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.     

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.     

A soluble phosphodiesterase in Leishmania donovani negatively regulates cAMP signaling by inhibiting protein kinase A through a two way process involving catalytic as well as non-catalytic sites

Intracellular cAMP level and cAMP mediated responses are elevated when Leishmania are exposed to macrophage phagolysosome conditions (37 °C and pH 5.5). Phosphodiesterases play major role in cAMP regulation and in the present study we have cloned and characterized a 2.1 kb cytosolic isoform of phosphodiesterase from Leishmania donovani (LdPDED) which plays important role in cAMP homeostasis when the promastigotes are exposed to macrophage phagolysome conditions for converting to axenic amastigotes. Domain characterization suggested the presence of two pseudo-substrate sites similar to the ones present in the regulatory subunit of cAMP-dependent protein kinase A (PKA) and a putative PKA phosphorylation site at T⁷⁰⁸ of C-terminus of LdPDED. Deletion constructs and site directed mutagenesis revealed the ability of LdPDED to interact with L. donovani PKA catalytic subunits (LdPKAC1 and LdPKAC2) resulting in inhibition of kinase activity in one hand and increase of phosphodiesterase activity through PKA mediated phosphorylation at putative phosphorylation site on the other hand. This study therefore identifies a unique phosphodiesterase in L. donovani which appears to regulate cAMP-dependent PKA signaling through a two way process.     

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.     

Trypanothione reductase activity is prominent in metacyclic promastigotes and axenic amastigotes of Leishmania amazonesis. Evaluation of its potential as a therapeutic target

The activity of trypanothione reductase in Leishmania amazonensis was evaluated and it was demonstrated that TR is expressed in the soluble fractions of infective promastigotes and amastigotes, while non-infective promastigotes expressed the enzyme at basal levels. This data allows an association of enzyme activity and the infective capacity of the parasite. We have also previously demonstrated that amidine compounds (N, N'-diphenyl-4-methoxy-benzamidine and pentamidine) were active against this parasite. Here, experiments concerning the effect of these compounds on TR activity, showed that both compounds significantly inhibited the enzyme. However, against glutathione reductase, only pentamidine showed a significant inhibitory action, suggesting an association with the toxic effects of this drug used in the clinic for the treatment 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.     

Trypanothione Reductase Activity is Prominent in Metacyclic Promastigotes and Axenic Amastigotes of Leishmania amazonesis. Evaluation of its Potential as a Therapeutic Target

The activity of trypanothione reductase in Leishmania amazonensis was evaluated and it was demonstrated that TR is expressed in the soluble fractions of infective promastigotes and amastigotes, while non-infective promastigotes expressed the enzyme at basal levels. This data allows an association of enzyme activity and the infective capacity of the parasite. We have also previously demonstrated that amidine compounds (N, N′-diphenyl-4-methoxy-benzamidine and pentamidine) were active against this parasite. Here, experiments concerning the effect of these compounds on TR activity, showed that both compounds significantly inhibited the enzyme. However, against glutathione reductase, only pentamidine showed a significant inhibitory action, suggesting an association with the toxic effects of this drug used in the clinic for the treatment of leishmaniasis.     

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.     

Cell surface carbohydrate of Leishmania mexicana amazonensis: differences between infective and non-infective forms

The cell surface carbohydrates of Leishmania mexicana amazonensis (amastigotes and promastigotes, both infective and non-infective forms) were comparatively analyzed by agglutination assay employing 28 highly purified lectins, and by binding assay using 125I-labeled lectins. Among the D-GalNAc binding lectins, Bandeiraea simplicifolia-I, Dolichos biflorus, Phaseolus vulgaris and Glycine max were highly specific for the amastigotes, while that from Maclura aurantiaca selectively agglutinated promastigotes. The lectins from Wistaria floribunda, Phaseolus lunatus (D-GalNAc), Arachis hypogaea (D-Gal) and Triticum vulgaris (D-GlcNAc) were selective for the infective forms (both amastigotes and promastigotes), not reacting with the non-infective ones. Conversely, no parasite agglutination occurred with the L-fucose binding lectins Lotus tetragonolobus and Ulex europaeus-I. Binding studies with 125I-labeled lectins from Wistaria floribunda, Triticum vulgaris and Arachis hypogaea were performed to find whether unagglutinated non-infective promastigotes might have receptors for these lectins, in which case absence of agglutination could be due to a peculiar arrangement of the receptors. These assays essentially confirmed the selectivity, demonstrated in the agglutination assays of these lectins for the infective promastigotes.     

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.     

MAP kinase activation by mu opioid receptor involves phosphatidylinositol 3-kinase but not the cAMP/PKA pathway.

The involvement of protein kinases was studied in mu opioid receptor activation of mitogen-activated protein (MAP) kinase using cells transfected with the receptor clone. The cAMP/protein kinase A (PKA) pathway is known to be the major biochemical pathway for mu opioid receptor signaling. However, our data showed that stimulating adenylyl cyclase or activating PKA had no effect on mu receptor enhancement of MAP kinase activity, suggesting that the cAMP/PKA pathway is not involved in mediating the mu receptor activation of MAP kinase. Inhibition of phosphatidylinositol (PI) 3-kinase reduced mu receptor enhancement of MAP kinase activity, suggesting PI 3-kinase involvement. Together, these results show that cross-talk between the mu opioid receptor and the MAP kinase cascade is not mediated by the cAMP/PKA pathway, but involves PI 3-kinase.     

Leishmania infantum: stage-specific activity of pentavalent antimony related with the assay conditions

The determination of the intrinsic sensitivity of Leishmania strains to pentavalent antimonials in clinical trials, before treatment is begun, is essential in order to avoid failures and to allow alternative drugs to be chosen. A comparative study of SbV activity on promastigotes, axenic amastigote-like cells, and intracellular amastigotes of Leishmania infantum, when administered in the form of meglumine antimoniate and free, in hydrochloric solution, was performed. Results indicate that the conditions under which the promastigotes were cultured affect the IC(50) obtained, although results were homogeneous when the products were assayed on axenic-like and intracellular amastigotes. The IC(50) obtained for SbV in the form of meglumine antimoniate or in hydrochloric solution on promastigotes cultured in Schneider's medium depends on the growth rate of the culture and therefore could be regulated by modifying the fetal calf serum concentration in the medium. The pH of the culture medium strongly affected the activity of meglumine antimoniate but not that of the SbV hydrochloric solution on promastigotes cultured in Schneider's medium. This influence of pH was observed to a much lesser extent when promastigotes were cultured on M199 or RPMI media. In homogeneous culture conditions, which included the regulation of the promastigote growth rate through the heat-inactivated fetal calf serum concentration in the medium and the dilution of the meglumine antimoniate with Schneider's medium at pH 6.5, the activity of SbV, free or in the form of meglumine antimoniate, was the same in promastigotes, intracellular amastigotes, and axenic amastigote-like cells.     

Geranylgeraniol enhances testosterone production via the cAMP/protein kinase A pathway in testis-derived I-10 tumor cells

Testosterone levels in men decrease with age; this decline has been linked to various diseases and can shorten life expectancy. Geranylgeraniol (GGOH) is an isoprenoid found in plants that plays an important role in several biological processes; however, its role in steroidogenesis is unknown. Here, we report that GGOH enhances the production of testosterone and its precursor progesterone in testis-derived I-10 tumor cells. GGOH induced protein kinase A (PKA) activity and increased cAMP levels and was found to regulate cAMP/PKA signaling by activating adenylate cyclase without altering phosphodiesterase activity. GGOH also stimulated mRNA and protein levels of steroidogenic acute regulatory protein, a downstream effector in the cAMP/PKA pathway. These results demonstrate that GGOH enhances steroidogenesis in testis-derived cells by modulating cAMP/PKA signaling. Our findings have potential applications for the development of therapeutics that increase testosterone levels in aging men.     

In vitro sodium nitroprusside-mediated toxicity towards Leishmania amazonensis promastigotes and axenic amastigotes

Leishmania parasites survive despite exposure to the toxic nitrosative oxidants during phagocytosis by the host cell. In this work, the authors investigated comparatively the resistance of Leishmania amazonensis promastigotes and axenic amastigotes to a relatively strong nitrosating agent that acts as a nitric oxide (NO) donor, sodium nitroprusside (SNP). Results demonstrate that SNP is able to decrease, in vitro, the number of L. amazonensis promastigotes and axenic amastigotes in a dose-dependent maner. Promastigotes, cultured in the presence of 0.25, 0.5, and 1 mmol L(-1) SNP for 24 h showed about 75% growth inhibition, and 97-100% when the cultures were treated with >2 mmol L(-1) SNP. In contrast, when axenic amastigotes were growing in the presence of 0.25-8 mM SNP added to the culture medium, 50% was the maximum of growth inhibition observed. Treated promastigotes presented reduced motility and became round in shape further confirming the leishmanicidal activity of SNP. On the other hand, axenic amastigotes, besides being much more resistant to SNP-mediated cytotoxicity, did not show marked morphological alteration when incubated for 24 h, until 8 mM concentrations of this nitrosating agent were used. The cytotoxicity toward L. amazonensis was attenuated by reduced glutathione (GSH), supporting the view that SNP-mediated toxicity triggered multiple oxidative mechanisms, including oxidation of thiols groups and metal-independent oxidation of biomolecules to free radical intermediates.     

Centrin gene disruption impairs stage-specific basal body duplication and cell cycle progression in Leishmania

Centrin is a calcium-binding cytoskeletal protein involved in the duplication of centrosomes in higher eukaryotes. To explore the role of centrin in the protozoan parasite Leishmania, we created Leishmania deficient in the centrin gene (LdCEN). Remarkably, centrin null mutants (LdCEN(-/-)) showed selective growth arrest as axenic amastigotes but not as promastigotes. Flow cytometry analysis confirmed that the mutant axenic amastigotes have a cell cycle arrest at the G(2)/M stage. The axenic amastigotes also showed failure of basal body duplication and failure of cytokinesis resulting in multinucleated "large" cells. Increased terminal deoxy uridine triphosphate nick end labeling positivity was observed in centrin mutant axenic amastigotes compared with wild type cells, suggesting the activation of a programmed cell death pathway. Growth of LdCEN(-/-) amastigotes in infected macrophages in vitro was inhibited and also resulted in large multinucleated parasites. Normal basal body duplication and cell division in the LdCEN knockout promastigote is unique and surprising. Further, this is the first report where disruption of a centrin gene displays stage-specific/cell type-specific failure in cell division in a eukaryote. The centrin null mutant defective in amastigote growth could be useful as a vaccine candidate against leishmaniasis.     

Calcium-Dependent Increases in Protein Kinase-A Activity in Mouse Retinal Ganglion Cells Are Mediated by Multiple Adenylate Cyclases

Neurons undergo long term, activity dependent changes that are mediated by activation of second messenger cascades. In particular, calcium-dependent activation of the cyclic-AMP/Protein kinase A signaling cascade has been implicated in several developmental processes including cell survival, axonal outgrowth, and axonal refinement. The biochemical link between calcium influx and the activation of the cAMP/PKA pathway is primarily mediated through adenylate cyclases. Here, dual imaging of intracellular calcium concentration and PKA activity was used to assay the role of different classes of calcium-dependent adenylate cyclases (ACs) in the activation of the cAMP/PKA pathway in retinal ganglion cells (RGCs). Surprisingly, depolarization-induced calcium-dependent PKA transients persist in barrelless mice lacking AC1, the predominant calcium-dependent adenylate cyclase in RGCs, as well as in double knockout mice lacking both AC1 and AC8. Furthermore, in a subset of RGCs, depolarization-induced PKA transients persist during the inhibition of all transmembrane adenylate cyclases. These results are consistent with the existence of a soluble adenylate cyclase that plays a role in calcium-dependent activation of the cAMP/PKA cascade in neurons.     

Role of Epac and protein kinase A in thyrotropin-induced gene expression in primary thyrocytes

cAMP pathway activation by thyrotropin (TSH) induces differentiation and gene expression in thyrocytes. We investigated which partners of the cAMP cascade regulate gene expression modulations: protein kinase A and/or the exchange proteins directly activated by cAMP (Epac). Human primary cultured thyrocytes were analysed by microarrays after treatment with the adenylate cyclase activator forskolin, the protein kinase A (PKA) activator 6-MB-cAMP and the Epac-selective cAMP analog 8-pCPT-2'-O-Me-cAMP (007) alone or combined with 6-MB-cAMP. Profiles were compared to those of TSH. Cultures treated with the adenylate cyclase- or the PKA activator alone or the latter combined with 007 had profiles similar to those induced by TSH. mRNA profiles of 007-treated cultures were highly distinct from TSH-treated cells, suggesting that TSH-modulated gene expressions are mainly modulated by cAMP and PKA and not through Epac in cultured human thyroid cells. To investigate whether the Epac-Rap-RapGAP pathway could play a potential role in thyroid tumorigenesis, the mRNA expressions of its constituent proteins were investigated in two malignant thyroid tumor types. Modulations of this pathway suggest an increased Rap pathway activity in these cancers independent from cAMP activation.     

Leishmania mexicana: Purine metabolism in promastigotes,axenic amastigotes,and amastigotes derived from vero cells

Leishmania mexicana mexicana promastigotes, axenic amastigotes, and amastigotes derived from Vero cells were examined for de novo purine synthesis and mechanisms of purine salvage. Both promastigotes and axenic amastigotes were incapable of de novo purine synthesis, as shown by the lack of [¹⁴C]formate and [¹⁴C]glycine incorporation into purine nucleotide pools. However, the ready incorporation of [¹⁴C]hypoxanthine, [¹⁴C]adenine, and [¹⁴C]guanine suggested that purine salvage pathways were operating. In addition, a significant percentage (?60%) of the total label from these purine precursors was associated with adenylate nucleotides. Nucleotide pool levels of axenic amastigotes were consistently greater but the specific activities were less than those of promastigotes, suggesting a slower rate of purine metabolism in the axenic amastigote form. Similar results were obtained from amastigotes isolated from infected Vero cells.