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 intracellular 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, beta-oxidation, amino acid catabolism, tricarboxylic acid cycle, mitochondrial respiration chain, and oxidative phosphorylation capacities are all up-regulated. 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.

Protozoans of the genus Leishmania are obligate intracellular parasites that cycle between the midgut of sandflies and the phagolysosomes of mammalian macrophages and therefore are exposed to extreme environmental changes. Recent evidence obtained from in vitro experiments indicate that such environmental changes trigger a developmental program in the parasites. Thus, following heat shock, promastigotes from certain Leishmania species differentiate to amastigotes. Promastigotes also respond to acidification of their environment by changing the expression of a number of genes. However, the combination of both low pH and high temperature induces the transformation of the promastigote to the amastigote in all Leishmania species examined to date. This review discusses the role of pH and heat shock in gene regulation and its contribution to the differentiation processes in Leishmania spp. Cycling between cold-blooded insect vectors and the warm-blooded mammalian host is not unique to Leishmania spp., but typical to most parasitic protozoa. It is therefore likely that the mechanism of stress-induced differentiation is shared by other mammalian parasites.

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Respiring intact cells maintained an internal pH more alkaline by 0.63 -0.75 unit than that of the milieu at extracellular pH 7, both in growth medium and KCl solutions. The ApH decreased when respiration was inhibited by anaerobiosis or in the presence of KCN.The AGH, established by EDTA/valinomycin-treated cells, was constant (122 -129 mV) over extracellular potassium concentration of 0.01 mM -1 mM. At the lower potassium concentration A $ (110-120mV) was the predominant component, and at the higher concentration d p H increased to 0.7 units (42 mV). At 150 mM potassium A,EH was reduced to 70 mV mostly due to a d pH component of 0.89 (53 mV). The interchangeability of the A fiH components is consistent with an electrogenic proton pump and with potassium serving as a counter ion in the presence of valinomycin. Indeed both parameters of A ,EH decreased in the presence of carbonylcyanide p-trifluoromethoxyphenylhydrazone.The highest ApH of 2 units was observed in the intact cells at pH 6; increasing the extracellular pH decreased the ApH to 0 at pH 7.65 and to -0.51 at pH 9. A similar pattern of dependence of A pH on extracellular pH was observed in EDTA/valinomycin-treated cells but the d $ was almost constant over the whole range of extracellular pH values (6 -8) implying electroneutral proton movement.Potassium is specifically required for respiration of EDTA-treated E. coli K12 cells since other monovalent or divalent cations could not replace potassium and valinomycin was not required.It is established that an electrochemical proton gradient (dpH) is built up through energization across energy-conserving membranes of eucaryotic organelles -chloroplasts and mitochondria (reviews in [l] to [4]). The methods employed for determining AfiH in microscopic systems, recently critically reviewed [4], include determinations of the pH gradient (dpH) and the potential gradient (All/) across the membrane, since both parameters contribute to ApH according to the relation : Mitchell [5] suggested that this proton gradient is of primary importance in the mechanism of biological energy conversion.In the procaryotic cell, the cytoplasmic membrane is the site of energy coupling. Upon energization, outward translocation of protons from whole bacterial cells has been demonstrated showed that upon glycolysis intact cells of Strepto-COCCUS faecalis maintain a more alkaline internal pH (0.5-1 unit higher than the medium) and a potential of 150-200 mV across the membrane with the interior negative [lo]. Since both these parameters of dDH were not determined simultaneously, the magnitude of dpH and the relationship between its components can only be indirectly estimated for these cells. A membrane potential of about 140 mV was recently estimated in respiring E. coli cells [Ill but the ApH was not determined. While in chromatophore fractions obtained from photosynthetic bacteria A$ and dpH contribute equally to the ApH [12,13], in membraneous vesicles obtained from E. coli only A$ was detected [14,15].Recent studies showed that an a...

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-amastigote 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.

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The standard treatment of human visceral leishmaniasis involves the use of pentavalent antimony (Sb V ). Its mechanism of action is unknown because of the limited information available about intracellular antimony metabolism and about the genes that regulate these processes. Herein, flow injection-inductively coupled plasma mass spectrometry (ICP-MS), flow injection hydride generation ICP-MS, and ion chromatography ICP-MS were used to measure antimony accumulation and intracellular metabolism in the human protozoan parasite Leishmania donovani. V reduction has yet to be identified, and it may or may not be enzymatic. This is the first description of intracellular Sb V reducing activity in Leishmania as well as in any prokaryotic or eukaryotic cell.Leishmania donovani is the major causative agent of visceral leishmaniasis. In nature, the parasite exists either as an extracellular promastigote found in the alimentary tract of sandflies or as an obligatory intracellular amastigote found in phagolysosomes of mammalian macrophages(1-3). During the last several years, a number of laboratories have utilized axenic culture of L. donovani amastigotes for the direct evaluation of cell biological and biochemical processes in the amastigote, devoid of the host macrophage (4 -7,8,9,10). This technique has also been used to investigate the mechanism of drug action and resistance as well as for screening of potential new drugs(11-13). The treatment of choice of human visceral leishmaniasis is the administration of pentavalent antimony (Sb V )-containing drugs such as sodium stibogluconate (Pentostam, Wellcome, Beckenham, UK) or meglumine antimoniate (Glucantime, Rhone-Poulenc, Paris, France). Despite extensive use of these compounds over the last decades, the mechanism of action remains unclear.It has been hypothesized that Sb V is not toxic to Leishmania, but rather that it is enzymatically reduced, probably by the host macrophage, to Sb III , the form of antimony that is highly Leishmania-toxic (14 -18). In contrast, it has been shown that Sb V is directly toxic to axenic amastigotes (12,13,19), thus negating the necessity of the macrophage for Sb V reduction. Furthermore, these data imply that either the parasite reduces Sb V to Sb III intracellularly, and thus Sb III is directly toxic to amastigotes, or that both oxidation states of antimony are active against Leishmania amastigotes. The modes of action of the two oxidation states of antimony (Sb III and Sb V ) on Leishmania are, as yet, not fully understood.Several groups have shown obligatory cross-resistance between Sb V , Sb III , and arsenite (As III ) in Leishmania tarentolae, Leishmania major, Leishmania mexicana, L. donovani and Leishmania infantum (11,[20][21][22]. In contrast, it has been suggested that, at least in L. donovani, such cross-resistance does not necessarily occur (12). Furthermore, it has been demonstrated that, when bound to organic compounds, structural similarities exist between Sb III and Sb V (23). For example, the trivalent antimony comp...

Leishmania donovani is an intracellular protozoan parasite that causes kala-azar in humans. During infection the extracellular insect forms (promastigotes) undergo rapid differentiation to intracellular amastigotes that proliferates in phagolysosomes of mammalian macrophages. We used microarraybased expression profiling to investigate the time-course of changes in RNA abundance during promastigote-to-amastigote differentiation in a host-free system that mimics this process. These studies revealed that several hundred genes underwent an ordered progression of transient or permanent up-and down-regulation during differentiation. Genes that were permanently upregulated in amastigotes were enriched for transporters and surface proteins, but under-represented in genes involved in protein and other metabolism. Most of these changes occurred late in the differentiation process, when morphological differentiation was essentially complete. Downregulated genes were over-represented in those involved in cell motility, growth and/or maintenance, and these changes generally occurred earlier in the process. Genes that were transiently up-or downregulated during differentiation included those encoding heat shock proteins, ubiquitin hydrolases, RNA binding proteins, protein kinases, a protein phosphatase, and a histone deacetylase. These results suggest that changes in mRNA abundance may be important in signal transduction, as well as protein and mRNA turnover, during differentiation. In addition to these mRNA changes, other transcripts including one or more rRNAs and snoRNAs, and non-coding RNAs from several telomeres, also showed substantial changes in abundance during the differentiation process. This paper provides the first genome-scale quantitative analysis of gene expression during the transition from promastigotes to amastigotes and demonstrates the utility of the host-free differentiation system.