To survive extremely different environments, intracellular parasites require highly adaptable physiological and metabolic systems. Leishmania donovani extracellular promastigotes reside in a glucose‐rich, slightly alkaline environment in the sand fly vector alimentary tract. On entry into human macrophage phagolysosomes, promastigotes differentiate into intra‐cellular amastigotes. These cope with an acidic milieu, where glucose is scarce while amino acids are abundant. Here, we use an axenic differentiation model and a novel high‐coverage, comparative proteomic methodology to analyze in detail protein expression changes throughout the differentiation process. The analysis identified and quantified 21% of the parasite proteome across 7 time points during differentiation. The data reveal a delayed increase in gluconeogenesis enzymes, coinciding with a decrease in glycolytic capacity. At the same time, β‐oxidation, amino acid catabolism, tricarboxylic acid cycle, mitochondrial respiration chain, and oxidative phosphorylation capacities are all up‐regu‐lated. The results indicate that the differentiating parasite shifts from glucose to fatty acids and amino acids as its main energy source. Furthermore, glycerol and amino acids are used as precursors for sugar synthesis, compensating for lack of exogenous sugars. These changes occur while promastigotes undergo morphological transformation. Our findings provide new insight into changes occurring in single‐cell organisms during a developmental process.—Rosenzweig D., Smith, D., Opperdoes, F., Stern, S., Olafson, R. W., Zilberstein D. Retooling Leishmania metabolism: from sand fly gut to human macrophage. FASEB J. 22, 590–602 (2008)

For many years, mRNA abundance has been used as the surrogate measure of gene expression in biological systems. However, recent genome‐scale analyses in both bacteria and eukaryotes have revealed that mRNA levels correlate with steady‐state protein abundance for only 50–70% of genes, indicating that translation and post‐translation processes also play important roles in determining gene expression. What is not yet clear is whether dynamic processes such as cell cycle progression, differentiation, or response to environmental changes change the relationship between mRNA and protein abundance. Here, we describe a systems approach to interrogate promastigote‐to‐amas‐tigote differentiation in the obligatory intracellular parasitic protozoan Leishmania donovani. Our results indicate that regulation of mRNA levels plays a major role early in the differentiation process, while translation and post‐translational regulation are more important in the latter part. In addition, it appears that the differentiation signal causes a transient global increase in the rate of protein synthesis, which is subsequently down‐regulated by phosphorylation of α‐subunit of translation initiation factor 2. Thus, Leishmania dynamically changes the relationship between mRNA and protein abundance as it adapts to new environmental circumstances. It is likely that similar mechanisms play a more important role than previously recognized in regulation of gene expression in other organisms.—Lahav, T., Sivam, D., Volpin, H., Ronen, M., Tsigankov, P., Green, A., Holland, N., Kuzyk, M., Borchers, C., Zilberstein, D., Myler, P. J. Multiple levels of gene regulation mediate differentiation of the intracellular pathogen Leishmania. FASEB J. 25, 515–525 (2011). www.fasebj.org

Summary: We describe the first functional and molecular characterization of an amino acid permease (LdAAP3) from the human parasitic protozoan Leishmania donovani, the causative agent of visceral leishmaniasis in humans. This permease contains 480 amino acids with 11 predicted trans‐membrane domains. Expressing LdAAP3 in Saccharomyces cerevisiae mutants revealed that LdAAP3 codes for a high‐affinity arginine transporter (K_m 1.9 µM). LdAAP3 is highly specific for arginine as its transport was not inhibited by other amino acids or arginine‐related compounds. Using green fluorescence protein (GFP) fused to the N‐terminus of LdAAP3, this transporter was localized to the surface membrane of promastigotes. The GFP–LdAAP3 chimera mediated a threefold increase in arginine transport in promastigotes, indicating that it is active and confirmed that LdAAP3 codes for an arginine transporter in parasite cells as well. LdAAP3 is novel as it shares a high level of homology with amino acid permeases from other trypanosomatidae but almost none with permeases from other phyla. The results of this work suggest that LdAAP3 might play a role in host–parasite interaction.

Amino acid sensing is an intracellular function that supports nutrient homeostasis, largely through controlled release of amino acids from lysosomal pools. The intracellular pathogen Leishmania resides and proliferates within human macrophage phagolysosomes. Here we describe a new pathway in Leishmania that specifically senses the extracellular levels of arginine, an amino acid that is essential for the parasite. During infection, the macrophage arginine pool is depleted due to its use to produce metabolites (NO and polyamines) that constitute part of the host defense response and its suppression, respectively. We found that parasites respond to this shortage of arginine by up-regulating expression and activity of the Leishmania arginine transporter (LdAAP3), as well as several other transporters. Our analysis indicates the parasite monitors arginine levels in the environment rather than the intracellular pools. Phosphoproteomics and genetic analysis indicates that the arginine-deprivation response is mediated through a mitogen-activated protein kinase-2-dependent signaling cascade. Protozoa of the genus Leishmania are the causative agents of leishmaniasis in humans. These parasites cycle between promastigotes in the sand fly mid-gut and amastigotes in phagolysosome of mammalian macrophages. During infection, host cells up-regulate nitric oxide while/or parasites induce expression of host arginase, both of which use arginine as a substrate. These elevated activities deplete macrophage arginine pools, a situation that invading Leishmania must overcome since it is an essential amino acid. Leishmania donovani imports exogenous arginine via a mono-specific amino acid transporter (AAP3) and utilizes it primarily through the polyamine pathway to provide precursors for trypanothione biosynthesis as well as hypusination of eukaryotic translation Initiation Factor 5A. Here we report the discovery of a pathway whereby Leishmania sense the lack of environmental arginine and respond with rapid up-regulation in the expression and activity of AAP3, as well as several other transporters. Significantly, this arginine deprivation response is also activated in parasites during macrophage infection. Phosphoproteomic analyses of L. donovani promastigotes have implicated a mitogen-activated protein kinase 2 (MPK2)-mediated signaling cascade in this response, and L. mexicana mutants lacking MPK2 are unable to respond to arginine deprivation. The arginine-sensing pathway might play an important role in Leishmania virulence and hence serve as target for drug development.

Arginine is an essential amino acid for the human pathogen Leishmania but not to its host. Thus, the mechanism by which this protozoan parasite regulates cellular homeostasis of arginine is critical for its survival and virulence. In a previous study, we cloned and functionally characterized a high affinity arginine-specific transporter, LdAAP3, from Leishmania donovani. In this investigation, we have characterized the relationship between arginine transport via LdAAP3 and amino acid availability. Starving promastigotes for amino acids decreased the cellular level of most amino acids including arginine but also increased the abundance of both LdAAP3 mRNA and protein and up-regulated arginine transport activity. Genetic obliteration of the polyamine biosynthesis pathway for which arginine is the sole precursor caused a significant decrease in the rate of arginine transport. Cumulatively, we established that LdAAP3 expression and activity changed whenever the cellular level of arginine changed. Our findings have led to the hypothesis that L. donovani promastigotes have a signaling pathway that senses cellular concentrations of arginine and subsequently activates a mechanism that regulates LdAAP3 expression and activity. Interestingly, this response of LdAAP3 to amino acid availability in L. donovani is identical to that of the mammalian cation amino acid transporter 1. Thus, we conjecture that Leishmania mimics the host response to amino acid availability to improve virulence.