Abstract
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.
Highlights
Kinetoplastid parasites, belonging to the genus Leishmania, are responsible for a spectrum of diseases that affect over 12 million people worldwide, ranging from self-limiting cutaneous infections to disseminating mucocutaneous and lethal visceral infections [1,2](http://www.who.int/leishmaniasis/en/)
While the core metabolism of the readily cultivated insect 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 antiparasite therapies
We have used 13C-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
Summary
Kinetoplastid parasites, belonging to the genus Leishmania, are responsible for a spectrum of diseases that affect over 12 million people worldwide, ranging from self-limiting cutaneous infections to disseminating mucocutaneous and lethal visceral infections [1,2](http://www.who.int/leishmaniasis/en/). Following an initial proliferative phase, promastigotes differentiate to nondividing metacyclic stages that are the primary stage injected into the skin during a sand fly bite. Metacyclic promastigotes are phagocytosed by macrophages, either directly or after initial uptake by neutrophils, and are delivered to the mature phagolysosome compartment where they differentiate to non-motile amastigote stages [2]. Amastigotes are responsible for both acute as well as longterm latent infections that can lead to reactivation of disease years or decades after the primary infection, in immunocompromised individuals [2]. How these stages survive within the macrophage’s mature phagolysosome remains poorly defined [3]
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