Leishmania mexicana amazonensis: plasma membrane adenine nucleotide translocator and chemotaxis

Leishmania cannot synthesize purines de novo and rely on their host to furnish these compounds. To accomplish this, they possess multiple purine nucleoside and nucleobase transporters. Subcellular fractionation, immunohistochemical localization with anti-adenine nucleotide translocator (ANT) antibodies and surface biotinylation show that the mitochondrial ANT is also present in the plasma membrane of both promastigotes and amastigotes. Leishmania, however, do not appear to rely on this transporter to supplement their purine or energy requirements via preformed ATP from its host. Rather, Leishmania appear to use the plasma membrane ANT as part of a chemotaxis response. ATP is a chemorepellant for Leishmania and cells treated with atractyloside, an inhibitor of ANT, no longer exhibit negative chemotaxis for this compound.     

Inhibition of growth of Leishmania mexicana mexicana by Leishmania mexicana amazonensis during "in vitro" co-cultivation

Inhibition of one Leishmania subspecies by exometabolites of another subspecies, a phenomenon not previously reported, is suggested by our recent observations in cell cloning experiments with Leishmania mexicana mexicana and Leishmania mexicana amazonensis. Clones were identified using the technique of schizodeme analysis. The phenomenon observed is clearly relevant to studies of parasite isolation, leishmanial metabolism, cross-immunity and chemotherapy.     

The surface free energy of Leishmania mexicana amazonensis.

Surface charge of Leishmania mexicana amazonensis was investigated by direct zeta-potential determination and ultrastructural cytochemistry, and its surface tension was studied by measurements of the advancing contact angle formed by the parasite monolayers with drops of liquids of different polarities. Both virulent and avirulent promastigotes exhibited negatively charged surfaces with a zeta-potential of about -15 mV. Treatment of these cells with trypsin, alkaline phosphatase, or phospholipase C rendered their surfaces less negatively charged, whereas neuraminidase did not alter the parasite negativeness. Cytochemically, we could observe a reduction in the cationized ferritin binding after the parasite treatment with each of the former enzymes, but not with neuraminidase. The surface free energy of parasites was calculated by taken to account the London dispersion, the Keeson dipole-dipole, and the Debye dipole-induced forces, as well as the surface polarity of the parasites and their zeta-potentials, by considering their adhesion to polystyrene surfaces. The delta G values of -6.4 and -18.1 mJ.m-2 were obtained for avirulent and virulent promstigotes, respectively.     

Heat-shock protein 83 of Leishmania mexicana amazonensis is an abundant cytoplasmic protein with a tandemly repeated genomic arrangement

The 83-kDa heat-shock protein HSP83 is a highly abundant protein in Leishmania amastigotes and promastigotes exposed to elevated temperature. Antibodies against this protein were obtained by immunization with a synthetic peptide derived from a conserved region. These antibodies recognized both the denatured and the native form of the molecule and were used for immunofluorescence analysis. These experiments, together with analysis by cell fractionation, show that HSP83 is distributed in the cytoplasm of Leishmania parasites. The gene for HSP83 in Leishmania mexicana amazonensis has been cloned from a genomic library, and molecular characterization shows it is present in several copies of 4-kb repeats arranged in tandem.     

Granulocyte-macrophage colony-stimulating factor is a growth-factor for promastigotes of Leishmania mexicana amazonensis

In this paper we show that murine lung conditioned medium (LCM) displays, in addition to its already described colony-stimulating activity on bone marrow cells, a potent growth-stimulating activity on promastigotes of Leishmania mexicana amazonesis. Immunoprecipitation of LCM with an antibody specific for murine granulocyte-macrophage colony stimulating factor (GM-CSF) abrogates both activities, indicating that the leishmanial growth-promoting activity is due to the presence of GM-CSF on LCM. Furthermore, recombinant GM-CSF (rGM-CSF) added to the culture medium or to the immunoprecipitated LCM is able to respectively induce or to partially recover the growth-promoting activity of the LCM. Sequential in vitro passages of the parasite induces a progressive loss of sensitivity to the growth-factor. Parasite forms recently collected from lesions are significantly more responsive to the growth-factor than forms already adapted to grow in culture. Since it has been shown that several different microorganisms display receptors for vertebrate-like hormones and that GM-CSF is able to enhance a cutaneous leishmanial lesion, our results permit us to raise the hypothesis that a direct interaction between a host-derived hormone and a pathogenic microorganism can be of importance in defining the fate of an infection. The fact that GM-CSF is produced by cells that actively participate in a leishmanial infection (T-lymphocytes and macrophages) reinforces our hypothesis.     

Granulocyte-Macrophage Colony-Stimulating Factor is a Growth-Factor for Promastigotes of Leishmania mexicana amazonensis

ABSTRACT. In this paper we show that murine lung conditioned medium (LCM) displays, in addition to its already described colony-stimulating activity on bone marrow cells, a potent growth-stimulating activity on promastigotes of Leishmania mexicana amazonensis . Immunoprecipitation of LCM with an antibody specific for murine granulocyte-macrophage colony stimulating factor (GM-CSF) abrogates both activities, indicating that the leishmanial growth-promoting activity is due to the presence of GM-CSF on LCM. Furthermore, recombinant GM-CSF (rGM-CSF) added to the culture medium or to the immunoprecipitated LCM is able to respectively induce or to partially recover the growth-promoting activity of the LCM. Sequential in vitro passages of the parasite induces a progressive loss of sensitivity to the growth-factor. Parasite forms recently collected from lesions are significantly more responsive to the growth-factor than forms already adapted to grow in culture. Since it has been shown that several different microorganisms display receptors for vertebrate-like hormones and that GM-CSF is able to enhance a cutaneous leishmanial lesion, our results permit us to raise the hypothesis that a direct interaction between a host-derived hormone and a pathogenic microorganism can be of importance in defining the fate of an infection. The fact that GM-CSF is produced by cells that actively participate in a leishmanial infection (T-lymphocytes and macrophages) reinforces our hypothesis.     

Characterization of the species- and stage-specificity of two monoclonal antibodies against Leishmania amazonensis

Leishmania metacyclogenesis is associated with changes in morphology, gene expression, and structural alterations of the lipophosphoglycan (LPG), the promastigote most abundant surface glycolipid. Purification of metacyclics is accomplished using lectins or monoclonal antibodies (MAbs) that exploit stage-specific differences in the LPG. Besides, LPG displays extensive interspecies polymorphisms and is synthesized by promastigotes of all species investigated to date. In this work we studied the species- and stage-specificity of two MAbs (3A1-La and LuCa-D5) used to purify metacyclics of Leishmania amazonensis. Their ability to recognize different members of the Trypanosomatidae family was tested by direct agglutination, indirect immunofluorescence, and dot-blot analysis of LPG. We found that both MAbs were highly selective for L. amazonensis: 3A1-La recognized only promastigotes and LuCa-D5 labeled amastigote and promastigote stages of this species. These MAbs might be useful for Leishmania typing.     

Interleukin-2 production during murine infection by Leishmania mexicana amazonensis

Highly susceptible BALB/c mice, resistant C57B1/6 and their F1 progeny (BDF1) were infected subcutaneously in the foot pad with Leishmania mexicana amazonensis. At various times after infection, spleen or draining popliteal lymph node cells were assayed for their capacity to generate Interleukin-2 (I1-2) by Concanavalin A (ConA) stimulation. In both BALB/c and C57B1/6 strains there was a transient increase in their capacity to produce I1-2, from the 3rd to the 10th week post-infection. Return to pre-infection levels occurred between 13th to 16th week post-infection in all three strains. BALB/c mice always produced higher titers of I1-2 than C57B1/6, but such differences were statistically significant only at 3 and 10 weeks post-infection. BDF1 mice had titers similar to those observed in BALB/c mice. I1-2 production by ConA-stimulated lymph node cells was lower as compared to the spleen, but with a similar pattern among the three mice strains. Our data show that susceptibility to infection by L. mexicana amazonensis is not associated with deficient ConA-stimulated I1-2 production.     

Leishmania (Leishmania) amazonensis: purification and characterization of a promastigote serine protease

Pathogenic protozoan proteases play crucial roles in the host-parasite interaction, and its characterization contributes to the understanding of protozoan disease mechanisms. A Leishmania amazonensis promastigote protease was purified 36-fold, using aprotinin-agarose affinity chromatography and gel filtration high performance liquid chromatography, yielding a total recovery of 49%. The molecular mass of active enzyme obtained from native gel filtration HPLC and SDS-PAGE under conditions of reduction and non-reduction was 68 kDa, suggesting that the enzyme may exist as a monomer. The protease isoelectric point (pI) was around 4.45 and, as demonstrated by deglycosylation assay, it did not have any carbohydrate content. The optimal pH and temperature of the enzyme were 8.0 and 28 degrees C, respectively, determined using alpha-N-rho-tosyl-L-arginyl-methyl ester (L-TAME) as substrate. Assays of thermal stability indicated that 50% of the enzymatic activity was preserved after 4 min of pre-treatment at 42 degrees C and after 24 h of pre-treatment at 37 degrees C, both in the absence of substrate. Hemoglobin, bovine serum albumin (BSA), ovalbumin, and both gelatin and peptide substrates containing arginine in ester bound were hydrolyzed by 68 kDa protease. The insulin beta-chain was also hydrolyzed by the protease, and four peptidic bonds (L11-V12, E13-A14, L15-Y16, and Y16-L17) were susceptible to the 68-kDa protease action. Inhibition studies suggested that the enzyme belonged to a serine protease class inhibited by calcium ions and activated by manganese ions. These findings demonstrate that the L. amazonensis 68-kDa serine protease differs from those of other protozoan parasites.     

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.     

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.     

Regulated degradation of an endoplasmic reticulum membrane protein in a tubular lysosome in Leishmania mexicana

The cell surface of the human parasite Leishmania mexicana is coated with glycosylphosphatidylinositol (GPI)-anchored macromolecules and free GPI glycolipids. We have investigated the intracellular trafficking of green fluorescent protein- and hemagglutinin-tagged forms of dolichol-phosphate-mannose synthase (DPMS), a key enzyme in GPI biosynthesis in L. mexicana promastigotes. These functionally active chimeras are found in the same subcompartment of the endoplasmic reticulum (ER) as endogenous DPMS but are degraded as logarithmically growing promastigotes reach stationary phase, coincident with the down-regulation of endogenous DPMS activity and GPI biosynthesis in these cells. We provide evidence that these chimeras are constitutively transported to and degraded in a novel multivesicular tubule (MVT) lysosome. This organelle is a terminal lysosome, which is labeled with the endocytic marker FM 4-64, contains lysosomal cysteine and serine proteases and is disrupted by lysomorphotropic agents. Electron microscopy and subcellular fractionation studies suggest that the DPMS chimeras are transported from the ER to the lumen of the MVT via the Golgi apparatus and a population of 200-nm multivesicular bodies. In contrast, soluble ER proteins are not detectably transported to the MVT lysosome in either log or stationary phase promastigotes. Finally, the increased degradation of the DPMS chimeras in stationary phase promastigotes coincides with an increase in the lytic capacity of the MVT lysosome and changes in the morphology of this organelle. We conclude that lysosomal degradation of DPMS may be important in regulating the cellular levels of this enzyme and the stage-dependent biosynthesis of the major surface glycolipids of these parasites.     

The 6-phosphogluconate dehydrogenase of Leishmania (Leishmania) mexicana: gene characterization and protein structure prediction

6-Phosphogluconate dehydrogenase (6PGDH) is a key enzyme of the oxidative branch involved in the generation of NADPH and ribulose 5-phosphate. In the present work, we describe the cloning, sequencing and characterization of a 6PGDH gene from Leishmania (Leishmania) mexicana. The gene encodes a polypeptide chain of 479 amino acid residues with a predicted molecular mass of 52 kDa and a pI of 5.77. The recombinant protein possesses a dimeric quaternary structure and displays kinetic parameter values intermediate between those reported for Trypanosoma brucei and T. cruzi with apparent K(m) values of 6.93 and 5.2 μM for 6PG and NADP(+), respectively. The three-dimensional structure of the enzymes of Leishmania and T. cruzi were modelled from their amino acid sequence using the crystal structure of the enzyme of T. brucei as template. The amino acid residues located in the 6PGDH C-terminal region, which are known to participate in the salt bridges maintaining the protein dimeric structure, differed significantly among the enzymes of Leishmania, T. cruzi, and T. brucei. Our results strongly suggest that 6PGDH can be selected as a potential target for the development of new therapeutic drugs in order to improve existing chemotherapeutic treatments against these parasites.     

Depletion of secondary lysosomes in mouse macrophages infected with Leishmania mexicana amazonensis: a cytochemical study

Leishmania amastigotes lodge and multiply within parasitophorous vacuoles, which can fuse with secondary lysosomes of the host macrophages. This study examines the effect of infection with amastigotes of L. mexicana amazonensis on the secondary lysosomes of mouse macrophage cultures. The cultures were stained for the activities of two lysosomal enzyme markers, acid phosphatase and arylsulfatase, and the light microscopic observations were supplemented by electron microscopy. Nearly all noninfected macrophages contained numerous stained secondary lysosomes. The number of such lysosomes was markedly reduced 24 h postinfection, and the reduction persisted for at least 10 days. Stained secondary lysosomes reappeared after the amastigotes were destroyed by exposure of the cultures to phenazine methosulfate or by placing them at 37.5 degrees C. The depletion of lysosomes shown by cytochemical methods may reflect a high rate of fusion of the lysosomes with the parasitophorous vacuoles, exceeding the rate of formation of new secondary lysosomes. Alternatively, the parasites may inhibit the synthesis of lysosomal hydrolases, or the assembly or formation of primary or secondary lysosomes.     

Leishmania amazonensis: characterization of an ecto-phosphatase activity

We have characterized a phosphatase activity present on the external surface of Leishmania amazonensis, using intact living parasites. This enzyme hydrolyzes the substrate p-nitrophenylphosphate (p-NPP) at the rate of 25.70+/-1.17 nmol Pi x h(-1) x 10(-7)cells. The dependence on p-NPP concentration shows a normal Michaelis-Menten kinetics for this ecto-phosphatase activity present a V(max) of 31.93+/-3.04 nmol Pi x h(-1) x 10(-7)cells and apparent K(m) of 1.78+/-0.32 mM. Inorganic phosphate inhibited the ecto-phoshatase activity in a dose-dependent manner with the K(i) value of 2.60 mM. Experiments using classical inhibitor of acid phosphatase, such as ammonium molybdate, as well as inhibitors of phosphotyrosine phosphatase, such as sodium orthovanadate and [potassiumbisperoxo(1,10-phenanthroline)oxovanadate(V)] (bpV-PHEN), inhibited the ecto-phosphatase activity, with the K(i) values of 0.33 microM, 0.36 microM and 0.25 microM, respectively. Zinc chloride, another classical phosphotyrosine phosphatase inhibitor, also inhibited the ecto-phosphatase activity in a dose-dependent manner with K(i) 2.62 mM. Zinc inhibition was reversed by incubation with reduced glutathione (GSH) and cysteine, but not serine, showing that cysteine residues are important for enzymatic activity. Promastigote growth in a medium supplemented with 1mM sodium orthovanadate was completely inhibited as compared to the control medium. Taken together, these results suggest that L. amazonensis express a phosphohydrolase ectoenzyme with phosphotyrosine phosphatase activity.     

Leishmania mexicana amazonensis: development of a peptide tag useful for labeling and purifying biotinylated recombinant proteins

A number of peptide tags are available to facilitate the characterization of recombinant proteins. We have tested the bacterial oxaloacetate decarboxylase biotinylation domain for its efficacy in tagging recombinant proteins in vivo in Leishmania. To achieve efficient biotinylation, Leishmania also had to be co-transformed with the gene for bacterial biotin protein ligase (birA gene product). The recombinant chimeric protein could be detected on blots probed with avidin-horseradish peroxidase and purified on immobilized monomeric avidin resins.     

Leishmania mexicana: molecular cloning and characterization of enolase

The gene of Leishmania mexicana enolase was cloned and overexpressed in Escherichia coli as an active enzyme; the protein was biochemically analyzed. This enolase shares with enolases from other trypanosomatids the presence of three atypical residues, each with a reactive side group, near the active site, already described for the enzyme from Trypanosoma brucei. The natural enzyme was purified, using a three-step procedure, from a cytosolic fraction of L. mexicana promastigotes. The kinetic properties of the purified recombinant enzyme were similar to those of the natural enzyme. Both the recombinant and natural enzyme were inhibited by inorganic pyrophosphate. Subcellular localization analysis after differential centrifugation showed that the enzyme activity is only associated with the cytosolic fraction. However, an apparently inactive form of enolase was detected by Western blots in the microsomal fraction. Digitonin treatment of parasites and immunofluorescence studies with permeabilized and non-permeabilized parasites showed that enolase is also associated with membranes and it was found at the external face of the plasma membrane.