Interaction of sitamaquine with membrane lipids of Leishmania donovani promastigotes

Sitamaquine is an 8-aminoquinoline which is active by the oral route for the treatment of life-threatening visceral leishmaniasis caused by Leishmania donovani, with an IC50 of 29.2 microM against the promastigote form in vitro. At high concentration (100 microM), sitamaquine affected parasite motility, morphology and growth in a way that was only partially reversible. As a first approach to determine its mechanism of action, we describe the interaction of sitamaquine with parasite membrane components, representing the first barrier to be crossed by the drug. Analysis of the physicochemical interactions of sitamaquine with monolayers of phospholipids and sterols at the air-water interface showed that these interactions only occurred in the presence of anionic phospholipids. Thus, electrostatic interactions between positively charged sitamaquine and the negative polar headgroups are a pre-requisite for subsequent hydrophobic interactions between the sitamaquine aromatic ring and the alkyl chains of phospholipids leading to drug insertion into the monolayer.     

Leishmania donovani: identification of glycoproteins released by promastigotes during growth in vitro

Culture supernatants of metabolically labeled Leishmania donovani promastigotes were shown to contain approximately 40 electrophoretically distinct released protein compounds. Of these, approximately 20 were glycoproteins which contained terminal mannose residues, as judged by their specific binding to concanavalin A-agarose beads. Smaller subsets of the released glycoproteins were bound by agarose-conjugated Lens culinaris, Ricinus communis, and peanut lectins. Promastigote mannose-containing released glycoproteins were isolated by concanavalin A affinity chromatography and used to immunize a rabbit. This antiserum recognized the parasite-released mannose-containing glycoproteins, including the soluble acid phosphatase, both by immunoprecipitation from solution and in immunoblot analyses. In an antibody bridged enzyme assay this polyspecific serum was also capable of binding native acid phosphatase out of solution and bridging it to the denatured enzyme on SDS-PAGE transblots. Although this antiserum was raised against all 20 released glycoproteins, in agarose gels its major precipitin activity was against the secreted soluble acid phosphatase.     

Heme metabolism in promastigotes of Leishmania donovani

Promastigotes of Leishmania donovani (Dd-8 strain) showed presence of important key enzymes of heme synthesizing (d-aminolevulinic acid synthase and ferrochelatase) and degrading (heme oxygenase and biliverdin reductase) systems, classical leishmanicidal drugs viz allopurinol, amphotericin B, pentamidine and CDRI compound 93/202 inhibited the heme oxygenase activity of the parasite, whereas, -aminolevulinic acid synthase activity practically remained unaffected. The Km, Vmax ad pH values of heme oxygenase of promastigotes were found to be 1666 M hemin, 625 nmol of bilirubin formed h-1 mg protein-1 and 7.5 respectively. The findings suggest the presence and importance of heme metabolism in the de novo synthesis of different hemoproteins of the Leishmania parasite as well as the detoxification and its defence against biological insults.     

Effects of DL-alpha-difluoromethylornithine on Leishmania donovani promastigotes

Alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase, has been demonstrated to be an effective agent against a variety of parasitic protozoa but not against Leishmania spp. In this report, we show that Leishmania donovani promastigotes in continuous culture are sensitive to the growth inhibitory and cytotoxic effects of DFMO. Incubation of the promastigotes with DFMO obliterates intracellular putrescine pools and depletes spermidine concentrations, which correlates with the onset of growth inhibition. The effects of DFMO on the growth and the intracellular polyamine pools can be reversed completely by the addition of 10 microM putrescine to the culture medium. These results suggest that the treatment of leishmaniasis may be amenable to chemotherapeutic manipulation by DFMO.     

Arginine catabolism by Leishmania donovani promastigotes.

Leishmania donovani promastigotes were grown to late log phase, washed and resuspended in iso-osmotic buffer containing L-arginine, and the rate of urea formation was then measured under various conditions. Addition of glucose or mannose activated urea formation, whereas 2-deoxyglucose inhibited and 6-deoxyglucose had no effect. Addition of alanine or of alpha-aminoisobutyrate inhibited urea formation, alanine causing a greater inhibition than alpha-aminoisobutyrate. Addition of leucine, proline, glycine, or lysine had no effect on urea formation. The presence of glutamate also increased the rate of urea formation from arginine, but to a lesser extent than did glucose. The presence of both glucose and alanine caused no net change in urea formation, whereas the inhibitory effect of alanine exceeded the activating effect of glutamate, so that a small inhibition in the rate of urea formation occurred in the presence of both alanine and glutamate. Cells grown to 3-day stationary phase had a markedly reduced rate of arginine catabolism to urea, but the activating effect of glucose and the inhibitory effect of alanine were qualitatively similar to their effects on late log phase cells. Addition of water to cells suspended in buffer also inhibited urea formation, but this appeared to be due primarily to the release of alanine caused by the hypo-osmotic stress. Addition of mannitol to cells suspended in buffer caused a small inhibition of arginine catabolism. Addition of dibutyrylcyclic AMP, 3',5'-cyclic GMP, phorbol myristic acid, or A23187 had no effect on the rate of urea formation from arginine.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro tryptophan catabolism by Leishmania donovani donovani promastigotes.

Metabolism of tryptophan by promastigotes of Leishmania donovani donovani was investigated in cells suspended in a simple buffer solution supplemented with glucose. Metabolites from supernatant and lysed cell pellets were analyzed by capillary gas liquid chromatography and 13C nuclear magnetic resonance spectroscopy, with structural confirmation by gas liquid chromatography-mass spectrometry. Tryptophan does not appear to serve as a carbon energy source for L. d. donovani promastigotes since parasites could survive for only short periods in buffer containing tryptophan without glucose, levels of tricarboxylic acid cycle intermediates remained unchanged in the presence of added tryptophan and label from [13C]tryptophan was not detected in any of the intermediates. Leishmania d. donovani catabolized L-tryptophan via aminotransferase and aromatic lactate dehydrogenase reactions to form one major end product, indole-3-lactic acid. The activity of aromatic lactate dehydrogenase required manganese and was NADH-dependent in these organisms that lack lactate dehydrogenase. Promastigotes taken from the mid-log stage of growth produced higher concentrations of indole-3-lactic acid than those from the stationary stage. Conservation of a similar tryptophan catabolic pathway among four Leishmania species suggests the pathway is physiologically important to the parasites themselves.     

Characterization of the redox components of transplasma membrane electron transport system from Leishmania donovani promastigotes

An investigation has been made of the points of coupling of four nonpermeable electron acceptors e.g., alpha-lipoic acid (ALA), 5,5'-dithiobis (2-nitroaniline-N-sulphonic acid) (DTNS), 1,2-naphthoquinone-4-sulphonic acid (NQSA) and ferricyanide which are mainly reduced via an interaction with the redox sites present in the plasma membrane of Leishmania donovani promastigotes. ALA, DTNS, NQSA and ferricyanide reduction and part of O2 reduction is shown to take place on the exoplasmic face of the cell, for it is affected by external pH and agents that react with the external surface. Redox enzymes of the transplasma membrane electron transport system orderly transfer electron from one redox carrier to the next with the molecular oxygen as the final electron acceptor. The redox carriers mediate the transfer of electrons from metabolically generated reductant to nonpermeable electron acceptors and oxygen. At a pH of 6.4, respiration of Leishmania cells on glucose substrate shut down almost completely upon addition of an uncoupler FCCP and K+-ionophore valinomycin. The most pronounced effects on O2 uptake were obtained by treatment with antimycin A, 2-heptadecyl-4-hydroxyquinone-N-oxide, paracholoromercuribenzene sulphonic acid and trifluoperazine. Relatively smaller effects were obtained by treatment with potassium cyanide. Inhibition observed with respect to the reduction of the electron acceptors ALA, DTNS, NQSA and ferricyanide was not similar in most cases. The redox chain appears to be branched at several points and it is suggested that this redox chain incorporate iron-sulphur center, b-cytochromes, cyanide insensitive oxygen redox site, Na+ and K+ channel, capsaicin inhibited energy coupling site and trifluoperazine inhibited energy linked P-type ATPase. We analyzed the influence of ionic composition of the medium on reduction of electron acceptors in Leishmania donovani promastigotes. Our data suggest that K+ have some role for ALA reduction and Na+ for ferricyanide reduction. No significant effects were found with DTNS and NQSA reduction when Na+ or K+ was omitted from the medium. Stimulation of ALA, DTNS, NQSA and ferricyanide reduction was obtained by omitting Cl- from the medium. We propose that this redox system may be an energy source for control of membrane function in Leishmania cells.     

Leishmania donovani: cell-surface heparin receptors of promastigotes are recruited from an internal pool after trypsinization

With the use of [3H]heparin, we recently demonstrated that Leishmania donovani promastigotes express a cell-surface receptor that is specific for the glycosaminoglycan heparin (Mukhopadhyay et al. 1989, The Biochemical Journal, 264, 517-525.). Treatment of the parasite with trypsin abolishes 75-90% of this [3H]heparin-binding activity. When trypsinized promastigotes were resuspended in fresh culture medium in the absence and presence of cycloheximide (10 micrograms/ml), approximately 25-30% of the original heparin-binding capacity was restored within 1 hr, indicating that recruitment of receptors from an internal pool occurred without de novo protein synthesis. Scatchard analysis of the regenerated receptor revealed that the number of regenerated binding sites per cell was 2.3 x 10(5); these sites have a binding affinity of 6.7 x 10(-7) M. Like the native heparin receptors on the surface of freshly isolated cells, the receptors recruited after trypsinization are also highly specific for heparin, as a 25-fold excess of four other glycosaminoglycans displaced less than 10% of bound [3H]heparin from the trypsinized cells. The structural requirements of the ligand heparin, namely the number of monosaccharide units and degree of sulfation, were compared for both the native and regenerated receptor: for both receptors, oversulfated polysaccharide heparin fragments of at least six to eight sugar residues were most efficient at displacing [3H]heparin. The concentrations of oligosaccharide fragments required to displace 50% of [3H]heparin were 0.32 and 0.035 microM for the hexa- and octasaccharides, respectively. Colloidal gold-labeled heparin was bound to promastigotes and visualized by electron microscopy. This analysis revealed that the heparin bound almost exclusively to the flagella of control cells (not subjected to trypsin) and those which had regenerated receptor after trypsinization. The physiological significance of this heparin-binding activity on the surface of promastigotes is discussed.     

Transplasma membrane electron transport in Leishmania donovani promastigotes

Leishmania donovani promastigotes are capable of reducing certain electron acceptors with redox potential at pH 7 down to -125 mV; outside the plasma membrane promastigotes can reduce ferricyanide. Ferricyanide has been used as an artificial electron acceptor probe for studying the mechanism of transplasma membrane electron transport. Transmembrane ferricyanide reduction by L. donovani promastigotes was not inhibited by such mitochondrial inhibitors as antimycin A or cyanide, but it responded to inhibitors of glycolysis. Transmembrane ferricyanide reduction by Leishmania appears to involve a plasma membrane electron transport chain dissimilar to that of hepatocyte cells. As with other cells, transmembrane electron transport is associated with proton release, which may be involved in internal pH regulation. The Leishmania transmembrane redox system differs from that of mammalian cells in being 4-fold less sensitive to chloroquine and 12-fold more sensitive to niclosamide. Sensitivities to these drugs suggest that transplasma membrane electron transport and associated proton pumping may be targets for the drugs used against leishmaniasis.     

Proteomic approach for identification and characterization of novel immunostimulatory proteins from soluble antigens of Leishmania donovani promastigotes

Visceral leishmaniasis (VL) caused by Leishmania donovani is a major parasitic disease prevalent in endemic regions of Bihar in India. In the absence of good chemotherapeutic options, there is a need to develop an effective vaccine against VL which should be dependent on the generation of a T helper type 1 (Th1) immune response. We have shown that soluble proteins from promastigote of a new clinical isolate of L. donovani (2001) ranging from 68 to 97.4 kDa (F2 fraction), induce Th1 responses in the peripheral blood mononuclear cells of cured Leishmania patients and hamsters and also showed significant prophylactic potential. To understand the nature of F2 proteins, it was further characterized using 2-DE, MALDI-TOF and MALDI-TOF/TOF-MS. In all, 63 spots were cut from a CBB stained gel for analysis and data was retrieved for 52 spots. A total of 33 proteins were identified including six hypothetical/unknown proteins. Major immunostimulatory proteins were identified as elongation factor-2, p45, heat shock protein (HSP)70, HSP83, aldolase, enolase, triosephosphate isomerase, protein disulfideisomerase and calreticulin. This study substantiates the usefulness of proteomics in characterizing a complex protein fraction (F2) map of soluble L. donovani promastigote antigen identified as Th1 stimulatory for its potential as vaccine targets against VL.     

Mechanism of complement-independent and antibody-mediated killing of Leishmania donovani promastigotes

Immune serum raised against flagellar fraction of Leishmania donovani isolate UR6 has profound lethal effect on the in vitro growth of the parasite. Lethal effect of immune serum was also examined using two other isolates of L. donovani, namely DD8 and AG83. It was observed that immune serum is equally effective against UR6 and DD8 but has no effect on AG83 promastigotes. Parasite killing is mediated by Leishmania-specific antibodies in the absence of complement or any other factors present in rabbit serum. Results indicate that the lethal effect of immune serum is due to impairment in membrane function leading to inhibition in uptake of essential nutrients needed for growth and survival of parasites.     

Effects of long-term in vitro cultivation on Leishmania donovani promastigotes

Promastigotes of Leishmania donovani that had been subcultured in modified Tobie's medium for 2 to 3 years showed decreased infectivity and lack of virulence for hamsters and mice compared to newly transformed promastigotes. Amastigotes derived from these long-term promastigote cultures decreased in number rapidly in hamsters, but only slightly in mice, over a 48-day period. In cultured mouse and hamster macrophages infected in vitro, amastigotes derived from long-term cultures rapidly decreased to low numbers, which persisted. The same pattern was seen in macrophages treated with catalase, an inhibitor of the oxygen-dependent killing mechanism of the macrophage. Promastigotes from long-term cultures also differed from virulent first-passage promastigotes in size, growth patterns in Tobie's medium, and in the quantities of some of their antigens.     

Metabolic variation during development in culture of Leishmania donovani promastigotes

The genome sequencing of several Leishmania species has provided immense amounts of data and allowed the prediction of the metabolic pathways potentially operating. Subsequent genetic and proteomic studies have identified stage-specific proteins and putative virulence factors but many aspects of the metabolic adaptations of Leishmania remain to be elucidated. In this study, we have used an untargeted metabolomics approach to analyze changes in the metabolite profile as promastigotes of L. donovani develop during in vitro cultures from logarithmic to stationary phase. The results show that the metabolomes of promastigotes on days 3-6 of culture differ significantly from each other, consistent with there being distinct developmental changes. Most notable were the structural changes in glycerophospholipids and increase in the abundance of sphingolipids and glycerolipids as cells progress from logarithmic to stationary phase.     

Partial purification and characterization of a soluble protein kinase from Leishmania donovani promastigotes

A soluble protein kinase from the promastigote form of the parasitic protozoon Leishmania donovani was partially purified using DEAE-cellulose, Sephadex G-200 and phosphocellulose columns. The enzyme preferentially utilized protamine as exogenous phosphate acceptor. The native molecular mass of the enzyme was about 85 kDa. Mg2+ ions were essential for enzyme activity; other metal ions, e.g. Ca2+, Co2+, Zn2+ and Mn2+, could not substitute for Mg2+. cAMP, cGMP, Ca2+/calmodulin and Ca2+/phospholipid did not stimulate enzyme activity. The pH optimum of the enzyme was 7.0-7.5, and the temperature optimum 37 degrees C. The apparent Km for ATP was 60 microM. Phosphoamino acid analysis revealed that the protein kinase transferred the gamma-phosphate of ATP to serine residues in protamine. The thiol reagents p-hydroxymercuribenzoic acid, 5-5'-dithio-bis(2-nitrobenzoic acid) and N-ethylmaleimide inhibited enzyme activity; the inhibition by p-hydroxymercuribenzoic acid and 5-5'-dithio-bis(2-nitrobenzoic acid) was reversed by dithiothreitol.     

Fluidity-dependent Mg2(+)-ATPase activity in membranes from Leishmania donovani promastigotes.

The state of the lipid phase of the membrane plays a key role in the exposure of various receptors, antigens and enzymes on the membrane surface. The fluidity of membranes of Leishmania donovani promastigotes was monitored by two independent methods, i.e. influx of sterol from liposomes and removal of phospholipids by treatment with phospholipase C. The altered sterol/phospholipid ratio, in both cases, provided evidence that the activity of the functionally important membrane-bound enzyme Mg2(+)-ATPase is modulated by the state of the lipid phase of the membrane.     

Acid phosphatase activity of promastigotes of Leishmania donovani: a marker of virulence

Seven cloned lines of promastigotes of Leishmania donovani (UR 6) were isolated by limiting dilution. One clone, UR6-C25, failed to multiply inside the macrophages of line J774G8 and thus was labelled as avirulent. Another, UR6-C24, multiplied inside macrophages, had a virulence index as high as 93 +/- 9.8 and was thus labelled as highly virulent. The other five clones had variable degree of virulence indices ranging from 46.4 +/- 5.8 to 67.6 +/- 3.5. No significant difference in the degrees of attachment of virulent and avirulent populations of promastigotes to macrophages was observed, suggesting no difference in the ligand utilised by these populations for attachment to the macrophages. Acid phosphatase activity of cloned promastigotes correlated with the degree of virulence. These data suggest that acid phosphatase activity could be used as a marker to differentiate avirulent from virulent populations of promastigotes of L. donovani.     

Leishmania donovani: physicochemical, immunological, and biological characterization of excreted factor from promastigotes.

Leishmanial excreted factor (EF) from promastigote cultures was enriched from the crude product by differential precipitation with ammonium sulfate and perchloric acid, followed by column chromatography; and by boiling EF-antibody complex. Boiling destroyed the antibody, releasing the EF, which retained its ability to precipitate antibody. Enriched EF from Leishmania donovani promastigotes was found to be a highly negatively charged, carbohydrate-like material with a molecular weight approximating to 33,000, when monitored against a series of protein markers by gel filtration. Its ability to precipitate with antibody was unimpaired by boiling, lyophilization, pH changes from 1 to 11, treatment with high concentrations of NaCl, 10% phosphotungstic acid in 10% HCl, 0.6 M perchloric acid, 5% H₂SO₄, acetone, or dioxan. It did not absorb at wavelengths between 220 and 750 nm. Treatment with trypsin, Pronase, neuraminidase, and hyaluronidase did not affect its activity. Biochemical analysis showed that enriched EF contains carbohydrates but, at our level of detection, no protein, lipid, triglycerides, fatty acids, DNA, RNA, pentoses, amino sugars, sialic or uronic acid. Precipitation of EF by antibody was studied and the optimal molecular proportions for complete precipitation determined. EF-antibody complex, prepared at optimal proportions, and EF complexed with methylated bovine serum albumin, like EF alone, did not elicit antibody production in rabbits. EF in 0.5% phenol-saline elicited a delayed skin response of induration and erythema in guinea pigs cured of L. enriettii. Elevated temperature increased the release of EF from promastigotes, while the presence of trypsin acting at 37 C seemed to inhibit this effect slightly. Fractionation of mechanically broken promastigotes, by differential centrifugation and stepwise sucrose gradients, revealed a factor that precipitated rabbit antibody against whole promastigotes. This factor was associated with the soluble, organelle-free fraction and resembled EF when monitored by gel diffusion. This factor did not migrate when the complete extract from the broken promastigotes was run in immunoelectrophoresis. Boiling the extract for 5 min released a factor, which migrated to the anode. This factor appeared to be associated with another component in the promastigote, from which it dissociated on boiling. Boiling hamster tissues infected with leishmanial amastigotes, i.e., spleens containing L. donovani and epididymides containing L. tropica, also released factors similar to EF. These precipitated antibody in the same way, producing precipitation arcs that were continuous with those formed by EF from the homologous promastigotes. EF acted as a conditioner for culture promastigotes. Conditioned cultures showed maximal growth before similar, unconditioned cultures. However, both types of culture produced equal numbers of promastigotes per unit volume by the end of exponential growth.     

Leishmania donovani: Metabolite mapping of promastigotes using proton nuclear magnetic resonance spectroscopy

Proton nuclear magnetic resonance spectroscopy was used for studying the intracellular metabolite profile of promastigotes of Leishmania donovani. The major intracellular metabolites observed in the promastigotes were acetate, alanine, succinate, glycine, -glycerophosphorylcholine, acetoacetate, arginine and ethanol. A comparative study of the intracellular metabolite profile of promastigotes of different strains of L. donovani showed that, all the major intracellular metabolites were present in promastigotes of different strains. A quantitative estimation of metabolites showed a strain specific (Finger print) metabolite profile which can be used for strain/species identification/differentiation.