Role of hexosamine biosynthesis in Leishmania growth and virulence

Abstract

SummaryLeishmania parasites incorporate N-acetylglucosamine (GlcNAc) into surface-expressed glycosylphosphatidylinositol (GPI) glycolipids and N-linked glycans. To investigate whether these glycoconjugates are required for infectivity of promastigote and intracellular amastigote stages, we generated a Leishmania major mutant lacking the gene encoding glutamine : fructose-6-phosphate amidotransferase (GFAT). The L. major Dgfat mutant is unable to synthesize GlcN-6-phosphate de novo and is auxotrophic for GlcN or GlcNAc. GlcN starvation leads to the rapid depletion of dolichol-linked oligosaccharides and GPI precursors, hypersensitivity to elevated temperatures encountered in the mammalian host and eventual parasite death. Short-term tunicamycin treatment induces a similar hypersensitivity to temperature, indicating that N-linked glycans are required for thermotolerance and viability. L. major Dgfat promastigotes are unable to proliferate in ex vivo infected macrophages, demonstrating that GlcN(Ac) levels in the phagolysosome are low. In contrast, Dgfat amastigotes grow as well as wild-type amastigotes in macrophages and induce lesions in susceptible mice. These stages still require GlcN(Ac) for viability but can apparently scavenge all of their glucosamine requirements from the macrophage phagolysosome. These results highlight significant differences in the nutrient requirements of promastigote and amastigote stages and suggest that enzymes involved in UDP-GlcNAc biosynthesis are essential for pathogenesis in the mammalian host.

A Toxoplasma gondii Gluconeogenic Enzyme Contributes to Robust Central Carbon Metabolism and Is Essential for Replication and Virulence

“…The activity of ER glycosylation pathways can also be assessed by measuring the steady-state concentrations of dolichol-linked oligosaccharide and GPI glycolipid intermediates in crude microsomal preparations (Naderer et al, 2008). Microsomes were prepared from iDtgfbp2 parasites, with or without prior ATC treatment, and labeled with GDP-3 H-Man in order to tag pre-existing ER acceptors, such as dolichol-phosphate, dolichol-PP-GlcNAc 2 , and GlcN-PI (Naderer et al, 2008).…”

“…Microsomes were prepared from iDtgfbp2 parasites, with or without prior ATC treatment, and labeled with GDP-3 H-Man in order to tag pre-existing ER acceptors, such as dolichol-phosphate, dolichol-PP-GlcNAc 2 , and GlcN-PI (Naderer et al, 2008). Synthesis of dolichol phosphomannose and several GPI intermediates was markedly repressed in microsomes from ATC-treated iDtgfbp2 parasites ( Figure 5G, Figure S4C), suggesting a defect in apicoplast-dependent polyprenol synthesis in addition to, or separate from, downregulation of glycosylation pathways.…”

“…GDP-[2-3 H]-mannose labeling was done as described previously (Naderer et al, 2008). In brief, syringe-released and filter-purified tachyzoites were hyptonically permeabilized (1 mM HEPES [pH 7.4], 2 mM EGTA, 2 mM DTT, EDTA-free protease inhibitor cocktail [Roche]) and the cell-free lysates were incubated at 30 C with 3.5 Ci/mmol GDP-[2-3 H]-mannose for 35 min.…”

Isotopomer Profiling of Leishmania mexicana Promastigotes Reveals Important Roles for Succinate Fermentation and Aspartate Uptake in Tricarboxylic Acid Cycle (TCA) Anaplerosis, Glutamate Synthesis, and Growth

“…Glutamate is the precursor for other amino acids, such as proline and glutamine, that are both internalized at low rates under standard culture conditions. Glutamine is also required for pyrimidine and hexosamine biosynthesis (44,52), as well as the synthesis of the major thiols of these parasites, glutathione and trypanothione (53,54). Trypanothione participates in many anabolic and redox processes and can be conjugated to other metabolites necessitating continuous synthesis (53,54).…”

“…Although previous studies have shown that mannogen is rapidly mobilized under glucose starvation conditions (34), our data suggest that mannogen is also constitutively synthesized and catabolized under conditions of glucose excess. Mannogen turnover could provide a mechanism for regulating cytosolic hexose phosphate levels and fluxes through other essential metabolic pathways, such as the pentose phosphate pathway, amino sugar biosynthesis, and N-glycosylation (43,44).…”