Role of hexosamine biosynthesis in Leishmania growth and virulence

Leishmania parasites incorporate N-acetylglucosamine (GIcNAc) into surface-expressed glycosylphosphatidyl inositol (GPI) glycolipids and N-linked glycans. To investigate whether these glyco-conjugates 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 Delta gfat mutant is unable to synthesize GIcN-6-phosphate de novo and is auxotrophic for GIcN or GIcNAc. GIcN 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 Delta gfat promastigotes are unable to proliferate in ex vivo infected macrophages, demonstrating that GIcN(Ac) levels in the phagolysosome are low. In contrast, Delta gfat amastigotes grow as well as wild-type amastigotes in macrophages and induce lesions in susceptible mice. These stages still require GIcN(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-GIcNAc biosynthesis are essential for pathogenesis in the mammalian host.