Induction of a Stringent Metabolic Response in Intracellular Stages of Leishmania mexicana Leads to Increased Dependence on Mitochondrial Metabolism

Leishmania parasites alternate between extracellular promastigote stages in the insect vector and an obligate intracellular amastigote stage that proliferates within the phagolysosomal compartment of macrophages in the mammalian host. Most enzymes involved in Leishmania central carbon metabolism are constitutively expressed and stage-specific changes in energy metabolism remain poorly defined. Using C-13-stable isotope resolved metabolomics and (H2O)-H-2 labelling, we show that amastigote differentiation is associated with reduction in growth rate and induction of a distinct stringent metabolic state. This state is characterized by a global decrease in the uptake and utilization of glucose and amino acids, a reduced secretion of organic acids and increased fatty acid -oxidation. Isotopomer analysis showed that catabolism of hexose and fatty acids provide C4 dicarboxylic acids (succinate/malate) and acetyl-CoA for the synthesis of glutamate via a compartmentalized mitochondrial tricarboxylic acid (TCA) cycle. In vitro cultivated and intracellular amastigotes are acutely sensitive to inhibitors of mitochondrial aconitase and glutamine synthetase, indicating that these anabolic pathways are essential for intracellular growth and virulence. Lesion-derived amastigotes exhibit a similar metabolism to in vitro differentiated amastigotes, indicating that this stringent response is coupled to differentiation signals rather than exogenous nutrient levels. Induction of a stringent metabolic response may facilitate amastigote survival in a nutrient-poor intracellular niche and underlie the increased dependence of this stage on hexose and mitochondrial metabolism. Author SummaryLeishmania are sandfly-transmitted parasitic protozoa that cause a spectrum of important diseases in humans. While the core metabolism of the readily cultivated insect (promastigote) stage has been studied, much less is known about the metabolism of the obligate intracellular amastigote stage, which proliferates within the mature lysosome of mammalian macrophages and is the target of anti-parasite therapies. We have used C-13-tracing experiments to delineate the major pathways of carbon metabolism in different promastigote stages, as well as amastigote stages generated in culture and isolated from animal lesions. Both dividing and non-dividing promastigotes exhibited high metabolic activity, with excessive rates of glucose and amino acid consumption and secretion of metabolic end-products. In contrast, both amastigote stages exhibited a stringent metabolic phenotype, characterized by low levels of glucose and amino acid uptake and catabolism and increased catabolism of fatty acids. This phenotype was not induced by nutrient limitation, but is hard-wired into amastigote differentiation. This response may lead to increased dependence on hexose catabolism for anabolic pathways, as chemical inhibition of de novo glutamate and glutamine biosynthesis inhibited parasite growth in macrophages. This study highlights key aspects of amastigote metabolism that underpin their capacity to survive in macrophage phagolysosomes.