Evidence That Intracellular Stages of Leishmania major Utilize Amino Sugars as a Major Carbon Source

Abstract

Intracellular parasites, such as Leishmania spp, must acquire suitable carbon sources from the host cell in order to replicate. Here we present evidence that intracellular amastigote stages of Leishmania exploit amino sugars in the phagolysosome of mammalian macrophages as a source of carbon and energy. L. major parasites are capable of using N-acetylglucosamine and glucosamine as primarily carbon sources and contain key enzymes required for conversion of these sugars to fructose-6-phosphate. The last step in this pathway is catalyzed by glucosamine-6-phosphate deaminase (GND), which was targeted to glycosomes via a canonical C-terminal targeting signal when expressed as a GFP fusion protein. Mutant parasites lacking GND were unable to grow in medium containing amino sugars as sole carbohydrate source and rapidly lost viability, concomitant with the hyper-accumulation of hexosamine-phosphates. Expression of native GND, but not a cytosolic form of GND, in Δgnd parasites restored hexosamine-dependent growth, indicating that toxicity is due to depletion of glycosomal pools of ATP. Non-lethal increases in hexosamine phosphate levels in both Δgnd and wild type parasites was associated with a defect in promastigote metacyclogenesis, suggesting that hexosamine phosphate levels may influence parasite differentiation. Promastigote and amastigote stages of the Δgnd mutant were unable to replicate within macrophages and were either completely cleared or exhibited reduced lesion development in highly susceptible Balb/c mice. Our results suggest that hexosamines are a major class of sugars in the macrophage phagolysosome and that catabolism of scavenged amino sugars is required to sustain essential metabolic pathways and prevent hexosamine toxicity.

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

“…A dependence on carbohydrate metabolism is supported by the presence of multiple sugar kinases in the Leishmania genomes (3) and may reflect the abundance of sugars in the sandfly mid-gut, particularly at later stages of infection when infected female sandflies feed on plants and sugar-rich honeydews (56). Intriguingly, hexose uptake and catabolism has also been shown to be essential for the survival and proliferation of L. mexicana and L. major amastigotes in the mammalian host (6,5) despite the fact that the macrophage phagolysosome appears to be a sugar-poor niche (39). Although the macrophage phagolysosome is expected to have relatively high levels of amino acids, amastigotes may still be limited in their capacity to salvage key amino acids such as glutamate, glutamine, and proline.…”

“…GFP-expressing parasites were immobilized onto poly-L-lysine-coated coverslips and visualized by fluorescence microscopy (Zeiss Axioplan 2 microscope). Images were compiled in Photoshop (6).…”

N-Acetylglucosamine (GlcNAc) Induction of Hyphal Morphogenesis and Transcriptional Responses in Candida albicans Are Not Dependent on Its Metabolism

“…2). Another example of this type of inhibition was reported for Leishmania, in which the GlcNAc sensitivity of a gnd mutant lacking glucosamine deaminase was attributed to depletion of ATP (35). The inhibitory effect of GlcNAc on the Leishmania gnd mutant appears to be distinct from that seen for the C. albicans mutants because it could be overcome by adding alternative carbon sources, such as glycerol.…”

Role of Cytosolic Glyceraldehyde-3-Phosphate Dehydrogenase in Visceral Organ Infection by Leishmania donovani

“…Since GAPDH is also able to catalyze the reverse reaction from 3-phospho-D-glyceroyl phosphate into GAP in gluconeogenesis. The observation in this study that the L. donovani cGAPDH-null mutant had reduced infectivity in visceral organs suggests that cGAPDH could be involved in glycolysis and/or gluconeogenesis pathways when L. donovani resides as an amastigote in the macrophage phagolysosome, where the sugar levels are relatively low (47,51).…”

“…However, several studies have shown that glucose and/or amino sugars are required for the amastigote stage, though they might be mainly needed for the formation of glycoconjugates (46)(47)(48)(49). Leishmania amastigotes are able to utilize amino sugars in the phagolysosome of mammalian macrophages as a source of carbon and energy (47). Interestingly, gene deletion study of the gluconeogenic enzyme fructose-1,6-bisphosphatase (FBP) showed Leishmania also requires gluconeogenesis for its virulence (50).…”