. Its application was demonstrated in quantitating the number of LPG repeat units from several species of Leishmania as well as from two life-cycle stages of these organisms.

Leishmania braziliensis: a novel mechanism in the lipophosphoglycan regulation during metacyclogenesis.

During metacyclogenesis of Leishmania in its sand fly vector, the parasite differentiates from a noninfective, procyclic form to an infective, metacyclic form, a process characterised by morphological changes of the parasite and also biochemical transformations in its major surface lipophosphoglycan (LPG). This lipid-anchored polysaccharide is polymorphic among species with variations in sugars that branch off the conserved Gal(beta1,4)Man(alpha1)-PO4 backbone of repeat units and the oligosaccharide cap. Lipophosphoglycan has been implicated as an adhesion molecule that mediates the interaction with the midgut epithelium of the sand fly in the subgenus Leishmania. This paper describes the LPG structure for the first time in a species from the subgenus Viannia, Leishmania (Viannia) braziliensis. The LPG from the procyclic form of L. braziliensis was found to lack side chain sugar substitutions. In contrast to other species from the subgenus Leishmania, metacyclic forms of L. braziliensis makes less LPG and add 1-2 (beta1-3) glucose residues that branch off the disaccharide-phosphate repeat units of LPG. Thus, this represents a novel mechanism in the regulation of LPG structure during metacyclogenesis.

Leishmania tropica: intraspecific polymorphisms in lipophosphoglycan correlate with transmission by different Phlebotomus species.

Lipophosphoglycan (LPG) is a dominant surface molecule of Leishmania promastigotes which has been shown to be critical for parasite-sand fly vector interactions. To provide additional evidence for its importance in transmission, the LPGs from three Leishmania tropica strains that differ in their capability to infect sand flies, were biochemically characterized. One of these strains, ISER/IL/98/LRC-L747, was isolated from a Phlebotomus sergenti female collected in the Judean Desert close to Jerusalem. The other strains originated from a different focus in the Galilee region of northern Israel. One was isolated from a patient (MHOM/IL/02/Ofri-LRC-L863) and the other from a naturally infected Phlebotomus arabicus female (IARA/IL/00/Amnunfly1-LRC-L810). The LPG structures of the isolates from the Galilee (L863 and L810) were similar to each other, but differed in the LPG repeat units from the Judean Desert isolate (L747). The terminal sugar in the side chains of the repeat units of LPG purified from L863 and L810 was beta-galactose and was not capped with glucose, unlike L747 and a previously characterized L. tropica strain from Iraq (L36). Since L810 was isolated from P. arabicus and L747 from P. sergenti, variations in the structure of their LPGs may explain their capacity to infect different sand fly species.

The LPG1 gene family of Leishmania major.

In Leishmania major, the core of the abundant surface lipophosphoglycan (LPG) is structurally related to that of the smaller glycosylinositolphospholipids (GIPLs) in containing galactosylfuranose (Gal(f)) residues in a Gal(f) (beta1, 3)Man motif. However, deletion of the putative Gal(f)-transferase (Gal(f)T) LPG1 affected Gal(f) incorporation in LPG but not GIPLs. We hypothesized that the presumptive GIPL Gal(f)-transferases could be homologous to LPG1, and identified three related genes in the L. major genome. These were termed LPG1L, LPG1R, and LPG1G, the latter of which was found in three identical copies located at the telomeres of chromosomes 5, 19, and 32 based on Leishmania genome project data. Neither LPG1 nor its homologues LPG1L and LPG1R were involved in the biosynthesis of GIPLs, as an lpg1(-)/lpg1l(-)/lpg1r(-) triple knockout (the first such in Leishmania) grew normally and made wild-type levels of Gal(f) -containing GIPLs. In contrast, overexpression of these three led to elevated galactose incorporation in glycoproteins. Gal(f)-containing glycoproteins had not been described in Leishmania but occur at high levels in other closely related trypanosomatids including Trypanosoma cruzi, Crithidia, Leptomonas, and Endotrypanum, and LPG1L and LPG1R homologs were detected in these species. These data suggest that the glyco-synthetic capabilities of Leishmania and perhaps other trypanosomatids may be larger than previously thought, with some activities being 'cryptic' in different lineages and potentially serving as reservoirs for glycoconjugate variation during evolution. Future tests will address whether the LPG1G family encodes the hypothesized GIPL-specific Gal(f)T.

Ether phospholipids and glycosylinositolphospholipids are not required for amastigote virulence or for inhibition of macrophage activation by Leishmania major.

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 GPI-anchored 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 (detergent-resistant 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.