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.     

Two separate growth phases during the development of Leishmania in sand flies: implications for understanding the life cycle

The life cycle of Leishmania alternates between two main morphological forms: intracellular amastigotes in the mammalian host and motile promastigotes in the sand fly vector. Several different forms of promastigote have been described in sandfly infections, the best known of these being metacyclic promastigotes, the mammal-infective stages. Here we provide evidence that for Leishmania (Leishmania) mexicana and Leishmania (Leishmania) infantum (syn. chagasi) there are two separate, consecutive growth cycles during development in Lutzomyia longipalpis sand flies involving four distinct life cycle stages. The first growth cycle is initiated by procyclic promastigotes, which divide in the bloodmeal in the abdominal midgut and subsequently give rise to non-dividing nectomonad promastigotes. Nectomonad forms are responsible for anterior migration of the infection and in turn transform into leptomonad promastigotes that initiate a second growth cycle in the anterior midgut. Subsequently, leptomonad promastigotes differentiate into non-dividing metacyclic promastigotes in preparation for transmission to a mammalian host. Differences in timing, prevalence and persistence of the four promastigote stages were observed between L. mexicana and L. infantum in vivo, which were reproduced in cultures initiated with lesion amastigotes, indicating that development is to some extent governed by a programmed series of events. A new scheme for the life cycle in the subgenus Leishmania (Leishmania) is proposed that incorporates these findings.     

Activity of an engineered synthetic killer peptide on Leishmania major and Leishmania infantum promastigotes

This study was undertaken to analyze the effect of an engineered, killer decapeptide (KP) on Leishmania major and Leishmania infantum promastigotes. The KP was synthesized on the basis of the sequence of a recombinant, single-chain anti-idiotypic antibody acting as a functional internal image of a yeast killer toxin. The evaluation of in vitro inhibitory activity of KP on L. major and L. infantum, release of intracellular green fluorescent protein (GFP) molecules by L. major, DNA fragmentation, and ultrastructural analysis (TEM) of L. infantum upon KP treatment were performed. KP presented antiproliferative and leishmanicidal activity with LC(50)/1 day of 58 and 72 microM for L. major and L. infantum, respectively. A dose-dependent decrease in proliferation and increase of killing of promastigotes was seen after KP treatment. No DNA fragmentation in L. infantum promastigotes or release of intracellular GFP molecules on peptide treatment of a GFP expressing L. major clone was demonstrated. Moreover the plasma-membrane was not disrupted, but, by TEM analysis, intracellular damage was observed.     

Synthesis and antileishmanial activity of 3-(alpha-azolylbenzyl)indoles

The goal of the present study was to evaluate several azolyl-substituted indoles as new antileishmanial agents. Ten 3-(alpha-azolylbenzyl)indoles have been synthesized using Friedel-Crafts acylation as a key-step. All the target compounds were found to display high levels of activity when tested against Leishmania mexicana promastigotes in vitro. The most active compounds, showing an IC50 < 1 microM, were 5-bromo-1-ethyl-3-[(2,4-dichlorophenyl)(1H-imidazol-1-yl)methyl]-1H-indole 15 and its triazole analogue 17. Four representative compounds 15, 17, 22 and, 23 were also tested against intracellular amastigotes of L. mexicana using ketoconazole and meglumine antimoniate as reference compounds, the results of which are 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.     

Developmentally regulated expression of a cell surface class I nuclease in Leishmania mexicana

Leishmania mexicana, like other trypanosomatid parasites, is a purine auxotroph and must obtain these essential nutrients from its sandfly and mammalian hosts. A single copy gene encoding its unique externally oriented, surface membrane, purine salvage enzyme 3'-nucleotidase/nuclease, was isolated. Structural features of the deduced protein included: an endoplasmic reticulum-directed signal peptide, several conserved class I catalytic and metal co-factor (Zn(2+)) binding domains, transmembrane anchor sequence and a C-terminal cytoplasmic tail. 3'-Nucleotidase/nuclease gene (mRNA) and protein (enzyme activity) expression were examined in three different L. mexicana developmental forms: procyclic promastigotes, metacyclic promastigotes and amastigotes. Results of both approaches demonstrated that the 3'-nucleotidase/nuclease was a stage-specific enzyme, being expressed by promastigote forms (stages restricted to the insect vector), but not by amastigotes (which produce disease in mammalian hosts). Starvation of these parasites for purines resulted in the significant up-regulation of both 3'-nucleotidase/nuclease mRNA and enzyme activity in promastigotes, but not in amastigotes. These results underscore the critical role that the 3'-nucleotidase/nuclease must play in purine salvage during the rapid multiplicative expansion of the parasite population within its insect vector. To our knowledge, the L. mexicana 3'-nucleotidase/nuclease is the first example of a nutrient-induced and developmentally regulated enzyme in any parasitic protozoan.     

Leishmania species: mechanisms of complement activation by five strains of promastigotes

The interaction of fresh serum with promastigotes of Leishmania major, L. donovani, L. mexicana mexicana, L. mexicana amazonensis, and L. braziliensis guyanensis results in lysis of all strains tested with either fresh human or guinea pig serum at 37 C for 30 min. Lysis does not occur in the cold and requires divalent cations and complement that is active hemolytically. Serum deficient in the eighth component of complement is not lytic. Lysis of L. major, L. mexicana, and L. braziliensis proceeds fully in human serum containing EGTA/Mg2+ or in guinea pig serum deficient in the fourth complement component. These species consume only small amounts of C4 from human serum and do not require calcium to optimally bind C3. The data indicate that all are activators of the alternative complement pathway and that the classical pathway is not required for the lysis of these organisms. Promastigotes of L. donovani, in contrast, activate the classical pathway. The presence of calcium is required for both optimal C3 binding and parasite lysis, and L. donovani promastigotes consume C4 when incubated in human serum. In high concentrations, human serum agglutinates all tested Leishmania spp. The agglutinating factor does not require divalent cations, is heat stable, and works at 4 C, suggesting that it is an antibody. This "naturally occurring" antibody cross reacts with all Leishmania spp. and agglutinates them. The adsorption of serum with any Leishmania species or with beads that are Protein A coated, removes the agglutinogen. This factor causes a slight enhancement in alternative pathway activation by L. major and mediates the classical activation by L. donovani. In adsorbed serum, L. donovani promastigotes only weakly activate the alternative complement pathway. Increased concentrations of adsorbed serum are therefore necessary for lysis to proceed. The titer can be partially restored by the addition of heat inactivated serum. Using purified components of the classical cascade, we are unable to visualize surface bound C3 on L. donovani promastigotes unless heat inactivated serum is also present. We conclude that all Leishmania spp. promastigotes are susceptible to lysis by normal serum independent of antibody. The presence of small amounts of naturally occurring antibody in human serum enhances the susceptibility of L. donovani promastigotes to lysis by activating the classical complement pathway.     

Purine phosphoribosyltransferases of Leishmania mexicana mexicana and other flagellate protozoa

Amastigotes and cultured promastigotes of Leishmania mexicana mexicana and L. m. amazonensis, cultured promastigotes of L. donovani and L. tarentolae, and the culture forms of Crithidia fasciculata, Herpetomonas muscarum muscarum and H. m. ingenoplastis all possessed four phosphoribosyltransferase (PRTase) activities: adenine PRTase, hypoxanthine PRTase, guanine PRTase and xanthine PRTase. The enzymes of L. m. mexicana required divalent cations for activity; Mn2+ or Co2+ produced maximal activity in most cases. Hypoxanthine PRTase, guanine PRTase and xanthine PRTase from all organisms were sedimentable in part, suggesting that they may occur within glycosomes. The enzymes of L. m. mexicana cultured promastigotes were inhibited by a range of purine analogues.     

Leishmania amazonensis: effects of heat shock on ecto-ATPase activity

In this work we demonstrated that promastigotes of Leishmania amazonensis exhibit an Mg-dependent ecto-ATPase activity, which is stimulated by heat shock. The Mg-dependent ATPase activity of cells grown at 22 and 28 degrees C was 41.0+/-5.2 nmol Pi/h x 10(7)cells and 184.2+/-21.0 nmol Pi/h x 10(7)cells, respectively. When both promastigotes were pre-incubated at 37 degrees C for 2h, the ATPase activity of cells grown at 22 degrees C was increased to 136.4+/-10.6 nmol Pi/h x 10(7) whereas that the ATPase activity of cells grown at 28 degrees C was not modified by the heat shock (189.8+/-10.3 nmol Pi/h x 10(7)cells). It was observed that Km of the enzyme from cells grown at 22 degrees C (Km=980.2+/-88.6 microM) was the same to the enzyme from cells grown at 28 degrees C (Km=901.4+/-91.9 microM). In addition, DIDS (4,4'-diisothiocyanatostilbene 2,2'-disulfonic acid) and suramin, two inhibitors of ecto-ATPases, also inhibited similarly the ATPase activities from promastigotes grown at 22 and 28 degrees C. We also observed that cells grown at 22 degrees C exhibit the same ecto-phosphatase and ecto 3'- and 5'-nucleotidase activities than cells grown at 28 degrees C. Interestingly, cycloheximide, an inhibitor of protein synthesis, suppressed the heat-shock effect on ecto-ATPase activity of cells grown at 22 degrees C were exposed at 37 degrees C for 2h. A comparison between the stimulation of the Mg-dependent ecto-ATPase activity of virulent and avirulent promastigotes by the heat shock showed that avirulent promastigotes had a higher stimulation than virulent promastigotes after heat stress.     

Analysis of Leishmania proteinases reveals developmental changes in species-specific forms and a common 68-kDa activity

Abstract The proteinases of three species of Leishmania have been analysed by electrophoresis. Amastigotes of L. mexicana mexicana have several high-activity, low- M _r cysteine proteinases which are absent from log-phase promastigotes of L. m. mexicana and from all developmental stages of the other species analysed ( L. donovani and L. major ). Low-activity, low- M _r proteinases were present in populations of stationary-phase promastigotes of L. m. mexicana . All three species of Leishmania had higher M _r proteinases, a number of which showed developmental regulation, some of them being stage-specific. Significantly, at all stages of the life cycle in all three species a 68-kDa proteinase was apparent. In its size, sensitivity to inhibitors and ability to bind concanavalin A-agarose, this resembles the major surface protein thought to be present in all Leishmania species and which has recently been reported to possess proteinase activity in L. major promastigotes.     

Leishmania tropica: cysteine proteases are essential for growth and pathogenicity

Leishmania parasites are responsible for a diverse collection of diseases of humans and other animals. Cysteine proteases are putative virulence factors of leishmania parasites. There are differences in the susceptibility of specific stages in different Leishmania species to cysteine protease inhibitors. Here, we establish a key role of cysteine proteases in growth, viability, and pathogenicity of Leishmania tropica by using a specific cysteine protease inhibitor (N-Pip-F-hF-VS Phenyl). Reduction or arrest of promastigote growth occurred at inhibitor concentration of 5 and 100 microM, respectively. This shows an essential role for cysteine proteases in viability and growth of L. tropica promastigotes. It confirms that the promastigote stage of L. tropica more closely resembles that of Leishmania major than that of Leishmania mexicana, which is refractory to this inhibitor. Pathogenicity of L. tropica amastigotes in mice, as assessed by footpad swelling, was also reduced by treatment with the cysteine protease inhibitor. This suggests that cysteine proteases are essential for pathogenicity of L. tropica amastigote in mammalian host, similar to both L. major and L. mexicana.     

Functional and structural damage in Leishmania mexicana exposed to the cationic peptide dermaseptin.

The effect of dermaseptin (DS), a 34 amino acid residue cationic peptide isolated from Phyllomedusa sauvagii skin, has been studied on promastigotes of Leishmania mexicana growing in vitro. Within 5 min of incubation in the presence of DS, the flagellated parasites lost their motility. DS inhibited promastigote growth by 50% at a concentration of 3 microM and by 100% at 10 microM. Immunocytochemical, freeze fracture, label fracture and electron microscopic observations have shown that the amphipathic peptide generates perturbations of the lipid bilayer leading to altered permeability of the surface membrane and death of the parasite.     

The oxidative response of rabbit peritoneal neutrophils to leishmanias and other trypanosomatids

Luminometry has been used to measure the respiratory burst of rabbit peritoneal neutrophils that is elicited by different forms and species of Leishmania and Herpetomonas. Mid-log phase and metacyclic promastigotes of L. major evoked large responses; that due to metacyclics was lower and slower, but they also bound in smaller numbers than mid-log phase cells. Promastigotes of L. mexicana mexicana also stimulated a large respiratory burst whereas amastigotes elicited little or none. Leishmania donovani promastigotes and culture forms of H. muscarum muscarum and H. m. ingenoplastis all evoked large responses by neutrophils. There was, however, very little response to L. mexicana mexicana promastigotes, L. donovani promastigotes or H. muscarum muscarum when they were added in large numbers. This 'inhibition' was not apparent with L. major.     

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.     

In vitro effect of an alkaloid isolated from Amphimedon viridis (Porifea) on promastigots of Leishmania mexicana

The dose dependent antiproliferative effect of an alkaloidal substance extracted from the sponge Amphimedon viridis was tested on Leishmania mexicana promastigotes. Sponges were collected in Isla Larga, Venezuela (10 degrees 20' 20" - 10 degrees 24" N, 64 degrees 19' - 64 degrees 22' W), cut and dipped in methanol for vacum filtering extraction every 24 hr. The aqueous extract was separated by chromatography over silica gel. The parasites were from the Venezuelan NR strain. Their growth rate was reduced by 50 % with a dose of 10 microg/ml in 48 hr, whilst concentrations of 30 and 40 microg/ml induce leishmanicidal action after 110 and 20 min, respectively. Lysis is preceded by an immediate increase in cellular volume associated with progressive damage of cellular content and the destruction of organelles. These findings suggest that one important factor associated with the antiproliferative effect of this alkaloidal substance on L. mexicana promastigotes is the loss of the plasma membrane selective permeability.     

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.     

Electrophoretic analysis of the polypeptides of Leishmania amastigotes and promastigotes

The polypeptides of Leishmania mexicana mexicana (M379), L. m. amazonensis (LV78), L. major (LV39) and L. d. donovani (LV39) amastigotes and cultured promastigotes have been analysed by SDS-polyacrylamide gel electrophoresis. The polypeptide banding patterns of the promastigotes of the four species were quite similar, but distinct differences were detected between those of amastigotes. The results suggest that the various species of Leishmania are adapted differently for survival and growth in the mammalian host. The polypeptides of L. m. mexicana amastigotes were very rapidly hydrolysed unless protected by the cysteine proteinase inhibitor leupeptin.     

Synthesis and characterization of [Au(dppz)2]Cl3. DNA interaction studies and biological activity against Leishmania (L) mexicana

[Au(dppz)(2)]Cl(3) was synthesized by the reaction of HAuCl(4) in excess of the dypirido[3,2-a: 2,3-c]phenazine (dppz) ligand. This complex was characterized by elemental analysis, fast atom bombardment (FAB) mass, NMR, UV-visible and IR spectroscopies. DNA-gold complex interactions were studied by spectroscopic titrations, viscosity measurements and electrophoretical assays. These studies showed that the gold complex interacts with DNA by intercalation mode. These observations, led us to carry out biological tests on cultures of promastigotes of Leishmania (L) mexicana. [Au(dppz)(2)]Cl(3) induced a dose dependent antiproliferative activity with minimal inhibitory concentration (MIC) of 3.4nM and lethal doses LD(26) of 17nM for 48h. These findings suggest that a very potent leishmanicidal activity could be associated to the cellular processes involving parasite DNA, constituting a new promising chemotherapeutic alternative in the search for definitive leishmaniasis cure.     

Synthesis and in vitro antileishmanial activity of 5-substituted-2'-deoxyuridine derivatives

We report herein the synthesis and the in vitro antileishmanial evaluation of 5-substituted-2'-deoxyuridine nucleosides. The most active compound against Leishmania donovani promastigotes was Thia-dU (3a) with an IC50 =3 microM. This compound exhibited the same activity as zidovudine (3'-azido-2'-deoxythymidine) used as nucleoside reference compound. Considering the cytotoxicity of synthetic compounds on peritoneal murine macrophages, the most toxic compound was MeThio-dU (3d) with a MTC at 10 microM. Only Methia-dU (3b) was active against intramacrophagic amastigotes with an IC50 =6.5 microM. This latter can now be evaluated in vivo, for further investigations through structure-based drug design.     

Expression of Cysteine Proteinases by Metacyclic Promastigotes of Leishmania mexicana

ABSTRACT. The expression of cysteine proteinases by metacyclic promastigotes of Leishmania mexicana was investigated using gelatin polyacrylamide gel electrophoresis. Two prominent bands were detected which distinguished metacyclics from multiplicative promastigotes, lacking detectable cysteine proteinase activity, and amastigotes, with a distinct banding pattern composed of multiple enzymes. A correlation between relative activity of the metacyclic-specific bands and the prevalence of metacyclics was found both during the growth cycle in vitro as metacyclogenesis occurred, and by comparison of stationary phase populations from consecutive subpassages in vitro. Irreversible inhibition of the metacyclic activities using N-benzyloxycarbonyl-phenylalanyl-alanyl diazomethane did not inhibit metacyclic to amastigote transformation in vitro. These activities provide a useful biochemical marker for the metacyclic promastigotes of L. mexicana .