The protozoan parasite Leishmania mexicana proliferates within macrophage phagolysosomes in the mammalian host. In this study we provide evidence that a novel class of intracellular ␤1-2 mannan oligosaccharides is important for parasite survival in host macrophages. Mannan (degree of polymerization 4 -40) is expressed at low levels in non-pathogenic promastigote stages but constitutes 80 and 90% of the cellular carbohydrate in the two developmental stages that infect macrophages, non-dividing promastigotes, and lesionderived amastigotes, respectively. Mannan is catabolized when parasites are starved of glucose, suggesting a reserve function, and developmental stages having low mannan levels or L. mexicana GDPMP mutants lacking all mannose molecules are highly sensitive to glucose starvation. Environmental stresses, such as mild heat shock or the heat shock protein-90 inhibitor, geldanamycin, that trigger the differentiation of promastigotes to amastigotes, result in a 10 -25-fold increase in mannan levels. Developmental stages with low mannan levels or L. mexicana mutants lacking mannan do not survive heat shock and are unable to differentiate to amastigotes or infect macrophages in vitro. In contrast, a L. mexicana mutant deficient only in components of the mannose-rich surface glycocalyx differentiates normally and infects macrophages in vitro. Collectively, these data provide strong evidence that mannan accumulation is important for parasite differentiation and survival in macrophages.Leishmania species are sandfly-transmitted protozoan parasites that cause a number of human diseases, ranging from self-healing cutaneous lesions to fatal visceral infections, afflicting more than 12 million people worldwide (www.who.int/ inf-fs/en/fact116.html). These parasites develop within the midgut of the sandfly vector, initially as rapidly dividing procyclic promastigotes and subsequently as non-dividing, but highly virulent, metacyclic promastigotes. Upon transmission to the mammalian host, metacyclic promastigotes invade macrophages and differentiate to the amastigote stage that proliferates within the phagolysosomal compartment. Parasite survival within the highly acidic and hydrolytic environment of the phagolysosome is likely to involve the induction of a number of biochemical processes, such as the activation of a heat shock response (1) and the stage-specific expression of specific nutrient transporters (2).It has recently been reported that one or more mannose (Man)-containing molecules are essential for L. mexicana survival in macrophages and infectivity in animals (3, 4). Specifically, targeted deletion of genes involved in GDP-Man synthesis, such as phosphomannomutase and GDP-Man pyrophosphorylase (GDP-MP), 1 did not affect parasite growth in rich culture medium but completely attenuated infectivity in macrophages and in highly susceptible BALB/c mice (3). The GDP-Man-negative mutant, GDPMP, was shown to be deficient in a range of cell surface and secreted mannose-containing molecules, including several abunda...

SUMMARY Apicomplexa are parasitic protozoa that cause important human diseases including malaria, cryptosporidiosis and toxoplasmosis. The replication of these parasites within their target host cell is dependent on both salvage as well as de novo synthesis of fatty acids. In T. gondii, fatty acid synthesis via the apicoplast-localized FASII is essential for pathogenesis, while the role of two other fatty acid biosynthetic complexes remains unclear. Here we demonstrate that the ER-localized fatty acid elongation (ELO) is essential for parasite growth. Conditional knock-down of the non-redundant hydroxyacyl-CoA dehydratase and enoyl-CoA reductase enzymes in the ELO pathway severely repressed intracellular parasite growth. 13C-glucose and 13C-acetate labeling and comprehensive lipidomic analyses of these mutants showed a selective defect in synthesis of unsaturated long and very long chain fatty acids (LCFAs and VLCFAs) and depletion of phosphatidylinositol and phosphatidylethanolamine species containing unsaturated LCFAs and VLCFAs. This requirement for ELO pathway was by-passed by supplementing the media with specific fatty acids, indicating active, but inefficient import of host fatty acids. Our experiments highlight a gap between the fatty acid needs of the parasite and availability of specific fatty acids in the host cell that the parasite has to close using a dedicated synthesis and modification pathway.

Pathogenic species of Mycobacteria and Corynebacteria, including Mycobacterium tuberculosis and Corynebacterium diphtheriae, synthesize complex cell walls that are rich in very long-chain mycolic acids. These fatty acids are synthesized on the inner leaflet of the cell membrane and are subsequently transported to the periplasmic space as trehalose monomycolates (TMM), where they are conjugated to other cell wall components and to TMM to form trehalose dimycolates (TDM). Mycobacterial TMM, and the equivalent Corynebacterium glutamicum trehalose corynomycolates (TMCM), are transported across the inner membrane by MmpL3, or NCgl0228 and NCgl2769, respectively, although little is known about how this process is regulated. Here, we show that transient acetylation of the mycolyl moiety of TMCM is required for periplasmic export. A bioinformatic search identified a gene in a cell wall biosynthesis locus encoding a putative acetyltransferase (M. tuberculosis Rv0228/C. glutamicum NCgl2759) that was highly conserved in all sequenced Corynebacterineae. Deletion of C. glutamicum NCgl2759 resulted in the accumulation of TMCM, with a concomitant reduction in surface transport of this glycolipid and syntheses of cell wall trehalose dicorynomycolates. Strikingly, loss of NCgl2759 was associated with a defect in the synthesis of a minor, and previously uncharacterized, glycolipid species. This lipid was identified as trehalose monoacetylcorynomycolate (AcTMCM) by mass spectrometry and chemical synthesis of the authentic standard. The in vitro synthesis of AcTMCM was dependent on acetyl-CoA, whereas in vivo [(14)C]-acetate pulse-chase labeling showed that this lipid was rapidly synthesized and turned over in wild-type and genetically complemented bacterial strains. Significantly, the biochemical and TMCM/TDCM transport phenotype observed in the ΔNCgl2759 mutant was phenocopied by inhibition of the activities of the two C. glutamicum MmpL3 homologues. Collectively, these data suggest that NCgl2759 is a novel TMCM mycolyl acetyltransferase (TmaT) that regulates transport of TMCM and is a potential drug target in pathogenic species.

The cell surface of the parasitic protozoan Leishmania mexicana is coated by glycosylphosphatidylinositol (GPI)-anchored glycoproteins, a GPI-anchored lipophosphoglycan and a class of free GPI glycolipids. To investigate whether the anchor or free GPIs are required for parasite growth we cloned the L.mexicana gene for dolichol-phosphate-mannose synthase (DPMS) and attempted to create DPMS knockout mutants by targeted gene deletion. DPMS catalyzes the formation of dolichol-phosphate mannose, the sugar donor for all mannose additions in the biosynthesis of both the anchor and free GPIs, except for a α1-3-linked mannose residue that is added exclusively to the free GPIs and lipophosphoglycan anchor precursors. The requirement for dolichol-phosphatemannose in other glycosylation pathways in L.mexicana is minimal. Deletion of both alleles of the DPMS gene (lmdpms) consistently resulted in amplification of the lmdpms chromosomal locus unless the promastigotes were first transfected with an episomal copy of lmdpms, indicating that lmdpms, and possibly GPI biosynthesis, is essential for parasite growth. As evidence presented in this and previous studies indicates that neither GPI-anchored glycoproteins nor lipophosphoglycan are required for growth of cultured parasites, it is possible that the abundant and functionally uncharacterized free GPIs are essential membrane components.

Glycosylphosphatidylinositol (GPI) glycolipids are major cell surface constituents in the Leishmania parasites. Distinct classes of GPI are present as membrane anchors for several surface glycoproteins and an abundant lipophosphoglycan as well as being the major glycolipids (GIPLs) in the plasma membrane. In this study we have identified putative precursors for the protein and lipophosphoglycan anchors and delineated the complete pathway for GIPL biosynthesis in Leishmania mexicana promastigotes. Based on the structural analyses of these GPI intermediates and their kinetics of labeling in vivo and in cell-free systems, we provide evidence that the GIPLs are the products of an independent biosynthetic pathway rather than being excess precursors of the anchor pathways. First, we show that the similar glycan head groups of the GIPL and protein/ lipophosphoglycan anchor precursors are assembled on two distinct pools of PI corresponding to 1-O-(C18:0)alkyl-2-stearoyl-PI and 1-O-(C24:0/C26:0)-2-stearoyl-PI, respectively. These PI species account for 20 and 1% of the total PI pool, respectively, indicating a remarkable specificity in their selection. Second, analysis of the flux of intermediates through these pathways in vivo and in a cell-free system suggests that the GIPL and anchor pathways are independently regulated. We also show that GIPL biosynthesis requires fatty acid remodeling, in which the sn-2 stearoyl chains are replaced with myristoyl or lauroyl chains. Fatty acid remodeling is dependent on CoA and ATP and occurs on pre-existing but not on de novo synthesized GIPLs. We suggest that the compartmentalization of different GPI pathways may be important in regulating the species and stage-specific expression of different GPI structures in these parasites.