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 13C-stable isotope resolved metabolomics and 2H2O 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 β-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.

Leishmania parasites proliferate within nutritionally complex niches in their sandfly vector and mammalian hosts. However, the extent to which these parasites utilize different carbon sources remains poorly defined. In this study, we have followed the incorporation of various 13 C-labeled carbon sources into the intracellular and secreted metabolites of Leishmania mexicana promastigotes using gas chromatography-mass spectrometry and C]alanine uptake and catabolism. TCA cycle anaplerosis is apparently needed to sustain glutamate production under standard culture conditions. Specifically, inhibition of mitochondrial aconitase with sodium fluoroacetate resulted in the rapid depletion of intracellular glutamate pools and growth arrest. Addition of high concentrations of exogenous glutamate alleviated this growth arrest. These findings suggest that glycosomal and mitochondrial metabolism in Leishmania promastigotes is tightly coupled and that, in contrast to the situation in some other trypanosomatid parasites, the TCA cycle has crucial anabolic functions.Leishmania spp. are parasitic protozoa that cause a spectrum of disease in humans, ranging from self-limiting cutaneous infections to disseminating infections (mucocutaneous and visceral leishmaniasis) that can lead to severe morbidity and death (1). Approximately 12 million people are infected worldwide, resulting in more than 50,000 deaths each year (2). There are no vaccines against any of these diseases, and current drug therapies are both limited and, in many cases, are being undermined by widespread resistance (2). Although the genomes of several Leishmania species have now been sequenced and key aspects of metabolism intensively studied, major gaps exist in our understanding of central carbon metabolism in these divergent eukaryotes (3, 4). Given the importance of intermediary metabolism for parasite growth and protection against host microbicidal processes, detailed dissection of Leishmania carbon metabolism may reveal new therapeutic targets (4 -6).Leishmania develop as extracellular flagellated promastigote stages within the digestive tract of the sandfly vector. This stage is transmitted to the mammalian host when the female sandfly host takes a blood meal and is rapidly phagocytosed by neutrophils and macrophages (1). Promastigotes internalized into the phagolysosome compartment of macrophages differentiate into the obligate intracellular amastigote stage that perpetuates infection in the mammalian host. Promastigote stages are readily cultivated in vitro and have been the focus of most metabolic studies. Like the intensively studied insect (procyclic) stage of the related trypanosomatid, Trypanosoma brucei (7,8), Leishmania promastigotes are thought to catabolize glucose via glycolysis, with the initial enzymes in this pathway being largely or exclusively compartmentalized in modified peroxisomes, termed glycosomes (9, 10). The glycosomal catabolism of glucose to 1,3-bisphosphoglycerate requires an investment of both ATP and NAD ϩ , and the rate at whic...