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.     

Leishmania major LmACR2 is a pentavalent antimony reductase that confers sensitivity to the drug pentostam

Arsenicals and antimonials are first line drugs for the treatment of trypanosomal and leishmanial diseases. To create the active form of the drug, Sb(V) must be reduced to Sb(III). Because arsenic and antimony are related metalloids, and arsenical resistant Leishmania strains are frequently cross-resistant to antimonials, we considered the possibility that Sb(V) is reduced by a leishmanial As(V) reductase. The sequence for the arsenate reductase of Saccharomyces cerevisiae, ScAcr2p, was used to clone the gene for a homologue, LmACR2, from Leishmania major. LmACR2 was able to complement the arsenate-sensitive phenotype of an arsC deletion strain of Escherichia coli or an ScACR2 deletion strain of Saccharomyces cerevisiae. Transfection of Leishmania infantum with LmACR2 augmented Pentostam sensitivity in intracellular amastigotes. LmACR2 was purified and shown to reduce both As(V) and Sb(V). This is the first report of an enzyme that confers Pentostam sensitivity in intracellular amastigotes of Leishmania. We propose that LmACR2 is responsible for reduction of the pentavalent antimony in Pentostam to the active trivalent form of the drug in Leishmania.     

Developmental regulation of proline transport in Leishmania donovani

Leishmania donovani are the causative agents of kala azar in humans. These organisms cycle between the proline-rich environment of the sand fly vector (extracellular promastigotes) and the sugar-rich condition in the mammalian host (intracellular amastigotes). Parasites have adapted to these extreme changes in proline concentrations: promastigotes utilize proline as a carbon source, whereas amastigotes utilize sugars and fatty acids. Previous studies have suggested that promastigotes and amastigotes express distinct proline transporters. However, the information available on these transporters is limited. In this work, proline transport was investigated in axenic L. donovani cultures. Three transport systems were identified: cation-dependent and -independent proline transporters in promastigotes (systems A and B, respectively) and a single cation-independent transporter in amastigotes (system C). Systems A and C have broad specificity to almost all amino acids and obtain optimum activity at acidic pH ranges (pH 6 and 5, respectively). System B is more specific to proline, as it is inhibited by only five amino acids. Temperature response analyses indicated that the transporters of both promastigotes and amastigotes perform best at 37 degrees C. The activity of system A during parasite differentiation was assessed. The transport activity of system A disappeared 3 days after promastigotes were induced to differentiate into amastigotes. In these cells, elevated temperature and acidic pH each suppressed the activity of system A. When amastigotes were induced to differentiate back into promastigotes, system A resumed its activity 24 h after differentiation was initiated. In conclusion, L. donovani obtain proline transport systems that are stage specific, regulated by both pH and temperature. This paper constitutes the first investigation of amino acid transport in axenic L. donovani.     

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).     

Imaging host cell-Leishmania interaction dynamics implicates parasite motility, lysosome recruitment, and host cell wounding in the infection process

Leishmania donovani causes human visceral leishmaniasis. The parasite infectious cycle comprises extracellular flagellated promastigotes that proliferate inside the insect vector, and intracellular nonmotile amastigotes that multiply within infected host cells. Using primary macrophages infected with virulent metacyclic promastigotes and high spatiotemporal resolution microscopy, we dissect the dynamics of the early infection process. We find that motile promastigotes enter macrophages in a polarized manner through their flagellar tip and are engulfed into host lysosomal compartments. Persistent intracellular flagellar activity leads to reorientation of the parasite flagellum toward the host cell periphery and results in oscillatory parasite movement. The latter is associated with local lysosomal exocytosis and host cell plasma membrane wounding. These findings implicate lysosome recruitment followed by lysosome exocytosis, consistent with parasite-driven host cell injury, as key cellular events in Leishmania host cell infection. This work highlights the role of promastigote polarity and motility during parasite entry.     

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.     

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.     

Dual action of antimonial drugs on thiol redox metabolism in the human pathogen Leishmania donovani

Despite extensive use of antimonial compounds in the treatment of leishmaniasis, their mode of action remains uncertain. Here we show that trivalent antimony (Sb(III)) interferes with trypanothione metabolism in drug-sensitive Leishmania parasites by two inherently distinct mechanisms. First, Sb(III) decreases thiol buffering capacity by inducing rapid efflux of intracellular trypanothione and glutathione in approximately equimolar amounts. Second, Sb(III) inhibits trypanothione reductase in intact cells resulting in accumulation of the disulfide forms of trypanothione and glutathione. These two mechanisms combine to profoundly compromise the thiol redox potential in both amastigote and promastigote stages of the life cycle. Furthermore, we demonstrate that sodium stibogluconate, a pentavalent antimonial used clinically for the treatment for leishmaniasis, induces similar effects on thiol redox metabolism in axenically cultured amastigotes. These observations suggest ways in which current antimony therapies could be improved, overcoming the growing problem of antimony resistance.     

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.     

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.     

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.     

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.     

Leishmania mexicana: enzyme activities of amastigotes and promastigotes and their inhibition by antimonials and arsenicals

A major difference between the metabolism of Leishmania species amastigotes and cultured promastigotes was found in the area of CO2 fixation and phosphoenolpyruvate metabolism. Malate dehydrogenase (EC 1.1.1.37) and phosphoenolpyruvate carboxykinase (EC 4.1.1.49) were at much higher activities in amastigotes than promastigotes of both L. m. mexicana and L. donovani, whereas the reverse was true of pyruvate kinase (EC 2.7.1.40). Pyruvate carboxylase (EC 6.4.1.1) and malic enzyme (carboxylating) (EC 1.1.1.40) could not be detected in L. m. mexicana amastigotes. Promastigotes of L. m. mexicana had a high NAD-linked glutamate dehydrogenase activity in comparison to amastigotes, whereas NADP-linked glutamate dehydrogenase activity was detected only in amastigotes. Leishmania m. mexicana culture promastigotes were killed in vitro by the trivalent antimonial Triostam (LD50, 20 micrograms/ml) and the trivalent arsenical melarsen oxide (LD50, 20 micrograms/ml), but they were unaffected by Pentostam. Neither antimonial drug significantly inhibited leishmanial hexokinase (EC 2.7.1.2), phosphofructokinase (EC 2.7.1.11), pyruvate kinase, malate dehydrogenase or phosphoenolpyruvate carboxykinase, whereas melarsen oxide was a potent inhibitor of all the enzymes tested except phosphoenolpyruvate carboxykinase.     

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.     

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.     

Differential survival of Leishmania donovani amastigotes in human monocytes

Leishmania donovani is an important intracellular protozoal pathogen of man; it is found solely within macrophages in its amastigote stage in humans, and exists in its extracellular, flagellated promastigote stage in the sandfly, its arthropod vector. To determine if either stage of L. donovani was capable of surviving within monocytes--the oxidatively active precursors of tissue macrophages--interactions of the parasite with human monocytes were studied in vitro. Amastigotes and promastigotes were ingested to a comparable degree by monocytes; whereas 79% of promastigotes were killed within 48 hr, however, amastigotes survived and multiplied threefold over 5 days. Promastigotes, which have been shown to be sensitive to hydrogen peroxide-peroxidase-halide microbicidal mechanisms, elicited a phagocytic oxidative burst that was 49% of the response to serum-opsonized zymosan, as assessed by luminol-enhanced chemiluminescence. NBT was reduced to formazan in 71% of monocytes exposed to promastigotes. The death of promastigotes within monocytes could be attributed at least in part to oxidative microbicidal mechanisms because there was no significant decrease in the number of cell-associated parasites in monocytes from donors with chronic granulomatous disease of childhood. In contrast to promastigotes, amastigotes survived within monocytes, despite eliciting an oxidative response that was 27% of the response produced by serum-opsonized zymosan; this response was not significantly different from that produced by promastigotes. In a phagocyte-free system, amastigotes were found to be sevenfold more resistant than were promastigotes to the lethal effects of hydrogen peroxide. The survival of L. donovani in human monocytes is thus dependent on the parasite stage; promastigotes are ingested, they elicit an oxidative burst, and the majority are killed by oxidative microbicidal mechanisms, whereas amastigotes are ingested and survive to parasitize human monocytes successfully, despite eliciting a phagocytic oxidative burst.     

A novel signal sequence negative multimeric glycosomal protein required for cell cycle progression of Leishmania donovani parasites

Expression of the intracellular form amastigote specific genes in the Leishmania donovani parasite plays a major role in parasite replication in the macrophage. In the current work, we have characterized a novel hypothetical gene, Ld30b that is specifically transcribed in the intracellular stage of the parasite. The recombinant Ld30b protein exists as a pentamer in solution as identified by native-PAGE and size exclusion gel chromatography. Structural analysis using circular dichroism and molecular modeling indicate that Ld30b belongs to family of cAMP-dependent protein kinase type I-alpha regulatory subunit. Co-localization immunofluorescence microscopy and western blot analyses (using anti-Ld30b antibody and anti-hypoxanthine-guanine phosphoribosyl transferase, a glycosome marker) on the isolated parasite glycosome organelle fractions show that Ld30b is localized in glycosome, though lacked a glycosome targeting PTS1/2 signal in the protein sequence. Episomal expression of Ld30b in the parasite caused the arrest of promastigotes and amastigotes growth in vitro. Cell cycle analysis using flow cytometry indicates that these parasites are arrested in ‘sub G0/G1’ phase of the cell cycle. Single allele knockout of Ld30b in the parasite similarly attenuated its growth by accumulation of cells in the S phase of cell cycle, thus confirming the probable importance of appropriate level of protein in the cells. Studying such intracellular stage expressing genes might unravel novel regulatory pathways for the development of drugs or vaccine candidates against leishmaniasis.     

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.     

Anti-parasite activity of nucleoside analogues: the metabolism of carbocyclic inosine in promastigotes of Leishmania tropica and Leishmania donovani and its activity against amastigotes of Leishmania donovani in vitro

Carbocyclic inosine is a potent inhibitor for the growth of the promastigote form of Leishmania tropica and Leishmania donovani. In culture, the EC50 values of carbocyclic inosine are 8.3 X 10(-8) and 1.3 X 10(-7) M for the promastigotes of L. tropica and L. donovani, respectively. On the other hand, it is less toxic towards mouse mammary tumor FM3A cells: the EC50 value is 2.7 X 10(-4)M. Carbocyclic inosine is metabolized by Leishmania promastigotes to give carbocyclic adenosine-5'-triphosphate(aristeromycin-5'-triphosphate) and carbocyclic guanosine-5'-triphosphate. This metabolic conversion provides a mechanism for the parasite-selective toxicity of carbocyclic inosine. Carbocyclic inosine was found to be active against L. donovani amastigotes in an in vivo-like cultivation in vitro.