Dan Zilberstein

Retooling Leishmania metabolism: from sand fly gut to human macrophage

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)

CHARACTERIZATION OF DEVELOPMENTALLY-REGULATED ACTIVITIES IN AXENIC AMASTIGOTES OF LEISHMANIA DONOVANI

Leishmania donovani is an obligatory intracellular parasite which cycles between the midgut of sand flies (extracellular promastigote) and the phagolysosomes of mammalian macrophages (intracellular amastigote). Promastigotes have been readily cultured, whereas axenic cultures of amastigotes have only recently been developed. A new method for in vitro differentiation of L. donovani promastigotes into amastigotes is presented, in which promastigotes are exposed to environmental changes that mimic the in vivo process. First, promastigotes are subjected to 37 degrees C + 5% CO2 for 24 h, and then are shifted to pH 5.5. Under these conditions, differentiation is completed within 120 h. In the reverse process, amastigotes are induced to differentiate back to promastigotes by transferring them to promastigote growth conditions (medium 199 at pH 7.4 and 26 degrees C). Axenic amastigotes closely resemble animal-derived amastigotes. They manifest all seven proteins of the amastigote-specific A2 gene family. They down-regulate lipophosphoglycan (LPG) synthesis and do not express it on their surface. LPG is up-regulated 2 h after inducing amastigotes to differentiate to promastigotes. Within 6 h, parasites resume the promastigote level of this molecule, although differentiation is completed only after 48 h. Axenic amastigotes also express amastigote-like metabolic activities of proline uptake, as well as thymidine and proline incorporation. In conclusion, the results indicate that the method developed for in vitro differentiation of L. donovani promastigotes to amastigotes is efficient and yields organisms resembling animal-derived amastigotes. Being able to induce in vitro differentiation of L. donovani provides us with an excellent tool to study Leishmania development and differentiation.

Visceral leishmaniasis: elimination with existing interventions

The worlds burden of infectious diseases can be substantially reduced by more-effective use of existing interventions. Advances in case detection, diagnosis, and treatment strategies have made it possible to consider the elimination of visceral leishmaniasis in the Indian subcontinent. The priority must now be to effectively implement existing interventions at the community level by actively finding cases in endemic villages and treating them with single-dose liposomal amphotericin B at primary-health-care centres. Once the elimination target of one case per 10,000 population has been reached, combination therapies involving miltefosine and paromomycin can be introduced to ensure long-term availability of several drugs for visceral leishmaniasis and to protect against resistance.

Multiple levels of gene regulation mediate differentiation of the intracellular pathogen Leishmania

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

Post-translational modification of cellular proteins during Leishmania donovani differentiation.

The pathogenic intracellular parasites Leishmania donovani cycle between sand fly gut and the human macrophage phagolysosome, differentiating from extracellular promastigotes to intracellular amastigote forms. Using isobaric tagging for relative and absolute quantifications (iTRAQ/LC‐MS/MS) proteomic methodology, we recently described the ordered gene expression changes during this process. While protein abundance changes in Leishmania were documented, little is known about their PTMs. Here we used iTRAQ to detect protein phosphorylation, methylation, acetylation, and glycosylation sites throughout differentiation. We found methylation of arginines, aspartic acids, glutamic acids, asparagines, and histidines. Detected acetylation sites included serines and protein N‐terminal acetylations on methionines, serines, alanines, and threonines. Phosphorylations were detected on serines and threonines, but not tyrosines. iTRAQ identified novel fucosylation sites as well as hexosylations. We observed quantity changes in some modifications during differentiation, suggesting a role in L. donovani intracellular development. This study is the first high‐throughput analysis of PTM sites dynamics during an intracellular parasitic development.

A novel high‐affinity arginine transporter from the human parasitic protozoan Leishmania donovani

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 (Km 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.

Transport of Nutrients and Ions across Membranes of Trypanosomatid Parasites

Publisher Summary This chapter discusses the membrane transport mechanisms in Leishmania and Trypanosoma. It focuses on the transport of four groups of substrates: amino acids, sugars, protons, and calcium and discusses the structure and function of the membrane transporters of these substances . Leishmania and Trypanosoma are protozoan parasites that live inside mammalian hosts, either intracellularly or extracellularly. The occurrence of a controlled transport of nutrients and ions into and from parasite cells is important for the maintenance of intracellular homeostasis. The level of expression and function of such membrane transporters is therefore critical for parasite survival inside their hosts. A regulation of intracellular pH and proton motive force is discussed in Leishmania and (2) T. brucei . Catabolism of D-glucose in Trypanosoma and Leishmania occurs in closed organelles called glycosomes. These organelles contain most of the glycolytic enzymes, including hexokinase, the enzyme responsible for the first step in glycolysis. The glycosomal membrane serves as a permeability barrier to most glycolytic intermediates. Substrate recognition by D-glucose transport systems of T. brucei trypanosomes and Leishmania are summarized diagrammatically.

Arginine Homeostasis and Transport in the Human Pathogen Leishmania donovani

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.

Growth at acidic pH induces an amastigote stage-specific protein in Leishmania promastigotes

This paper constitutes the first investigation into the effect of pH on phenotypic expression in Leishmania. The advantage of our experimental system is that it makes use of the extracellular (promastigote) form, which can be readily grown in culture medium, as a model to study parasite development in vivo

Regulation of l-proline transport in Leishmania donovani by extracellular pH

We have previously shown that Leishmania donovani promastigotes adapted to long-term culture at acidic pH can serve as a model to study parasite development in a lysosomal-like environment. In this study we investigated the effect of growth pH on L. donovani L-proline transport systems. Reducing the pH of the growth medium causes an up to 7-fold decrease in the extent of L-proline transport. Transport resumes after switching the culture from pH 4.5 to pH 7 for 48 h by a protein synthesis-dependent process. The pH optimum for transport changes from 7.5 in promastigotes grown at pH 7 to 5.5 in cells grown at pH 4.5. In addition, kinetic analysis of L-proline transport showed that Vmax in pH 4.5-grown L. donovani promastigotes is one-tenth that of cells grown at pH 7 (4.5 and 44.7 nmol min-1 (10(8) cells)-1, respectively). The apparent Km for L-proline in pH 4.5 promastigotes is one-half of the Km in pH 7 cells (0.30 and 0.65 mM, respectively). In contrast to L-proline transport, D-glucose transport demonstrates a growth pH-independent activity: Km and Vmax as well as optimum pH of transport are similar in promastigotes grown at either pH 7 or pH 4.5. Taken together, the results indicate that in L. donovani, expression and activity of L-proline transport is regulated by culture pH. The pH-dependent expression of L-proline transporters may be of physiological significance during the promastigote-amastigote transition.