Over-expression of Leishmania major MAP kinases reveals stage-specific induction of phosphotransferase activity

During the infectious cycle, protozoan parasites undergo various developmental transitions and switch virulence factors in response to extracellular signals in insect vectors and human hosts. Despite the importance of environmental sensing in parasite pathogenicity, little is known about the pathways that transduce extracellular signals into stage-specific gene expression. Here, we used a transgenic approach to gain insight into localisation and activity of three green fluorescence protein (GFP)-tagged Leishmania major mitogen-activated protein kinases, LmaMPK4, 7 and 10. The GFP-LmaMPKs in both L. major and Leishmania donovani transgenic lines showed predominant cytoplasmic localisation and the over-expression had no effect on promastigote morphology, growth and the ability to differentiate into stationary-phase metacyclics for L. major and axenic amastigotes for L. donovani. We isolated the GFP-tagged MPKs from parasite extracts and tested their phosphotransferase activity across various culture conditions. For all three GFP-LmaMPKs, kinase activity was low or absent in promastigote extracts but significantly increased in L. major promastigotes after exposure to pH 5.5 and 34 degrees C, and in axenic L. donovani amastigotes. Enhanced activity correlated with increased GFP-LmaMPK phosphorylation as judged by phospho-specific fluorescent staining of the immuno-precipitated kinases. We could extend these findings to the endogenous LmaMPK10, which accumulated in the phospho-protein fraction of axenic amastigotes but not promastigotes, and thus follows the stage-specific phosphorylation profile of episomally expressed GFP-LmaMPK10. These results provide evidence for the functional conservation of Leishmania MAP kinases in parasite environmental sensing and underscore the potential of transgenic approaches to gain insight into signaling events during the Leishmania life cycle.     

Generation of Leishmania donovani axenic amastigotes: their growth and biological characteristics

In this report, we describe an in vitro culture system for the generation and propagation of axenic amastigotes from the well characterised 1S-CL2D line of Leishmania donovani. Fine structure analyses of these in vitro-grown amastigotes demonstrated that they possessed morphological features characteristic of L. donovani tissue-derived amastigotes. Further, these axenic amastigotes (LdAxAm) were shown to synthesise and release a secretory acid phosphatase isoform similar to that produced by intracellular amastigotes. Such LdAxAm also expressed surface membrane 3'-nucleotidase enzyme activity similar to that of tissue-derived amastigotes. Moreover, LdAxAm, in contrast to promastigotes, expressed significant levels of the amastigote-specific A2 proteins. In addition, LdAxAm, derived from long term cultures of Ld 1S-CL2D promastigotes, had significant infectivity for both human macrophages in vitro and for hamsters in vivo. Thus, the in vitro culture system described herein provides a useful tool for the generation of large quantities of uniform populations of axenic amastigotes of the L. donovani 1S-CL2D line. The availability of such material should greatly facilitate studies concerning the cell and molecular biology of this parasite developmental stage.     

Life in vacuoles--nutrient acquisition by Leishmania amastigotes

Leishmania have a digenetic life cycle, involving a motile, extracellular stage (promastigote) which parasitises the alimentary tract of a sandfly vector. Bloodfeeding activity by an infected sandfly can result in transmission of infective (metacyclic) promastigotes to mammalian hosts, including humans. Leishmania promastigotes are rapidly phagocytosed but may survive and transform into non-motile amastigote forms which can persist as intracellular parasites. Leishmania amastigotes multiply in an acidic intracellular compartment, the parasitophorous vacuole. pH plays a central role in the developmental switch between promastigote and amastigote stages, and amastigotes are metabolically most active when their environment is acidic, although the cytoplasm of the amastigote is regulated at near-neutral pH by an active process of proton extrusion. A steep proton gradient is thus maintained across the amastigote surface and all membrane processes must be adapted to function under these conditions. Amastigote uptake systems for glucose, amino acids, nucleosides and polyamines are optimally active at acidic pH. Promastigote uptake systems are kinetically distinct and function optimally at more neutral environmental pH, indicating that membrane transport activity is developmentally regulated. The nutrient environment encountered by amastigotes is not well understood but the parasitophorous vacuole can fuse with endosomes, phagosomes and autophagosomes, suggesting that a diverse range of macromolecules will be present. The parasitophorous vacuole is a hydrolytic compartment in which such material will be rapidly degraded to low molecular weight components which are typical substrates for membrane transporters. Amastigote surface transporters must compete for these substrates with equivalent host transporters in the membrane of the parasitophorous vacuole. The elaboration of accumulative transporters with high affinity will be beneficial to amastigotes in this environment. The influence of environmental pH on membrane transporter function is discussed, with emphasis on the potential role of a transmembrane proton gradient in active, high affinity transport.     

Leishmania donovani: long-term culture of axenic amastigotes at 37 degrees C

L. donovani promastigotes were subjected to heat treatment yielding an axenic amastigote stage which was long-term cultured at 37 degrees C. No differences were observed between the growth rates of axenic amastigotes and promastigotes. Flow cytometry-derived DNA histograms of axenic amastigotes and promastigotes were typical of exponentially growing cell populations. Moreover, axenic amastigotes were metabolically active as evidenced by the release of an immunoprecipitable extracellular acid phosphatase (SAcP) into their culture supernatant. Cell transformation was confirmed by transmission electronmicroscopic examination of thin sections and extended by fracture-flip survey which allowed differentiation of cell membranes. The ultrastructure and nanoanatomy of axenic amastigotes was identical to that of intracellular amastigotes. The production of large amounts of heat-shock axenic amastigotes suitable for biochemical and biological studies of differentiation in Leishmania donovani may have important implications in the development of prevention and/or treatment strategies.     

Enzymes of carbohydrate metabolism in Leishmania donovani amastigotes

A method for the isolation of Leishmania donovani amastigotes from infected hamster spleen and liver tissues is described. Over 85% of the isolated amastigotes were viable as judged by acridine orange-ethidium bromide staining and in vitro transformation to the promastigote form. A comprehensive survey of the enzymes of carbohydrate metabolism in L. donovani amastigotes and promastigotes was conducted. Amastigotes and promastigotes possess all of the enzymes of the Embden-Meyerhof pathway, hexose monophosphate shunt, and tricarboxylic acid cycle. Cell-free extracts of both forms demonstrate an active glutamate dehydrogenase, thus linking activity which permits entry of pyruvate into the tricarboxylic acid cycle. Both forms demonstrate an active glutamate dehydrogenase, thus linking amino acid metabolism with carbohydrate metabolism. Pyruvate carboxylase, the enzyme responsible for replenishment of C4 acids by heterotrophic CO2 fixation into pyruvate, was also demonstrable in the tissue and insect forms. In general, activities of promastigote enzymes are higher than the amastigote enzymes. Differences between the vertebrate (amastigote) and invertebrate (promastigote) forms in their potential to utilize carbohydrates as substrates would appear to be quantitative rather than qualitative.     

Identification of amastigote-specific antigens of Leishmania donovani using kala-azar patient sera

Antigenic characterization of the soluble fraction of axenic amastigotes of Leishmania donovani ( strain Dd8, causative agent of Indian kala-azar) and their comparison with promastigotes is reported. The axenic amastigotes were assessed for their immunological status employing anti-A2 monoclonal antibody which is extremely specific for L. donovani amastigotes. SDS-PAGE of 35[S] methionine labeled proteins of the two parasite stages exhibited few stage specific and some conserved antigens in both the stages. An increased synthesis of heat shock proteins was observed in axenic amastigotes. Western blot experiments employing sera of kala azar positive patients identified immunodominent antigens of 116,83,26 and 12 kDa in axenic amastigotes which were not present in promastigotes. These amastigote stage specific antigens may have immense potential in immunodiagnosis and prophylaxis of kala-azar.     

Development of Luciferase Expressing Leishmania donovani Axenic Amastigotes as Primary Model for In Vitro Screening of Antileishmanial Compounds

The development of new therapeutic leads against leishmaniasis relies primarily on screening of a large number of compounds on multiplication of clinically irrelevant transgenic promastigotes. The advent of the successful in vitro culture of axenic amastigotes allows the development of transgenic axenic amastigotes as a primary screen which can test compounds in a high throughput mode like promastigotes, still representative of the clinically relevant mammalian amastigotes stage. The present study reports the development of luciferase-tagged axenic amastigotes of Leishmania donovani, the causative agent of Indian Kala-azar, for in vitro drug screening. Luciferase expressing promastigotes were transformed to axenic amastigotes at a low pH and high temperature without the loss of luciferase expression. As compared to transgenic promastigotes, the luciferase expressing axenic amastigotes exhibited more sensitivity to antileishmanial drugs, particularly to pentavalent antimony (~2.8-fold) and also to the test compounds. Hence, the developed luciferase expressing axenic amastigotes make an ideal choice for high throughput drug screening for antileishmanial compounds.     

Rapid fluorescent assay for screening drugs on Leishmania amastigotes

A rapid fluorescent viability assay employing alamarBlue was optimized for use with Leishmania axenic amastigotes, the stage of the parasite responsible for disease pathology. The activity of two protein kinase inhibitors, Staurosporine and H-89, as well as Amphotericin B, on promastigotes and amastigotes of Leishmania donovani and Leishmania tropica was compared. Both protein kinase inhibitors inhibited promastigote growth at lower concentrations than amastigotes, while the GI(50) for Amphotericin B on both stages was similar. This assay only requires a limited number of axenic amastigotes (50,000 cells/well) and can be used to rapidly screen large chemical or natural product libraries for activity against amastigotes.     

Characterization of metacaspases with trypsin-like activity and their putative role in programmed cell death in the protozoan parasite Leishmania

In this report, we have characterized two metacaspases of Leishmania donovani, L. donovani metacaspase-1 (LdMC1) and LdMC2. These two proteins show 98% homology with each other, and both contain a characteristic C-terminal proline-rich domain. Both genes are transcribed in promastigotes and axenic amastigotes of L. donovani; however, LdMC1 shows increased mRNA levels in axenic amastigotes. An anti-LdMC antibody was obtained and showed reactivity with a single approximately 42-kDa protein band in both promastigote and axenic amastigote parasite whole-cell lysates by Western blotting. Pulse-chase experiments suggest that LdMCs are not synthesized as proenzymes, and immunofluorescence studies show that LdMCs are associated with the acidocalcisome compartments of L. donovani. Enzymatic assays of immunoprecipitated LdMCs show that native LdMCs efficiently cleave trypsin substrates and are unable to cleave caspase-specific substrates. Consistently, LdMC activity is insensitive to caspase inhibitors and is efficiently inhibited by trypsin inhibitors, such as leupeptin, antipain, and N(alpha)-tosyl-L-lysine-chloromethyl ketone (TLCK). In addition, our results show that LdMC activity was induced in parasites treated with hydrogen peroxide, a known trigger of programmed cell death (PCD) in Leishmania and that parasites overexpressing metacaspases are more sensitive to hydrogen peroxide-induced PCD. These findings suggest that Leishmania metacaspases are not responsible for the caspase-like activities reported in this organism and suggest a possible role for LdMCs as effector molecules in Leishmania PCD.     

Aspects of the cytological activity of edelfosine, miltefosine, and ilmofosine in Leishmania donovani

To discover the mode of action of alkyl-lysophospholipids in Leishmania donovani, we studied the effects of edelfosine, miltefosine, and ilmofosine on intracellular pH, the parasite's cell cycle, and the induction of apoptosis. The effect of the alkyl-lysophospholipids was combined with that of inhibitors of some pumps and exchange regulators of intracellular pH (Na+/ H+; Cl-/CO- 3; and the Na+/K+ ATPase). The effect of the 3 alkyl-lysophospholipids on intracellular pH was indirect; the primary action occurred in the parasite's cell membrane. To determine intracellular pH, we used flow cytometry for the macrophages and axenic amastigotes and spectrofluorometry for the promastigote forms. Apoptosis and the cell cycle were studied by flow cytometry. Treatment of the extracellular promastigote form of L. donovani with the 3 alkyl-lysophospholipids induced death by apoptosis, whereas in the infected cell they caused necrosis rather than apoptosis. Miltefosine and ilmofosine at doses of 38 microM caused G2/M cell cycle inhibition in L. donovani promastigotes.     

Quantitative proteome profiling informs on phenotypic traits that adapt Leishmania donovani for axenic and intracellular proliferation

Protozoan parasites of the genus Leishmania are important human pathogens that differentiate inside host macrophages into an amastigote life cycle stage. Although this stage causes the pathogenesis of leishmaniasis, only few proteins have been implicated in amastigote intracellular survival. Here we compare morphology, infectivity and protein expression of L. donovani LD1S grown in host free (axenic) culture, or exclusively propagated in infected hamsters, with the aim to reveal parasite traits absent in axenic but selected for in hamster-derived amastigotes through leishmanicidal host activities. Axenic and splenic amastigotes showed a striking difference in virulence and the ability to cause experimental hepato-splenomegaly in infected hamsters. 2D-DIGE analysis revealed statistically significant differences in abundance for 152 spots, with 14 spots showing fivefold or higher abundance in splenic amastigotes. Proteins identified by MS analysis include the anti-oxidant enzyme tryparedoxin peroxidase, and enzymes implicated in protein and amino acid metabolism. Analysis of parasite growth in vitro in minimal medium demonstrated increased survival of hamster-derived compared with axenic parasites under conditions that mimic the nutrient poor, cytotoxic phagolysosome. Thus, our comparative proteomics analysis sheds important new light on the biochemistry of bona fide amastigotes and informs on survival factors relevant for intracellular L. donovani infection.     

Quinone-Amino Acid Conjugates Targeting Leishmania Amino Acid Transporters

The aim of the present study was to investigate the feasibility of targeting Leishmania transporters via appropriately designed chemical probes. Leishmania donovani, the parasite that causes visceral leishmaniasis, is auxotrophic for arginine and lysine and has specific transporters (LdAAP3 and LdAAP7) to import these nutrients. Probes 1–15 were originated by conjugating cytotoxic quinone fragments (II and III) with amino acids (i.e. arginine and lysine) by means of an amide linkage. The toxicity of the synthesized conjugates against Leishmania extracellular (promastigotes) and intracellular (amastigotes) forms was investigated, as well their inhibition of the relevant amino acid transporters. We observed that some conjugates indeed displayed toxicity against the parasites; in particular, 7 was identified as the most potent derivative (at concentrations of 1 µg/mL and 2.5 µg/mL residual cell viability was reduced to 15% and 48% in promastigotes and amastigotes, respectively). Notably, 6, while retaining the cytotoxic activity of quinone II, displayed no toxicity against mammalian THP1 cells. Transport assays indicated that the novel conjugates inhibited transport activity of lysine, arginine and proline transporters. Furthermore, our analyses suggested that the toxic conjugates might be translocated by the transporters into the cells. The non-toxic probes that inhibited transport competed with the natural substrates for binding to the transporters without being translocated. Thus, it is likely that 6, by exploiting amino acid transporters, can selectively deliver its toxic effects to Leishmania cells. This work provides the first evidence that amino acid transporters of the human pathogen Leishmania might be modulated by small molecules, and warrants their further investigation from drug discovery and chemical biology perspectives.     

Leishmanicidal activity of stearylamine-bearing liposomes in vitro

Liposomes consisting of stearylamine (SA) and egg yolk phosphatidylcholine (PC) were studied for their cytotoxic activity against freshly transformed promastigotes and intracellular amastigotes of Leishmania donovani, the causative agent of visceral leishmaniasis. More than 99% of the parasites of strain AG83 were killed within 60 min by treatment with 22 mol% SA-PC liposomes (132 microg/ml total lipids). This was further confirmed by incubating the liposome-treated promastigotes at 22 C for 96 hr. The killing activity of the liposomes progressively decreased with lowering lipid concentration. However, weak cytotoxic activity was still detected at 6.6 microg/ml lipids. Leishmanicidal activity of the liposomes became stronger with increasing SA content but was reduced with the incorporation of cholesterol in the liposomes. A similar cytotoxic effect was observed on other Indian strains of L. donovani, for example PKDL and DD8, as well as on species such as Leishmania donovani S1, Leishmania donovani infantum, Leishmania tropica, Leishmania amazonensis, and Leishmania mexicana. However, freshly transformed promastigotes appeared to be more susceptible than the ones subcultured. The strong leishmanicidal activity of SA-PC liposomes was also demonstrated toward intracellular L. donovani amastigotes. The SA-bearing vesicles could effectively inhibit the growth and multiplication of the parasites within the macrophages. The cytolytic activity of these liposomes on leishmanial parasites and low toxicity on host macrophages may, thus, find application in the therapy of leishmaniasis.     

Arylanthranilodinitriles: a new biaryl class of antileishmanial agents

A series of anthranilodinitrile-based biaryls were synthesized and evaluated in vitro against extracellular promastigotes and intracellular amastigotes of Leishmania donovani. Among various screened compounds, a biaryl with trifluoromethyl group 5f showed 83% inhibition against promastigotes and 70% inhibition against amastigotes of L. donovani at 8 and 20microg/mL concentrations, respectively.     

Programmed cell death in the unicellular protozoan parasite Leishmania.

In the present study we have demonstrated some features characterizing programmed cell death (PCD) in the unicellular protozoan parasite Leishmania donovani, the causative agent of visceral Leishmaniasis. We report that PCD is initiated in stationary phase cultures of promastigotes and both in actively growing cultures of axenic amastigotes and promastigotes upon treatment with anti Leishmanial drugs (Pentostam and amphotericin B). However, the two cell types respond to antileishmanial drugs differently. The features of PCD in L. donovani promastigotes are nuclear condensation, nicked DNA in the nucleus, DNA ladder formation, increase in plasma membrane permeability, decrease in the mitochondrial membrane potential (DeltaPsi m) and induction of a PhiPhiLux (PPL)-cleavage activity. PCD in both stationary phase culture and upon induction by amphotericin B resulted first in the decrease of mitochondrial membrane potential followed by simultaneous change in plasma membrane permeability and induction of PPL-cleavage activity. Of the total PPL-cleavage activity, several caspase inhibitors inhibited a significant amount (21-34%). Inhibitors of cathepsin or calpain did not inhibit PPL-cleavage activity. Taken together this study demonstrates that the characteristic features of PCD exist in unicellular protozoan Leishmania donovani. The implication of PCD on the Leishmania pathogenesis is discussed.     

Anti-leishmanial activity of neolignans from Virola species and synthetic analogues

Surinamensin, a neolignan isolated from Virola surinamensis, 3,4,5-trimethoxy-8-[2',6'-dimethoxy-4'-(E)-propenylphenoxy]-phenylpropane, a neolignan isolated from Virola pavonis, and 25 of its synthetic analogues or correlated substances with ether linkages and their corresponding C-8 sulphur and nitrogen analogues, were tested for activity against Leishmania donovani amastigotes and promastigotes in vitro. Some were active against L. donovani promastigotes at 30 microM but inactive against intracellular amastigotes. The natural neolignan from V. pavonis was active against promastigotes at 100 microM. The highest selective activity was found in those compounds with sulphur bridges. The beta-ketosulfide (3,4-dimethoxy)-8-(4'-methylthiophenoxy)-propiophenone produced 42% inhibition of L. donovani amastigotes in the liver of BALB/c mice at 100 mg/kg given once daily for five consecutive days (P>0.05).     

Leishmania donovani: amastigote inhibition and mode of action of berberine

Berberine, an alkaloid from Berberis aristata Linnaeus, may be a useful drug for the treatment of visceral leishmaniasis. In both the 8-day and long-term models of Leishmania donovani infection in hamsters, it markedly diminished the parasitic load and proved to be less toxic than pentamidine. It rapidly improved the hematological picture of infected animals. Like pentamidine, it inhibited in vitro multiplication of amastigotes in macrophage culture and their transformation to promastigotes in cell free culture. Manometric studies showed that both drugs had inhibitory action on both the endogenous and the glucose-stimulated respiration of amastigotes. They inhibited incorporation of [14C]adenine, [14C]uracil, and [3H]thymidine into nucleic acids, and of [14C]leucine into the protein of amastigotes, indicating an inhibitory action on macromolecular biosynthesis. They also decreased deoxyglucose uptake. Using spectrophotometric, spectrofluorimetric, and circular dichroism techniques, berberine was found to interact in vitro with nuclear DNA from L. donovani promastigotes.     

A novel sucrose/H+ symport system and an intracellular sucrase in Leishmania donovani

The flagellated form of pathogenic parasitic protozoa Leishmania, resides in the alimentary tract of its sandfly vector, where sucrose serves as a major nutrient source. In this study we report the presence of a sucrose transport system in Leishmania donovani promastigotes. The kinetics of sucrose uptake in promastigotes are biphasic in nature with both high affinity K(m) (K(m) of ～ 75 μM) and low affinity K(m) (K(m)～ 1.38 mM) components. By contrast the virulent amastigotes take up sucrose via a low affinity process with a K(m) of 2.5mM. The transport of sucrose into promastigotes leads to rapid intracellular acidification, as indicated by changes in the fluorescence of the pH indicator 2',7'-bis-(2-carboxyethyl)-5-(6) Carboxyfluorescein (BCECF). In experiments with right side-out plasma membrane vesicles derived from L. donovani promastigotes, an artificial pH gradient was able to drive the active accumulation of sucrose. These data are consistent with the operation of a H(+)-sucrose symporter. The symporter was shown to be independent of Na(+) and to be insensitive to cytochalasin B, to the flavonoid phloretin and to the Na(+)/K(+) ATPase inhibitor ouabain. However, the protonophore carbonylcyanide P- (trifluromethoxy) phenylhydrazone (FCCP) and a number of thiol reagents caused significant inhibition of sucrose uptake. Evidence was also obtained for the presence of a stable intracellular pool of the sucrose splitting enzyme, sucrase, in promastigote stage parasites. The results are consistent with the hypothesis that L. donovani promastigotes take up sucrose via a novel H(+)-sucrose symport system and that, on entering the cell, the sucrose is hydrolysed to its component monosaccharides by an intracellular sucrase, thereby providing an energy source for the parasites.     

Species and stage specificity of Leishmania donovani antigens.

Monoclonal antibodies D2 and D13 were produced in mice using Leishmania donovani promastigote membrane fractions. To study the species and stage specificity of the antigens recognized by these antibodies, we examined amastigotes prepared in vitro and cultured promastigotes by indirect immunofluorescence with monoclonal antibodies D2 and D13. Monoclonal antibody D2 showed weak reactivity for 9 of 9 strains of L. donovani complex promastigotes and 8 of 9 amastigotes. In contrast, only 2 of 22 strains from other complexes yielded equivocal reactions. Monoclonal antibody D13, however, had much broader reactivity. D13 reacted with all the promastigotes and amastigotes of L. donovani complex isolates as well as with 10 of 22 promastigotes and 8 of 13 amastigotes from other complexes. The high degree of species specificity seen with monoclonal antibody D2 provides a rationale for further study of this antibody and its purified antigen for parasite identification and serodiagnosis of visceral leishmaniasis. The strong fluorescent signal noted with D13 and the presence of the D13 epitope on all L. donovani complex parasites supports studies on its role as an antigen in immunoprophylaxis of visceral leishmaniasis.