Leishmania mexicana: purine-metabolizing enzymes of amastigotes and promastigotes

Cultured promastigote and isolated amastigote forms of Leishmania mexicana mexicana have been surveyed for the presence of enzymes involved in purine metabolism. Quantitative but not qualitative differences between the enzymes of two forms were discovered. There were found to be significant differences between the enzyme content of L. m. mexicana and that reported for L. donovani. Extracts of both parasite forms of L. m. mexicana were found to have higher levels of adenine deaminase (EC 3.5.4.2) and guanine deaminase (EC 3.5.4.3) than adenosine deaminase (EC 3.5.4.4). There appeared to be two distinct nucleosidases (EC 3.2.2.1), one active on nucleosides, the other on deoxynucleosides. Phosphorylase (EC 2.4.2.1) could be detected only in the catabolic direction. Nucleotidases were present, but were more active on 3' (EC 3.1.3.6)- than 5' (EC 3.1.3.5)-nucleotides. Phosphoribosyltransferase (EC 2.4.2.7,.8 and .22) and nucleoside kinase (EC 2.7.1.20) activities were detected in both forms. Nucleotide-interconverting enzymes were found to be present, with IMP dehydrogenase (EC 1.2.1.14) being the most active. Cell fractionation experiments revealed that, in the promastigote, enzyme separation within the parasite may play an important part in regulating cellular purine metabolism.     

Effects of hyperbaric oxygen on Leishmania amazonensis promastigotes and amastigotes

In the present study, we evaluated the effects of hyperbaric oxygen (HBO) exposure in both Leishmania amazonensis life stages (promastigotes and amastigotes) and on macrophage cultures infected with the parasite. HBO treatment protocols, which can be tolerated by humans and animals, induced irreversible metabolic damage and affected parasite morphology, growth and ability to transform. The observation that the antioxidant N-acetylcysteine (NAC) prevents some of these deleterious effects indicated an involvement of oxidative stress during parasite HBO exposure. In addition, HBO exposed L. amazonensis-infected macrophage cultures showed reduction of the percentage of infected cells and of the number of intracellular parasites per cell. Thus, the demonstration that HBO, a therapy used in the management of different diseases, is toxic for both L. amazonensis life stages and can alter macrophage susceptibility to the infection encourages further studies of this therapy in animal models of Leishmania infection.     

Proteophosphoglycans of Leishmania

Proteophosphoglycans are an expanding family of highly glycosylated Leishmania proteins with many unusual and some unique structural features. The novel protein-glycan linkage in proteophosphoglycans - phosphoglycosylation of Ser by lipophosphoglycan-like structures - emerges as a major form of protein glycosylation in Leishmania. Here, Thomas Ilg reviews the chemical structure, the ultrastructure, the genes and the potential functions of different members of this novel family of parasite glycoproteins.     

Leishmania mexicana: subcellular distribution of enzymes in amastigotes and promastigotes

Glycosomes and mitochondrial vesicles from cultured promastigotes of Leishmania mexicana mexicana have been separated using isopycnic centrifugation on linear sucrose gradients. Hexokinase (EC 2.7.1.2), glucose phosphate isomerase (EC 5.3.1.9), phosphofructokinase (EC 2.7.1.11), glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12), and phosphoenolpyruvate carboxykinase (EC 4.1.1.49) were recovered largely in association with glycosomes (density; 1.215 g/ml). Phosphoglycerate kinase (EC 2.7.2.3) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49) had some small glycosomal activity, but were mostly recovered in the soluble fractions. Malate dehydrogenase (EC 1.1.1.37) showed a broad peak corresponding to that of the mitochondrial marker oligomycin-sensitive ATPase (EC 3.6.1.4) (density; 1.190 g/ml). Glutamate dehydrogenase (EC 1.4.1.3) and alanine aminotransferase (EC 2.6.1.2) both showed small mitochondrial peaks, but most of the activities were recovered elsewhere on the gradient and in the soluble fractions. The subcellular location of enzymes in L.m. mexicana amastigotes was investigated by following the release of soluble enzymes from digitonin-treated amastigotes. This revealed distinct cytosolic, mitochondrial, and glycosomal compartments. The findings give an insight into the organization and control of L.m. mexicana promastigote and amastigote energy metabolism.     

Characterization of developmentally-regulated nucleases in promastigotes and amastigotes of Leishmania mexicana

Abstract Nitrate induced the expression of a membrane-bound nitrate reductase in the strict anaerobe Geobacter metallireducens . A fraction from a DEAE cellulose column which showed nitrate reductase activity contained polypeptides of M _r, 18, 36 and 43 K and three c type cytochromes ( M _r 28, 46 and 68 K). Western and Southern blot analysis revealed no homology between the nitrate reductase from G. metallireducens and the nitrate reductases from respiratory ( Escherichia coli ) and denitrifying bacteria ( Pseudomonas stutzeri, Pseudomonas aeruginosa ) which were shown to be related. These data, in addition to this organism's inability to use fumarate or formate, suggest that its nitrate reductase is novel.     

Purification and characterization of proteolytic enzymes of Leishmania mexicana mexicana amastigotes and promastigotes

Leishmania mexicana mexicana amastigote and promastigote soluble proteinases were purified using gel filtration and ion exchange chromatography. For the amastigotes, two main proteinase activity peaks were separated with both methods. These accounted for approximately 10% and 90% of the total activity. Characterization of the two activities for substrate specificity and sensitivity to inhibitors indicated that the major peak from both column methods contained enzymes with the characteristics of cysteine proteinases. SDS-polyacrylamide gel electrophoresis of the enzyme from the major peak purified by gel filtration revealed one polypeptide with a molecular weight in the region of 31 000. In contrast, the activity of the minor peak eluted from the columns was of higher molecular weight (67 000) and was similar to metalloproteinases. Purification of the soluble proteinases in the promastigote of L. m. mexicana produced only one activity peak from both column techniques. This activity (mol. wt 67 000) corresponded to the high molecular weight proteinase of the amastigote. The purified proteinases were active on 4-nitroanilide and 7-amino-4-methylcoumarin derivatives of various small peptides. The high molecular weight proteinases of both amastigotes and promastigotes were similarly active against most of the peptides, suggesting a low specificity of the enzymes. In contrast, the low molecular weight amastigote proteinases were particularly active against two of the substrates, namely BZ-Pro-Phe-Arg-Nan and Z-Phe-Arg-MCA. These results indicate that a highly active, substrate-specific, soluble proteinase, with characteristics of a cysteine proteinase, is produced upon transformation of the L. m. mexicana promastigote to amastigote. The discovery and characterization of this enzyme offers opportunities for the development of new antileishmanial agents.     

Nuphar lutea: In vitro anti-leishmanial activity against Leishmania major promastigotes and amastigotes

Several anti-leishmanial drugs of choice are of plant origin. Many of the available drugs against the disease are toxic and in certain cases parasite drug resistance is developed. The development of new compounds is urgently required.Aims of the studyTo determine the leishmanicidal activity of the Nuphar lutea plant extract against Leishmania major in vitro.Materials and methodsThe leishmanicidal activity of methanolic plant extract against L. major free living promastigotes and intracellular amastigotes was evaluated, using microscopic examinations and the enzymatic XTT assay.ResultsMethanolic extract of N. lutea was highly effective against both Leishmania promastigotes and L. amastigotes (IC₅₀=2±0.12 μg/ml; ID₅₀=0.65±0.023 μg/ml; LD₅₀=2.1±0.096 μg/ml, STI=3.23). The extract at 1.25 μg/ml totally eliminated the intracellular parasites within 3 days of treatment. Also, a synergistic anti-leishmanial activity was demonstrated with N. lutea extract combined with the anti-leishmanial drug – paromomycin. The partially purified N. lutea active component was found to be a thermo-stable alkaloid(s) with no electrical charge and is resistant to boiling and to methanol, dichloromethane and xylene treatment.ConclusionsThe present study suggests that N. lutea might be a potential source of anti-leishmanial compounds.     

Leishmania donovani: a chemically defined medium suitable for cultivation and cloning of promastigotes and transformation of amastigotes to to promastigotes

A chemically defined medium using commercially available alpha-MEM supplemented with hemin, HEPES, L-glutamine, D-glucose, folic acid, D-biotin and adenine supports the luxuriant growth and propagation of Leishmania donovani promastigotes. A peak parasite population of about 7.0 x 10(7)/ml at stationary phase and a population doubling time of 11.4 h for high-subpassage promastigotes were obtained. The medium was suitable for transformation of isolated amastigotes from infected hamster spleen. Promastigotes could be detected by culturing kala-azar patients' bone-marrow aspirate or spleen puncture material in this medium. Four out of six freshly transformed isolates gradually adapted and grew well in this medium. Macroscopic colonies appeared on agar plates prepared with the medium within 16-20 days after inoculation. The cloning efficiency was increased about five-fold by glycerol supplementation.     

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.     

Effect of amidine derivatives on nitric oxide production by Leishmania amazonensis promastigotes and axenic amastigotes.

The effects of pentamidine isethionate (reference drug) and N,N'-diphenyl-4-methoxy-benzamidine (test compound) on NO. production by Leishmania amazonensis promastigotes and axenic amastigotes were investigated by measuring nitrite, a by-product of nitric oxide released into culture supernatants. The NO. production by infective promastigotes was inhibited by OCH(3)-amidine in about 23.53% and by pentamidine in only 3.78%. In axenic amastigotes, the inhibition of NO. production by OCH(3)-amidine was significantly higher (52.94%; p=0.01) than that by pentamidine, which inhibited this radical production nonsignificantly (25.29%; p=0.1). The mechanism of amidine derivatives, as an antimicrobial agent, is unknown. However, other amidines, such as a diamidine (pentamidine), contain chemical structures shared by the guanidino group of the nitric oxide synthase substrate L-arginine, suggesting the possibility of an interaction with this enzyme or electronic factors (substituent constant) that alter physical and chemical properties significant for biological activity.     

Ca2+ signaling in the transformation of promastigotes to axenic amastigotes of Leishmania donovani

Three acidic phospholipases A₂ from Indian cobra (Naja naja naja) venom inhibited platelet aggregation in platelet rich plasma induced separately by ADP, collagen and epinephrine with different potencies. The order of inhibition was epinephrine > collagen > ADP. They did not inhibit platelet aggregation induced by arachidonic acid (10 M). The inhibition was dependent on concentration of the protein and the time of incubation of the phospholipases A₂ with platelet rich plasma. Parabromophenacyl bromide modified PLA₂ enzymes lost their enzymatic activity as well as platelet aggregation inhibition activity suggesting the involvement of catalytic function in platelet aggregation inhibitory activity.     

Axenic culture of Leishmania amastigotes

One of the future goals in Leishmania research will be to reproduce the entire life cycle oxenically, in vitro. In this article, Paul Bates reviews recent progress in the axenic culture of amastigotes and addresses some of the remaining problems associated with culture methods for both amostigote and promostigote forms.     

Proteomic analysis of antigens from Leishmania infantum promastigotes

Leishmaniasis is a zoonotic disease caused by the species of the genus Leishmania, flagellated protozoa that multiply inside mammalian macrophages and are transmitted by the bite of the sandfly. The disease is widespread and due to the lack of fully effective treatment and vaccination the search for new drugs and immune targets is needed. Proteomics seems to be a suitable strategy because the annotated sequenced genome of L. major is available. Here, we present a high-resolution proteome for L. infantum promastigotes comprising of around 700 spots. Western blot with rabbit hyperimmune serum raised against L. infantum promastiogote extracts and further analysis by MALDI-TOF and MALDI-TOF/TOF MS allowed the identification of various relevant functional antigenic proteins. Major antigenic proteins were identified as propionil carboxilasa, ATPase beta subunit, transketolase, proteasome subunit, succinyl-diaminopimelate desuccinylase, a probable tubulin alpha chain, the full-size heat shock protein 70, and several proteins of unknown function. In addition, one enzyme from the ergosterol biosynthesis pathway (adrenodoxin reductase) and the structural paraflagellar rod protein 3 (PAR3) were found among non-antigenic proteins. This study corroborates the usefulness of proteomics in identifying new proteins with crucial biological functions in Leishmania parasites.     

Lipids of Leishmania promastigotes.

A chromatographic analysis of lipids of cultured promastigotes of Leishmania donovani, L. braziliensis, L. mexicana, L. tropica, L. enriettii, L. hertigi, L. adleri, and L. tarentolae showed that total lipids were 2--15% of dry wt, and neutral and polar lipids were 14--55% and 45--86% of total lipids. Major lipid classes were as follows: sterol ester, triacylglycerol, sterol, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylcholine. Predominant fatty acids were 18:2 (n - 6) greater than 18:3 (n - 3) greater than 18:1 (n - 9) greater than 18:0 greater than 22:6 (n - 3) greater than 22:5 (n - 6) greater than 16:0 greater than 14:0 greater than 18:4 (n - 3) greater than 20:3 (n - 3). Some remarkable distributions of fatty acids among the phospholipid fractions were observed, as follows: diphosphatidylglycerol 18--33% 22:6 (n - 3); phosphatidylinositol 31--68% 18:1 (n - 9); phosphatidylcholine 13--41% 18:3 (n - 3). Alk-l-enyldiacyl glycerols, and alk-l-enylacyl and alkylacyl forms of phosphatidylethanolamine and of phosphatidylinositol were found, and their glyceryl ethers and fatty adehydes analyzed. Notable in the phosphatidylethanolamine of some leishmanias was a cyclopropane fatty acid (4--11%), identified as cis-9,10-methylene octadecanoic acid by chromatographic, and by mass and proton resonance spectrometric analyses. The comparative biochemistry of the cyclopropane fatty acid, characteristic of many prokaryotes, and of alpha-linolenic acid, characteristic of photosynthetic plants, are commented upon.     

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.     

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.