A comparative study of Leishmania mexicana amastigotes and promastigotes, enzyme activities and subcellular locations

Coombs, Graham H.; Craft, John A.; Hart, David T.

doi:10.1016/0166-6851(82)90021-4

Cited by 102 publications

(52 citation statements)

References 25 publications

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“…An in vivo metabolic conversion of pentavalent antimonial [Sb(V)] into trivalent ones [Sb(III)] was suggested more than 50 years ago by Goodwin and Page (15,16). This hypothesis was supported by the high toxicity of trivalent antimony against both parasite stages of different Leishmania species (10,14,26,31,34). Recently, we and other investigators have shown that axenically grown amastigotes of Leishmania represent a powerful model to investigate drug activity on the active and dividing population of the mammalian parasite stage (7,34).…”

mentioning

confidence: 64%

Antimonial-Mediated DNA Fragmentation inLeishmania infantumAmastigotes

Sereno

Holzmüller

Mangot

et al. 2001

Antimicrob Agents Chemother

140

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The basic treatment of leishmaniasis consists in the administration of pentavalent antimonials. The mechanisms that contribute to pentavalent antimonial toxicity against the intracellular stage of the parasite (i.e., amastigote) are still unknown. In this study, the combined use of several techniques including DNA fragmentation assay and in situ and cytofluorometry terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling methods and YOPRO-1 staining allowed us to demonstrate that potassium antimonyl tartrate, an Sb(III)-containing drug, was able to induce cell death associated with DNA fragmentation in axenic amastigotes of Leishmania infantum at low concentrations (10 g/ml). This observation was in close correlation with the toxicity of Sb(III) species against axenic amastigotes (50% inhibitory concentration of 4.75 g/ml). Despite some similarities to apoptosis, nuclease activation was not a consequence of caspase-1, caspase-3, calpain, cysteine protease, or proteasome activation. Altogether, our results demonstrate that the antileishmanial toxicity of Sb(III) antimonials is associated with parasite oligonucleosomal DNA fragmentation, indicative of the occurrence of late events in the overall process of apoptosis. The elucidation of the biochemical pathways leading to cell death could allow the isolation of new therapeutic targets.Leishmaniasis is a significant cause of morbidity and mortality in several countries. A vertebrate host is infected with flagellated extracellular promastigote forms via the bite of a sand fly. Promastigotes are rapidly transformed into nonflagellated amastigotes dividing actively within the mononuclear phagocytes of the vertebrate host. The basic treatment consists in the administration of sodium stibogluconate (Pentostam), meglumine (Glucantime), pentamidine, or amphotericin B. Treatment failure, especially for kala-azar, mucosal leishmaniasis, and diffuse cutaneous leishmaniasis is becoming a common problem in many areas where leishmaniasis is endemic. Immunological, physiological, or pharmacological deficiencies in the host are possible explanations for variations in clinical response (29). But there is evidence that inherent lack of susceptibility and (or) the development of resistance can also contribute to parasite unresponsiveness to drugs (13,18,23,28,39,40). The mode of action of pentavalent antimonials remains poorly understood (3, 4, 5). An in vivo metabolic conversion of pentavalent antimonial [Sb(V)] into trivalent ones [Sb(III)] was suggested more than 50 years ago by Goodwin and Page (15,16). This hypothesis was supported by the high toxicity of trivalent antimony against both parasite stages of different Leishmania species (10,14,26,31,34). Recently, we and other investigators have shown that axenically grown amastigotes of Leishmania represent a powerful model to investigate drug activity on the active and dividing population of the mammalian parasite stage (7, 34). We have shown that potassium antimonyl tartrate [containing Sb(III)] was generally...

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mentioning

confidence: 64%

Antimonial-Mediated DNA Fragmentation inLeishmania infantumAmastigotes

Sereno

Holzmüller

Mangot

et al. 2001

Antimicrob Agents Chemother

140

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“…Similarly, glycerol generated in the macrophage lysosomes from the degradation of triacylglycerols could serve as a glucogenic precursor for amastigotes. Because Leishmania parasites express aquaglyceroporins (48) and putative glycerol transporters (49) as well as the enzymes glycerol kinase and glycerol 3-phosphate dehydrogenase (35,41,50), they can import glycerol and convert it to dihydroxyacetone phosphate for entry into the gluconeogenic pathway. On the other hand, amastigotes up-regulate ␤-oxidation of fatty acids, a pathway that produces two carbon substrates, such as acetyl-CoA, to satisfy energy requirements (35).…”

Section: Discussionmentioning

confidence: 99%

Metabolic Changes in Glucose Transporter-deficient Leishmania mexicana and Parasite Virulence

Rodriguez‐Contreras

Landfear

2006

Journal of Biological Chemistry

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Leishmania mexicana are parasitic protozoa that express a variety of glycoconjugates that play important roles in their biology as well as the storage carbohydrate ␤-mannan, which is an essential virulence factor for survival of intracellular amastigote forms in the mammalian host. Glucose transporter null mutants, which are viable as insect form promastigotes but not as amastigotes, do not take up glucose and other hexoses but are still able to synthesize these glycoconjugates and ␤-mannan, although at reduced levels. Synthesis of these carbohydratecontaining macromolecules could be accounted for by incorporation of non-carbohydrate precursors into carbohydrates by gluconeogenesis. However, the significantly reduced level of the virulence factor ␤-mannan in the glucose transporter null mutants compared with wild-type parasites may contribute to the non-viability of these null mutants in the disease-causing amastigote stage of the life cycle.Leishmaniae are pathogenic protozoa of the order Kinetoplastida that cause important diseases in humans and other vertebrates (1). There are two principal stages in the parasite life cycle, (i) extracellular flagellated promastigotes that live within the alimentary tract of the sand fly vector and (ii) nonflagellated amastigotes that reside within the acidified phagolysosomal vesicles of vertebrate host macrophages. Both life cycle stages of Leishmania mexicana can be cultivated in vitro (2) facilitating biochemical and genetic studies. Uptake and metabolism of glucose in Leishmania species has been of considerable interest, as this sugar provides a major source of carbon and energy to these parasites (3). In L. mexicana, glucose catabolism is 10 -20-fold higher in the promastigotes compared with amastigotes (4). Furthermore, amastigotes transport glucose at a much lower rate than promastigotes and derive metabolic energy primarily from fatty acid oxidation (4, 5). The higher rates of glucose uptake and utilization by promastigotes in relation to amastigotes is consistent with the high concentrations of sugars that promastigotes experience from the plant sap ingested by the sand fly vector (6) compared with the apparently sugar-limited environment that amastigotes experience (7) in the macrophage phagolysosome.The L. mexicana genome encompasses a cluster of three glucose transporter genes, LmGT1, LmGT2, and LmGT3, which encode closely related isoforms (8). A L. mexicana glucose transporter "knock-out" line (⌬lmgt) (9), in which the entire cluster of glucose transporter genes was eliminated by targeted gene replacement, exhibited no detectable glucose transport activity. The ⌬lmgt promastigotes were able to grow, although at a reduced rate compared with that of wild-type cells. In contrast, ⌬lmgt cells exhibited dramatically reduced viability inside macrophages compared with wild-type parasites and were unable to grow as axenic amastigotes, demonstrating that glucose transporters are essential for amastigote viability. Furthermore, axenically cultured wild-type amastigotes re...

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“…Promastigotes were harvested by centrifugation at 5000 g for 5 min, then washed twice with PBS and resuspended in 100 mM Tris\HCl, pH 7.5, containing 1 mM EDTA and 250 mM sucrose. To ascertain whether the enzyme was particulate or soluble, parasites were lysed by three cycles of freezing (k170 mC) and thawing (25 mC) and centrifuged (12 000 g av for 10 min) to yield particulate ' P12 ' and soluble fractions [18]. Parasite breakage was assessed by phase-contrast microscopy.…”

Section: Parasite Lysis and Fractionationmentioning

confidence: 99%

Characterization of prenylated protein methyltransferase in Leishmania

Hasne

Lawrence

1999

Biochemical Journal

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Prenylated protein methyltransferase, an enzyme involved in the post-translational modification of many signalling proteins, has been characterized in a parasitic flagellated protozoan, Leishmania dono ani. The activity of this enzyme was monitored by the methylation of an artificial substrate, an S-prenylated cysteine analogue, with S-adenosyl--[methyl-$H]methionine as methyl donor. More than 85 % of the methyltransferase activity was associated with membranes. The enzyme methylates N-acetyl- Strans,trans-farnesyl--cysteine and N-acetyl-S-all-trans-geranylgeranyl--cysteine, but N-acetyl-S-trans,trans-geranyl--cysteine only very weakly. In contrast with the enzyme from mammals, the leishmanial enzyme had a greater affinity for the farnesylated substrate than for the geranylgeranylated one. Activity in itro

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A comparative study of Leishmania mexicana amastigotes and promastigotes, enzyme activities and subcellular locations

Cited by 102 publications

References 25 publications

Antimonial-Mediated DNA Fragmentation inLeishmania infantumAmastigotes

Antimonial-Mediated DNA Fragmentation inLeishmania infantumAmastigotes

Metabolic Changes in Glucose Transporter-deficient Leishmania mexicana and Parasite Virulence

Characterization of prenylated protein methyltransferase in Leishmania

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