Purine metabolism in Leishmania donovani amastigotes and promastigotes

Purine metabolism in Leishmania donovani amastigotes was found to be similar to that of promastigotes with the exception of adenosine metabolism. Adenosine kinase activity in amastigotes is approximately 50-fold greater than in promastigotes. Amastigotes deaminate adenosine to inosine through adenosine deaminase, an enzyme not present in promastigotes. Inosine is cleaved to hypoxanthine and phosphoribosylated by hypoxanthine-guanine phosphoribosyltransferase. Promastigotes cleave adenosine to adenine and deaminate adenine to hypoxanthine via adenase, an enzyme not present in amastigotes. Hypoxanthine is phosphoribosylated by hypoxanthine-guanine phosphoribosyltransferase.     

Stage-specific variations in lectin binding to Leishmania donovani.

Visceral leishmaniasis is caused by the dimorphic protozoan Leishmania donovani, which exists as an aflagellar amastigote within mammalian mononuclear phagocytes and as a flagellated extracellular promastigote in its sandfly vector. We have identified four plant lectins that bind to the L. donovani surface, and through these we have documented stage-specific differences in exposed surface carbohydrates. Concanavalin A bound to both promastigotes and amastigotes; binding was inhibited by mannose or alpha-methyl-mannoside, implying a mannose-containing residue on the surface of both parasite stages. Ricinus communis agglutinin, which binds to galactose-containing residues, also bound to both stages and was inhibited by lactose, implying a galactose-containing glycoconjugate on the parasite surface. Two other lectins, wheat germ agglutinin (WGA) and peanut agglutinin (PNA), exhibited stage specificity in their binding characteristics. Amastigotes bound WGA but not PNA. During the process of conversion from the amastigote to the promastigote stage, the WGA-binding glycoconjugate was lost, and a PNA-binding residue was newly displayed. WGA binding was inhibited by N-acetyl-D-glucosamine and was not altered by neuraminidase treatment, suggesting the presence of an exposed N-acetyl-D-glucosamine moiety on the amastigote surface. The PNA binding site is known to accommodate the oligosaccharide beta-D-galactose-(1----3)-N-acetyl-D-galactosamine; in our system, PNA may have identified an internal rather than a terminal galactose on the promastigote surface. Localized binding of WGA and PNA to the surface of intermediate phases of the parasite suggested inhomogeneous and changing surface characteristics during conversion from amastigote to promastigote stages. This evolution of L. donovani surface glycoconjugates may be important in the adaptation of the organism to its divergent mammalian host and arthropod vector environments.     

Elimination of Leishmania donovani amastigotes by activated macrophages.

Tissue macrophages are the obligatory host cells for Leishmania donovani, the causative agent of visceral leishmaniasis. In this study we sought to determine whether activated macrophages, as defined by the functional criterion of tumor cell cytotoxicity, were also able to exert a microbicidal effect on ingested L. donovani amastigotes. We found that mouse peritoneal macrophages activated by a variety of means exerted a cytotoxic effect on tumor cell targets but were not able to kill L. donovani amastigotes unless the infected macrophages were exposed continually to an activating stimulus. Corynebacterium parvum, Mycobacterium tuberculosis H37Ra, and lymphokine-activated peritoneal macrophages from C57BL/6J mice were cytotoxic for EMT6 tumor cell targets. However, L. donovani Sudan strain 1S amastigotes were not killed by these macrophages unless the activated state was maintained by daily addition of lymphokine to the infected monolayers for several days postinfection. The killing of amastigotes was dependent on the time of exposure to lymphokine, as well as on the concentration of lymphokine added to the culture.     

Characterization of a mutant Leishmania donovani deficient in adenosine kinase activity

From a mutagenized population of wildtype Leishmania donovani promastigotes, a clonal cell line, TUBA2, was isolated by virtue of its ability to survive and grow in 20 microM tubercidin (7-deazaadenosine). The TUBA2 clone was also 1000-fold less sensitive than the parental line to growth inhibition by formycin A, another cytotoxic adenosine analog. Parental and mutant cells, however, were equally sensitive to growth inhibition by formycin B, allopurinol riboside, and 6-thioguanosine. Mutant cell extracts, unlike those prepared from wildtype cells, did not phosphorylate radiolabelled adenosine, tubercidin, or formycin A. Intact adenosine kinase-deficient cells did not accumulate exogenous tubercidin or formycin A but incorporated [14C]adenosine at rates 25% of those found for parental cells. The uptake data suggest that adenosine kinase plays an important role in the metabolism of adenosine but indicate alternative metabolic pathways for this nucleoside. The metabolism of adenosine to the nucleotide level in TUBA2 cells appears to be initiated via deribosylation to adenine. Significant amounts of both adenosine hydrolytic and adenosine phosphorylytic activities have been detected in L. donovani promastigotes. Furthermore, L. donovani extracts could slowly catalyze the deamination of formycin A. The isolation and characterization of adenosine kinase-deficient cells has provided considerable insight into the function of the purine pathway in L. donovani.     

Generation of adenosine tri-phosphate in Leishmania donovani amastigote forms

Leishmania, the causative agent of various forms of leishmaniasis, is the significant cause of morbidity and mortality. Regarding energy metabolism, which is an essential factor for the survival, parasites adapt to the environment under low oxygen tension in the host using metabolic systems which are very different from that of the host mammals. We carried out the study of susceptibilities to different inhibitors of mitochondrial electron transport chain and studies on substrate level phosphorylation in wild-type L. donovani. The amastigote forms of L. donovani are independent on oxidative phosphorylation for ATP production. Indeed, its cell growth was not inhibited by excess oligomycin and dicyclohexylcarbodiimide, which are the most specific inhibitors of the mitochondrial Fo/F1-ATP synthase. In contrast, mitochondrial complex I inhibitor rotenone and complex III inhibitor antimycin A inhibited amastigote cell growth, suggesting the role of complex I and complex III in cell survival. Complex II appeared to have no role in cell survival. To further investigate the site of ATP production, we studied the substrate level phosphorylation, which was involved in the synthesis of ATP. Succinate-pyruvate couple showed the highest substrate level phosphorylation in amastigotes whereas NADH-fumarate and NADH-pyruvate couples failed to produce ATP. In contrast, NADPH-fumarate showed the highest rate of ATP formation in promastigotes. Therefore, we can conclude that substrate level phosphorylation is essential for the survival of amastigote forms of Leishmania donovani.     

Functional characterization of nucleoside transporter gene replacements in Leishmania donovani

Leishmania donovani express two nucleoside transporters of non-overlapping ligand selectivity. To evaluate the physiological role of nucleoside transporters in L. donovani, homozygous null mutants of the genes encoding the LdNT1 adenosine–pyrimidine nucleoside transporter and the LdNT2 inosine–guanosine transporter were created singly and in combination by single targeted gene replacement followed by selection for loss-of-heterozygosity. The mutant alleles were verified by Southern blotting, and the effects of gene replacement on transport phenotype were evaluated by rapid sampling transport measurements and by drug resistance profiles. The Δldnt1, Δldnt2, and Δldnt1/Δldnt2 mutants were all capable of proliferation in defined culture medium supplemented with any of a spectrum of purine nucleobases or nucleosides, except that a Δldnt2 lesion conferred an inability to efficiently salvage exogenous xanthosine, a newly discovered ligand of LdNT2. Each of the three knockout strains was viable as promastigotes and axenic amastigotes and capable of maintaining an infection in J774 and bone marrow-derived murine macrophages. These genetic studies demonstrate: (1) that L. donovani promastigotes, axenic amastigotes, and tissue amastigotes are viable in the absence of nucleoside transport; (2) that nucleoside transporters are not essential for sustaining an infection in mammalian host cells; (3) that the phagolysosome of macrophages is likely to contain purines that are not LdNT1 or LdNT2 ligands, i.e., nucleobases. Furthermore, the Δldnt1, Δldnt2, and Δldnt1/Δldnt2 knockouts offer a unique genetically defined null background for the biochemical and genetic characterization of nucleoside transporter genes and cDNAs from phylogenetically diverse species and of genetically manipulated LdNT1 and LdNT2 constructs.     

Characterization of plasma membrane bound inorganic pyrophosphatase from Leishmania donovani promastigotes and amastigotes

Background

Currently, a major problem in the management of visceral leishmaniasis or kala-azar, especially in the Indian subcontinent, is the growing unresponsiveness to conventional antimonial therapy. Membrane bound pyrophophatase (PPases) do not exist in plasma membrane from mammals. Thus, H⁺-PPases from Leishmania plasma membrane might be potential target in rational chemotherapy of the disease caused by Leishmania parasites.

Objective

To characterize the activities of inorganic pyrophophatase (PPase) in the plasma membrane of Leishmania donovani promastigote and amastigote.

Methods

Culture method of promastigote and amastigote were developed. We assayed PPase activity in isolated plasma membrane of L. donovani.

Results

We characterized K⁺-PPase present in the plasma membrane of Leishmania donovani and investigated its possible role in the survival of promastigote and amastigote. PPase activity was stimulated by K⁺ ions and sodium orthovanadate, inhibited by pyrophosphate analogs imidodiphosphate and alendronate, KF, DCCD, thiol reagent parachloromercuribenzenesulfonate (PCMBS), N-ethylmaliemide (NEM), phenylarsineoxide, ABC superfamily transport modulator verapamil and was also by F₁F_o-ATPase inhibitor quercetin.

Conclusion

We conclude that there are significant differences within promastigote, amastigote and mammalian host in cytosolic pH homeostasis to merit the inclusion of PPase transporter as putative targets for rational drug design.     

Proline transport in Leishmania donovani amastigotes: dependence on pH gradients and membrane potential.

Amastigotes of Leishmania donovani develop and multiply within the acidic phagolysosomes of mammalian macrophages. Isolated amastigotes are acidophilic; they catabolize substrates and synthesize macromolecules optimally at pH 5.5. Substrate transport in amastigotes has not been characterized. Here we show that amastigotes exhibit an uphill transport of proline (active transport) with an acid pH optimum (pH 5.5). It is dependent upon metabolic energy and is driven by proton motive force. Agents which selectively disturb the component forces of proton motive force, such as carbonyl cyanide chlorophenylhydrazone, nigericin and valinomycin, inhibit proline transport. Transport is sensitive to dicyclohexylcarbodiimide and insensitive to ouabain, demonstrating the involvement of a proton ATPase in the maintenance of proton motive force. It is suggested that the plasma membrane pH gradient probably makes the greatest contribution to proton motive force that drives substrate transport in the amastigote stage.     

Reaction kinetics and inhibition of adenosine kinase from Leishmania donovani

The reaction kinetics and the inhibitor specificity of adenosine kinase (ATP:adenosine 5'-phosphotransferase, EC 2.7.1.20) from Leishmania donovani, have been analysed using homogeneous preparation of the enzyme. The reaction proceeds with equimolar stoichiometry of each reactant. Double reciprocal plots of initial velocity studies in the absence of products yielded intersecting lines for both adenosine and Mg2+-ATP. AMP is a competitive inhibitor of the enzyme with respect to adenosine and noncompetitive inhibitor with respect to ATP. In contrast, ADP was a noncompetitive inhibitor with respect to both adenosine and ATP, with inhibition by ADP becoming uncompetitive at very high concentration of ATP. Parallel equilibrium dialysis experiments against [3H]adenosine and [gamma-32P]ATP resulted in binding of adenosine to fre enzyme. Tubercidin (7-deazaadenosine) and 6-methyl-mercaptopurine riboside acted as substrates for the enzyme and were found to inhibit adenosine phosphorylation competitively in vitro. 'Substrate efficiency (Vmax/Km)' and 'turnover numbers (Kcat)' of the enzyme with respect to specific analogs were determined. Taken together the results suggest that (a) the kinetic mechanism of adenosine kinase is sequential Bi-Bi, (b) AMP and ADP may regulate enzyme activity in vivo and (c) tubercidin and 6-methylmercaptopurine riboside are monophosphorylated by the parasite enzyme.     

Comparative proteome analysis of Leishmania donovani at different stages of transformation from promastigotes to amastigotes

To pass through its life cycle, protozoan parasites of the genus Leishmania have to differentiate from promastigotes to amastigotes. The molecular basis underiving this major transformation is poorly understood. One way to study this phenomenon is to isolate and characterize proteins that are specifically expressed in one of the two stages of the life cycle or during the stage differentiation. Using two-dimensional gel electrophoresis, we mapped the Leishmania donovani proteome during stage differentiation to identify stage-specific proteins and regulons. A protocol for extracting proteins of both promastigote and amastigote L. donovani cells was developed, which is compatible with isoelectric focusing. Up to 400 L. donovani protein spots were visualized on a silver-stained gel. Metabolic labeling of the cells was used to compare directly the protein synthesis pattern with the protein level pattern. The silver-stained images of L. donovani cells harvested on different days of stage differentiation were compared to the corresponding autoradiographs. A marked decrease in protein synthesis during stage differentiation from promastigotes to amastigotes was observed. The stained protein pattern as well as the protein pattern on the autoradiograph changed dramatically, especially after day 3 (about 24 h after pH shift) of transformation.     

The plasma membrane electrical gradient (membrane potential) in Leishmania donovani promastigotes and amastigotes.

The equilibrium distribution of tetraphenylphosphonium bromide was used to measure the membrane potential in Leishmania donovani amastigotes and promastigotes and to investigate mechanisms underlying the maintenance of membrane potential. At pH 7.0, membrane potential ranges between -90 and -113 mV. Increasing the external concentrations of hydrogen or potassium ions decreased membrane potential as did treatments with carbonylcyanide chlorophenylhydrazone or valinomycin. These observations are consistent with a membrane potential set by hydrogen and potassium ion diffusion gradients. Anaerobiosis lowered membrane potential, suggesting the involvement of ATPase(s) in maintaining membrane potential. Membrane potential was insensitive to treatment with ouabain, demonstrating the absence of a Na+/K(+)-ATPase. Treatment with dicyclohexylcarbodiimide caused a temporary hyperpolarization of the membrane suggesting the participation of a proton ATPase in the maintenance of membrane potential. Determination of the membrane potential makes it possible to quantitate the total proton motive force which is the force for active transport across the parasite membrane.     

Experimental selection of paromomycin and miltefosine resistance in intracellular amastigotes of Leishmania donovani and L. infantum

Although widespread resistance of Leishmania donovani and L. infantum against miltefosine (MIL) and paromomycin (PMM) has not yet been demonstrated, both run the risk of resistance selection. Unraveling the dynamics and mechanisms of resistance development is key to preserve drug efficacy in the field. In this study, resistance against PMM and MIL was experimentally selected in vitro in intracellular amastigotes of several strains of both species with different antimony susceptibility background. To monitor amastigote susceptibility, microscopic determination of IC₅₀-values and promastigote back-transformation assays were performed. Both techniques were also used to evaluate the susceptibility of field isolates from MIL-relapse patients. PMM-resistance could readily be selected in all species/strains, although promastigotes remained fully PMM-susceptible. Successful MIL-resistance selection was demonstrated only by promastigote back-transformation at increasing MIL-concentrations upon successive selection cycles. Important to note is that amastigotes with the MIL-resistant phenotype could not be visualized after Giemsa staining; hence, MIL-IC₅₀-values showed no shift. The same phenomenon was observed in a set of recent clinical isolates from MIL-relapse patients. This study clearly endorses the need to use intracellular amastigotes for PMM- and MIL-susceptibility testing. When monitoring MIL-resistance, promastigote back-transformation should be used instead of the standard Giemsa staining. In-depth exploration of the mechanistic background of this finding is warranted.     

Solubilization and kinetic characterization of mitochondrial adenosine triphosphatase from Leishmania donovani promastigotes

Oligomycin-sensitive particulate ATPase (MB ATPase) from L. donovani promastigotes was solubilized by chloroform treatment. Polyacrylamide gel electrophoresis revealed several protein bands, with the major one possessing ATPase activity. The solubilized enzyme had Mg2+-ATPase and Ca2+-ATPase but no K+-dependent alkaline phosphatase activity. The Mg2+-ATPase activity was stimulated by monovalent cations and was not sensitive to oligomycin. Hence it is referred to as F1 ATPase. It had optimum activity at pH 7.6 and 30 degrees C. The Arrhenius plot for MB ATPase was biphasic with activation energies (Ea) of 16.2 and 3.4 kcal mol-1, while F1 ATPase exhibited a linear plot with Ea = 10.1 kcal mol-1. Lineweaver-Burk plots were biphasic with Km values of 0.17 and 1.25 mM for MB ATPase and 0.18 and 1.33 mM for F1 ATPase. The enzyme could be preserved at -15 degrees C in Tris-SO2-(4)-EDTA-ATP-glycerol (t1/2 = 20 days).     

Phagocytosis of Leishmania donovani amastigotes is Rac1 dependent and occurs in the absence of NADPH oxidase activation

Macrophages produce little superoxide during phagocytosis of Leishmania donovani amastigotes. In this study, we characterized molecular events associated with L. donovani amastigotes uptake by mouse macrophages, to further define the mechanisms by which they are internalized without triggering superoxide production. Using transient transfections, we first showed that internalization of L. donovani amastigotes is mediated by the GTPases Rac1 and Arf6, of which Rac1 is recruited and retained on parasite-containing phagosomes. Next, we showed that, whereas internalization of amastigotes induced no superoxide release, co-internalization of serum-opsonized zymozan and amastigotes resulted in superoxide production. Furthermore, in co-internalization experiments, we detected superoxide production in over 95% of phagosomes containing IgG-opsonized SRBC compared to 5% of amastigote-harboring phagosomes. These results suggest that amastigotes evade the ability of macrophages to produce superoxide during phagocytosis. Consistently, we observed that amastigotes induced barely detectable phosphorylation of the NADPH oxidase component p47phox, leading to a defective phagosomal recruitment of p67phox and p47phox. Finally, we showed that amastigotes disrupt phagosomal lipid raft integrity, potentially interfering with NADPH oxidase assembly. Collectively, our results indicate that internalization of L. donovani amastigotes is a Rac1- and Arf6-dependent process that occurs in the absence of significant NADPH oxidase activation.     

Cytotoxicity of human serum for Leishmania donovani amastigotes: antibody facilitation of alternate complement pathway-mediated killing.

Mechanisms that mediate recovery from leishmanial infection have not been fully characterized but are generally believed to involve interactions between T lymphocytes and macrophages. A major role for serum-mediated effector mechanisms in the protection of humans from reinfection with Leishmania, however, has not been ruled out. In this report, amastigotes of L. donovani were incubated with dilutions of serum from normal subjects and from patients with kala-azar. Normal serum was cytotoxic for parasites at a dilution of greater than or equal to 1:20. Cytotoxicity did not occur in the presence of EDTA, was abolished by heating serum to 56 degrees C for 30 min, and was not diminished by prior adsorption of normal serum with parasites at 0 degree C. Killing proceeded normally in the presence of magnesium-ethylene glycol-bis(beta-aminoethyl ether)-N, N-tetraacetic acid, however, and was fully effected by C2-deficient serum. These studies indicated that killing of amastigotes, unlike that of promastigotes, was mediated via the alternate pathway of serum complement. In further studies, cytotoxicity of normal serum was enhanced three- to fivefold by factors in patient serum. This enhanced cytotoxicity also proceeded via the alternate complement pathway. Factors that enhanced cytotoxicity were characterized as parasite-specific immunoglobulin G: they eluted with immunoglobulin G on column chromatography, were adsorbed by immobilized staphylococcal protein A, and were not removed from the parasite surface by extensive washing. Thus, infection of individuals with L. donovani resulted in the production of a new, qualitatively and quantitatively distinct immune mechanism directed against the amastigote form of the parasite, namely, antibody-directed, alternate complement pathway-mediated cytotoxicity. These results provide a mechanistic framework for a role of humoral factors in human resistance to reinfection with L. donovani.     

Expression of a stage-specific lipophosphoglycan in Leishmania major amastigotes.

Amastigotes of Leishmania major were isolated from infected mice and radiolabeled for 2 h with [3H]galactose. An acidic [3H]glycoconjugate was extracted from a dilipidated residue fraction with the solvent water/ethanol/diethylether/pyridine/NH4OH (15:15:5:1:0.017). The radioactivity labeled glycoconjugate was found to possess the following characteristics that were similar to the lipophosphoglycan extractable from promastigotes: (i) migrated as a broad band upon electrophoresis on SDS polyacrylamide gels; (ii) deaminated with nitrous acid; and (iii) hydrolyzed with phosphatidylinositol-specific phospholipase C. Furthermore, analysis of the aqueous soluble material released by the latter enzyme revealed a negatively-charged [3H]polysaccharide intermediate in size compared to the analogous portions of LPG isolated from non-infective and metacyclic promastigotes. Most importantly, the [3H]polysaccharide was found to contain phosphate and was susceptible to mild acid hydrolysis, establishing that the intact molecule is a lipophosphoglycan. A structural difference, however, was found in the major, mild acid-generated fragment of the amastigote phosphoglycan, which was larger in size and not as anionic as the analogous fragment from the promastigote phosphoglycans. These results indicate that the amastigotes do express a lipophosphoglycan, but that it is structurally distinct from its promastigote counterparts.     

Roles of free GPIs in amastigotes of Leishmania

Glycosylated phosphatidylinositols (GPIs) are abundant cell surface molecules of the Leishmania. Amastigote-specific GPIs AmGPI-Y and AmGPI-Z, both ethanolamine (EtN)-containing glycolipids, were identified in Leishmania amazonensis. A paucity of GPI-anchored proteins in amastigotes of L. amazonensis made the kinetoplastid suitable for evaluating the importance of free (i.e. unconjugated to protein or polysaccharide) GPIs. A strain deficient in both AmGPI-Y and AmGPI-Z was produced by stable transfection of wild-type Leishmania with a GPI-phospholipase C gene. Phosphatidylinositol deficiency was not detected in the transfectants. GPI-deficient promastigotes infected murine macrophages in vitro and differentiated into amastigotes whose growth was arrested within the host cells. Cytostasis of amastigotes was also observed during axenic culture of GPI-deficient parasites. In a hamster model of leishmaniasis, GPI-deficient promastigotes produced smaller lesions with 20-fold fewer amastigotes than infections with control parasites. Together, these observations indicate that EtN-GPIs may be essential for amastigote viability, replication, and/or virulence. Implicit in these observations is the notion that drugs targeted against the GPI biosynthetic pathway might be of value in the management of human leishmaniasis.