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.     

Anti-parasite activity of nucleoside analogues in Leishmania tropica promastigotes

Many nucleoside analogs were screened for anti-protozoa activity on Leishmania tropica in an in vitro culture system. 3'-Deoxyinosine and several tubercidin derivatives were found to be potent inhibitors for growth of the promastigote form of L. tropica. EC50 value of 3'-deoxyinosine was 4.43 X 10(-7)M. This compound was remarkably less toxic towards mouse mammary tumor FM3A cells (EC50, 1.25 X 10(-4) M). 3'-Deoxyinosine is metabolized by Leishmania promastigote to give 3'-deoxyinosine-5'-monophosphate, 3'-deoxy-adenosine(cordycepin)-5'-mono, di-, and triphosphates. This means that Leishmania can aminate the 6-position of 3'-deoxyinosine-5'-monophosphate, thereby converting it into a highly toxic compound.     

Presence and properties of cAMP phosphodiesterase from promastigote forms of Leishmania tropica and Leishmania donovani

1. This paper reports the isolation and the partial purification of cAMP phosphodiesterase (EC 3.1.4.17) from the promastigote form of Leishmania tropica and a preliminary result from Leishmania donovani. 2. The activity of the fraction obtained from column chromatography was measured. 3. The effects of pH, temperature, time of incubation and various compounds on its activity in vitro were obtained. 4. Two peaks (I and II) exhibiting cyclic nucleotide phosphodiesterase activity were obtained. 5. Both activities were found to require the addition of Mg2+ ions for full effect. 6. The apparent Km values for the first and second peaks were 1.43 x 10(-3) M and 4.1 x 10(-3) M respectively. L. donovani shows only one peak of activity.     

Binding of Leishmania promastigotes to macrophages

Leishmania tropica promastigotes are easily attached to and engulfed by C3H peritoneal macrophages in vitro at 37 degrees C. Different sugars at 0.3-0.5 M inhibited in vitro the attachment of L. tropica promastigotes to C3H peritoneal macrophages with lactose (Gal-beta [1 leads to 4]Glc) being the most efficient. Inhibition of attachment is also affected by pre-treatment of promastigotes with galactose oxidase. Oligosaccharides extending from promastigote and amastigote cell surfaces contain an important proportion of non-reducing galactose as does the carbohydrate-rich factor (EF) excreted by promastigotes of L. tropica and L. donovani. This study suggests that Leishmania, an obligatory intracellular parasite, uses as a means of entering the host cell a cellular mechanism similar to that used in the removal of damaged cells from blood circulation. This mechanism is assumed to take advantage of the exposed sugars, particularly the exposed non-reducing galactose, on the parasite surface during the stage of attachment. Once the parasite is inside the cell, the EF it produces might have a protective function, being inhibitory to some of the host cell lysosomal enzymes.     

Superiority of hemoglobin to hemin for cultivation of Leishmania tropica promastigotes in serum-free media

Leishmania tropica promastigotes grew slowly but could be maintained for long periods in serum-free hemin-containing media formulated previously for other Leishmania species or in slightly simplified versions of these media. Replacement of hemin in the medium by hemoglobin resulted in a much longer log phase and a significant reduction in the doubling time. Cell counts in cultures started at 1 x 10(5) cells/ml increased 400-fold in less than 140 h in the hemoglobin-containing media. These media also proved suitable for growing L. donovani and L. enriettii promastigotes.     

Comparison of the protein kinase and acid phosphatase activities of five species of Leishmania

Promastigotes from log phase and stationary phase cultures of Leishmania donovani, L. braziliensis panamensis, L. tropica, L. major, and L. mexicana amazonensis were analyzed for their content of protein kinase and acid phosphatase activities. Cell surface, histone-specific protein kinase activity was 1.3- to 2.8-fold higher in stationary phase cells of all species except for L. tropica in which the activities of stationary and log phase cells were equal; L. mexicana amazonensis had the highest histone-specific protein kinase activity and L. donovani the lowest. When viable, motile promastigotes of all five species were incubated for 10 min with [gamma-32P]ATP and Mg2+ (10 mM) in the absence of exogenous histone acceptor; about one dozen proteins were phosphorylated in each case. Both log phase and stationary phase promastigotes of all five species extensively phosphorylated a 50-kDa protein that had the mobility of tubulin. Incubation of pure calf brain tubulin with [gamma-32P]ATP and purified L. donovani protein kinase resulted in extensive phosphorylation of the former. Highly infective metacyclic forms (PNA-) of L. major, isolated from a stationary culture using the peanut agglutinin (PNA), contained eight times more histone-specific protein kinase activity than noninfective log phase cells (PNA+). The PNA- and PNA+ forms of L. major both phosphorylated a 50-kDa protein when incubated with [gamma-32P]ATP and magnesium or manganese ions (10 mM); the 50-kDa protein was precipitated by anti-tubulin rabbit antibodies. Extracts of all five species contained large amounts of acid phosphatase activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prostaglandin production from arachidonic acid and evidence for a 9,11-endoperoxide prostaglandin H2 reductase in Leishmania

Lysates of Leishmania promastigotes can metabolise arachidonic acid to prostaglandins. Prostaglandin production was heat sensitive and not inhibited by aspirin or indomethacin. We cloned and sequenced the cDNA of Leishmania major, Leishmania donovani, and Leishmania tropica prostaglandin F(2alpha) synthase, and overexpressed their respective 34-kDa recombinant proteins that catalyse the reduction of 9,11-endoperoxide PGH(2) to PGF(2alpha). Database search and sequence alignment alignment showed that L. major prostaglandin F(2alpha) synthase exhibits 61, 99.3, and 99.3% identity with Trypanosoma brucei, L. donovani, and L. tropica prostaglandin F(2alpha) synthase, respectively. Using polymerase chain reaction amplification, Western blotting, and immunofluorescence, we have demonstrated that prostaglandin F(2alpha) synthase protein and gene are present in Old World and absent in New World Leishmania, and that this protein is localised to the promastigote cytosol.     

Prostaglandin production from arachidonic acid and evidence for a 9,11-endoperoxide prostaglandin H2 reductase in Leishmania

Lysates of Leishmania promastigotes can metabolise arachidonic acid to prostaglandins. Prostaglandin production was heat sensitive and not inhibited by aspirin or indomethacin. We cloned and sequenced the cDNA of Leishmania major, Leishmania donovani, and Leishmania tropica prostaglandin F(2alpha) synthase, and overexpressed their respective 34-kDa recombinant proteins that catalyse the reduction of 9,11-endoperoxide PGH(2) to PGF(2alpha). Database search and sequence alignment showed that L. major prostaglandin F(2alpha) synthase exhibits 61, 99.3, and 99.3% identity with Trypanosoma brucei, L. donovani, and L. tropica prostaglandin F(2alpha) synthase, respectively. Using polymerase chain reaction amplification, Western blotting, and immunofluorescence, we have demonstrated that prostaglandin F(2alpha) synthase protein and gene are present in Old World and absent in New World Leishmania, and that this protein is localised to the promastigote cytosol.     

Miltefosine-induced apoptotic cell death on Leishmania major and L. tropica strains

The aim of this study was to assess the cytotoxic effects of various concentrations of miltefosine on Leishmania major (MRHO/IR/75/ER) and L. tropica (MHOM/IR/02/Mash10) promastigotes and to observe the programmed cell death features. The colorimetric MTT assay was used to find L. major and L. tropica viability and the obtained results were expressed as 50% inhibitory concentration (IC50). Also, 50% effective doses (ED50) for L. major and L. tropica amastigotes were also determined. Annexin-V FLUOS staining was performed to study the cell death properties of miltefosine using FACS analysis. Qualitative analysis of the total genomic DNA fragmentation was performed by agarose gel electrophoresis. Furthermore, to observe changes in cell morphology, promastigotes were examined using light microscopy. In both strains of L. major and L. tropica, miltefosine induced dose-dependent death with features of apoptosis, including cell shrinkage, DNA laddering, and externalization of phosphatidylserine. The IC50 was achieved at 22 μM and 11 μM for L. major and L. tropica after 48 hr of incubation, respectively. ED50 of L. major and L. tropica amastigotes were 5.7 μM and 4.2 μM, respectively. Our results indicate that miltefosine induces apoptosis of the causative agent of cutaneous leishmaniasis in a dose-dependent manner. Interestingly, L. major did not display any apoptotic changes when it was exposed to miltefosine in concentrations sufficient to kill L. tropica.     

Leishmania major and Leishmania tropica: I the in vitro effects of an immunomodulator, S2-Complex

This study was undertaken to try to determine the possible anti-leishmanial activity of S2-Complex, an organic complex of copper chloride, ascorbic acid, and nicotinamide. The promastigotes, axenic amastigotes, and intracellular amastigotes of both Leishmania major and Leishmania tropica were incubated with different concentrations of S2-Complex. The EC50 for each form was calculated. Results show that all forms of the parasites were dose dependently inhibited by S2-Complex. The promastigotes of both parasites were the most resistant with highest EC50 followed by axenic amastigotes. While intracellular amastigotes were the most sensitive with the lowest EC50.These results indicate that S2-Complex has a direct anti-leishmanial effect. When mice were treated with S2-Complex or BCG for four days before harvesting the macrophages, and the macrophages infected with both L. major and L. tropica, they showed increased phagocytosis and increased parasite killing. The results of S2-Complex were not statistically different from the immunomodulating agent BCG. These results indicate that S2-Complex has an immunomodulating effect in addition to the direct anti-leishmanial effect.     

Leishmania donovani: characteristics of adenosine and inosine transporters in promastigotes of two different strains

The nucleoside transport characteristics of two strains of Leishmania donovani promastigotes were studied. Strain S1, growing in fully defined medium, and strain S2 (MHOM/ET/67/HA3) both transported adenosine and inosine, but only strain S1 transported uridine and thymidine. Competition studies in the presence of 100 microM of unlabeled adenosine, inosine, guanosine, 2'-deoxyadenosine, tubercidin, formycin B, 3'-deoxyinosine as well as uridine, thymidine and cytidine, with either 1 microM [3H]adenosine or [3H]inosine as permeant, were carried out. The inhibition profile with [3H]inosine as permeant was essentially identical in S1 and S2 promastigotes, indicating that the same inosine transporter was present in both strains. However, with [3H] adenosine as permeant, significant differences were noted between the two strains. Thus, only adenosine, 2'-deoxyadenosine, tubercidin, uridine, and thymidine were strongly inhibitory in S1 promastigotes, while essentially all nucleosides tested were effective in S2 promastigotes. This indicates that adenosine transport in S2 promastigotes seems to involve a transporter differing from that described for S1 promastigotes.     

The major surface protein of Leishmania promastigotes is a protease

The major surface protein of Leishmania promastigotes is evolutionarily conserved and is found in isolates of L. donovani, L. major, L. tropica, L. mexicana, and L. braziliensis. The data provided in this communication demonstrate that in L. major this integral membrane protein is a protease, which we now designate promastigote surface protease. The enzyme has an alkaline pH optimum and is active both in its detergent-solubilized form and at the surface of living or fixed promastigotes. A water-soluble form of promastigote surface protease is obtained following digestion with the phospholipase C responsible for the release of the variant surface glycoprotein of Trypanosoma brucei. Possible biological functions of promastigote surface protease during the life cycle of Leishmania parasites are discussed.     

Leishmania tropica and Leishmania donovani: solid phase radioimmunoassay using leishmanial excreted factor

A radioimmunoassay for the quantitative determination of anti-leishmanial excreted factor (EF) antibody in rabbit sera was developed. The assay, using Leishmania tropica and Leishmania donovani promastigotes EF, purified by either extraction with phenol followed by fractionation on a Sephadex G-100 column or by the dissociation of EF antibody complexes, was shown to be sensitive and reproducible. Using monospecific anti-EF antibodies, levels of as low as 0.06-0.12 micrograms/ml of anti-EF IgG could be detected. The specificity of the assay was assessed by inhibition with homologous and heterologous EF. Only minor cross-reactivity with heterologous EF was observed, and as little as 2.5 micrograms/ml of EF could be detected. Sera from kala-azar patients showed only 1.8-3.1 times more anti-EF activity, as compared with uninfected controls. No specificity was observed with sera from kala-azar patients with regard to the type of EF used. Almost the same activity was obtained with both EF from L. tropica and L. donovani. No anti-EF antibodies were detected in sera from patients with cutaneous leishmaniasis.     

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.     

Inhibition by Dications of in vitro growth of Leishmania major and Leishmania tropica: causative agents of old world cutaneous leishmaniasis

Old World cutaneous leishmaniasis is caused by infection with Leishmania major and Leishmania tropica. Pentamidine and related dications exhibit broad spectrum antiprotozoal activity. Based on the previously reported efficacy of these compounds against related organisms, 18 structural analogs of pentamidine were evaluated for in vitro antileishmanial activity, using pentamidine as the standard reference drug for comparison. Furan analogs and reversed amidine compounds were examined for activity against L. major and L. tropica promastigotes. The most active compounds against both Leishmania species were in the reversed amidine series. DB745 and DB746 exhibited the highest activity against L. major and DB745 was the most active compound against L. tropica. Both of these compounds exhibited 50% inhibitory concentrations (IC50) below 1 nM for L. major. Ten reversed amidines were also tested for their ability to inhibit growth in an axenic amastigote model. Nine of 10 reversed amidine analogs were active at concentrations below 1 nM. These results justify further study of dicationic compounds as potential new agents for treating cutaneous leishmaniasis.     

Monoclonal antibodies specific for Leishmania tropica. I. Characterization of antigens associated with stage- and species-specific determinants

Four monoclonal antibodies (T-1 through T-4), which were produced to membrane-enriched preparations of Leishmania tropica major promastigotes, reacted specifically with the members of L. tropica complex. The antibodies T-1 and T-4 react exclusively with L. t. major and L. t. minor. The remaining two monoclonal antibodies bind, in addition, to L. t. aethiopica and weakly to L. mexicana amazonensis. No significant cross-reactivity was observed with L. donovani, L. braziliensis braziliensis, and Trypanosoma cruzi. Antibodies T-1, T-2, and T-4 were found to be specific for the promastigote stage (insect) of L. tropica. Antibody T-3 reacts with both the amastigote and promastigote stages of the parasite. All of the monoclonal antibodies react with cell surface components on intact promastigotes. The protein antigens containing the species-specific determinants recognized by each of the four antibodies were identified by radioimmunoprecipitation of solubilized 125I-labeled L. t. major promastigotes. A single 50 kilodalton protein is recognized by clone T-4. T-1 recognized two high m.w. proteins (100 and 200 kilodaltons). These two antigens plus an additional protein of lower m.w. (70 kilodaltons) are also immunoprecipitated by the antibodies T-2 and T-3, demonstrating that species-specific determinants are present on several different cell surface proteins of L. t. major.     

Prophylactic immunization against experimental leishmaniasis. II. Further characterization of the protective immunity against fatal Leishmania tropica infection induced by irradiated promastigotes

The genetic vulnerability of BALB/c mice to Leishmania tropica (L. major) infection renders them incapable of controlling a primary cutaneous lesion that leads to uniformly fatal visceral disease. Potent, long-lasting protection involving both lesion healing and survival can be induced by repeated prophylactic i.v. immunization with gamma-irradiated (150K rad) L. tropica promastigotes. The effect is not dependent on continuing viability or cellular invasiveness of the irradiated parasites because their effective immunogenicity withstands heating at 56 degrees C for 1 hr. Immunity is not stage specific and encompasses both amastigote and promastigote challenges. Similar prophylaxis can be induced by immunization with heterologous irradiated L. donovani promastigotes. Repeated i.v. immunization with irradiated L. tropica promastigotes induces an antibody response in the isotype sequence M leads to G1/G3 leads to G2a/G2b leads to A with substantially higher titres than are found in response to the infection itself. Splenectomy before immunization drastically reduces this antibody response without incurring any impairment of the extent of protection. Passive transfer of large amounts (up to 10 ml) of hyperimmune serum (or isotype fractions thereof) throughout the first 8 wk of infection fails to arrest disease progression during this period. Despite the previously described lack of any detectable cutaneous DTH reactivity, which has hitherto correlated with protective cell-mediated immunity, the results obtained do not support attribution of an alternative causal role to the humoral response.     

Infectivity and virulence of Leishmania donovani promastigotes: a role for media,source, and strain of parasite

Transformation of promastigotes of Leishmania donovani strain AG83 from amastigotes derived from an infected animal was studied in three media, Schneider's Drosophila medium (SDM), Medium 199 (M199), and biphasic M199 (B-M199) with 10% fetal bovine serum. The media, SDM and B-M199, both supported a more efficient transformation of promastigotes in comparison with M199. Infectivity studies in hamsters and BALB/c mice showed that promastigotes isolated in B-M199 were several folds more infective than those obtained from M199. Comparison of the infectivity and virulence of promastigotes of AG83, with a recent isolate of kala-azar, SL94, harvested under similar conditions, revealed greater infectivity of SL94 for both macrophages and animal models. The present study demonstrates that the medium used for the conversion of amastigotes to promastigotes plays a major role in determining the infectivity of the freshly transformed L. donovani promastigotes in hamsters and BALB/c mice. The source and the strain of the parasite also influence the outcome of L. donovani infection.     

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.     

Leishmania mexicana: amastigote hydrolases in unusual lysosomes

Leishmania mexicana mexicana (M379) amastigotes were found to contain much higher activities than cultured promastigotes of five putative lysosomal enzymes: cysteine proteinase; arylsulfatase (EC 3.1.6.1); beta-glucuronidase (EC 3.2.1.31); DNase (EC 3.1.22.1), and RNase (EC 3.1.27.1). The release profiles of the first three of these enzymes from digitonin-permeabilized amastigotes suggests that they are located within organelles. Cytochemical staining for cysteine proteinase, using gold labeled antibodies and arylsulfatase, showed that both were present in large organelles previously named "megasomes." Comparative studies with L. mexicana amazonensis (LV78), L. donovani donovani (LV9), and L. major (LV39) revealed that L. mexicana amazonensis was similar to L. mexicana mexicana in possessing both high amastigote cysteine proteinase activity and large numbers of megasome organelles in amastigotes, whereas the other two species lacked both these features. The results suggest that the presence of numerous lysosome-like organelles in the amastigote is a characteristic of the L. mexicana group of parasites.