Localization of the plasma membrane and mitochondrial H(+)-ATPases in Leishmania donovani promastigotes.

Immunochemical methods were used to characterize the proton-translocating ATPases (H(+)-ATPases) of the plasma membrane and mitochrondrion of Leishmania donovani promastigotes. Antisera directed against the plasma membrane H(+)-ATPase of Saccharomyces cerevisiae reacted with a 66 kDa membrane protein of L. donovani promastigotes. By immunocytochemistry, the antiserum was shown to label the cell and flagellar surface of promastigotes as well as the Golgi apparatus and the membrane of intracellular organelles. The target antigen was shown to possess ATPase activity resembling the leishmanial H(+)-ATPase activity. Antisera raised against the beta-subunit of the F0F1-ATPase of Escherichia coli reacted with a 56 kDa protein in L. donovani promastigotes. Ultrastructurally, the anti-beta-subunit antibody was exclusively associated with the mitochondrion in these cells. This antiserum immunoprecipitates ATP hydrolytic activity typical of the F1 beta-subunit activity of the mitochondria of higher eukaryotes.     

Immunological identification of the alpha subunit of G13, a novel guanine nucleotide binding protein.

An antiserum (13CB) was generated against a synthetic peptide, HDNLKQLMLQ, which is predicted to represent the C-terminal decapeptide of the alpha subunit of the novel G-protein, G13. Competitive ELISA indicated that the antiserum reacted with this peptide but that it showed minimal ability to recognize peptides which represent the equivalent regions of the pertussis toxin-insensitive G-proteins, Gq + G11, G12, G15 + G16, GL1 (also called G14) as Gz, and well as other G-proteins. Immunoblots of human platelet membranes with antiserum 13CB identified a single 43-kDa polypeptide, and while this immunoreactivity was abolished by the presence of the cognate peptide it was not modified by the presence of peptides corresponding to the equivalent region of other G-proteins. Immunoreactivity corresponding to G13 alpha was detected in a range of cell types with human platelets having the highest levels of this polypeptide.     

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.     

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.     

Species and stage specificity of Leishmania donovani antigens.

Monoclonal antibodies D2 and D13 were produced in mice using Leishmania donovani promastigote membrane fractions. To study the species and stage specificity of the antigens recognized by these antibodies, we examined amastigotes prepared in vitro and cultured promastigotes by indirect immunofluorescence with monoclonal antibodies D2 and D13. Monoclonal antibody D2 showed weak reactivity for 9 of 9 strains of L. donovani complex promastigotes and 8 of 9 amastigotes. In contrast, only 2 of 22 strains from other complexes yielded equivocal reactions. Monoclonal antibody D13, however, had much broader reactivity. D13 reacted with all the promastigotes and amastigotes of L. donovani complex isolates as well as with 10 of 22 promastigotes and 8 of 13 amastigotes from other complexes. The high degree of species specificity seen with monoclonal antibody D2 provides a rationale for further study of this antibody and its purified antigen for parasite identification and serodiagnosis of visceral leishmaniasis. The strong fluorescent signal noted with D13 and the presence of the D13 epitope on all L. donovani complex parasites supports studies on its role as an antigen in immunoprophylaxis of visceral leishmaniasis.     

Arylanthranilodinitriles: a new biaryl class of antileishmanial agents

A series of anthranilodinitrile-based biaryls were synthesized and evaluated in vitro against extracellular promastigotes and intracellular amastigotes of Leishmania donovani. Among various screened compounds, a biaryl with trifluoromethyl group 5f showed 83% inhibition against promastigotes and 70% inhibition against amastigotes of L. donovani at 8 and 20microg/mL concentrations, respectively.     

Metacyclogenesis of Leishmania spp: species-specific in vitro transformation, complement resistance, and cell surface carbohydrate and protein profiles

Metacyclic (stationary) and logarithmic (log) forms of promastigotes of Leishmania donovani and Leishmania major were characterized in several ways. The highly active metacyclic forms were larger with more protein and less carbohydrate. The flagellum increased in length 2.4 times in L. major as compared to 1.8 times in L. donovani. Resistance to complement-mediated lysis by normal human serum of in vitro grown Leishmania promastigotes was related to the species, the growth phase in culture, and also the temperature. Metacyclic forms of both species had a much increased resistance to killing by normal serum at different temperatures. Differences in membrane-exposed carbohydrates were detected by fluorescein-conjugated lectins. Peanut agglutinin and Ulex agglutinin I differentiated log and stationary phase promastigotes of L. major. Higher amounts of acid phosphatase were demonstrated in the metacyclic phase. Differences in polypeptides were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two polypeptides of approximately 51 and 114 kDa were found exclusively in metacyclic promastigotes of both species, whereas 38- and 23-kDa polypeptides were lost or reduced during transformation from log to metacyclic phase promastigotes of L. donovani. In addition, a 75-kDa polypeptide was expressed only in metacyclic promastigotes of L. major.     

Wild-type Leishmania donovani promastigotes block maturation, increase integrin expression and inhibit detachment of human monocyte-derived dendritic cells--the influence of phosphoglycans.

The protective immune response against the parasite, including the role of dendritic cells (DC) in the course of infection, plays a fundamental role. This study shows that wild-type (WT) Leishmania promastigotes and specifically the phosphoglycans family of virulence-associated antigens inhibit human monocyte-derived dendritic cells (MoDC) maturation and detachment to distinct surfaces. Immature phagocytosis of Leishmania donovani promastigotes by immature MoDC results in the increased expression of CD11b and CD51, and inhibition of cell detachment to distinct surfaces, which was dependent on the presence of phosphoglycans. These findings demonstrate that phosphoglycans of WT L. donovani might also inhibit human DC migration to lymphoid organs.     

Wild-type Leishmania donovani promastigotes block maturation, increase integrin expression and inhibit detachment of human monocyte-derived dendritic cells – the influence of phosphoglycans

The protective immune response against the parasite, including the role of dendritic cells (DC) in the course of infection, plays a fundamental role. This study shows that wild-type (WT) Leishmania promastigotes and specifically the phosphoglycans family of virulence-associated antigens inhibit human monocyte-derived dendritic cells (MoDC) maturation and detachment to distinct surfaces. Immature phagocytosis of Leishmania donovani promastigotes by immature MoDC results in the increased expression of CD11b and CD51, and inhibition of cell detachment to distinct surfaces, which was dependent on the presence of phosphoglycans. These findings demonstrate that phosphoglycans of WT L. donovani might also inhibit human DC migration to lymphoid organs.     

Leishmanicidal activity of stearylamine-bearing liposomes in vitro

Liposomes consisting of stearylamine (SA) and egg yolk phosphatidylcholine (PC) were studied for their cytotoxic activity against freshly transformed promastigotes and intracellular amastigotes of Leishmania donovani, the causative agent of visceral leishmaniasis. More than 99% of the parasites of strain AG83 were killed within 60 min by treatment with 22 mol% SA-PC liposomes (132 microg/ml total lipids). This was further confirmed by incubating the liposome-treated promastigotes at 22 C for 96 hr. The killing activity of the liposomes progressively decreased with lowering lipid concentration. However, weak cytotoxic activity was still detected at 6.6 microg/ml lipids. Leishmanicidal activity of the liposomes became stronger with increasing SA content but was reduced with the incorporation of cholesterol in the liposomes. A similar cytotoxic effect was observed on other Indian strains of L. donovani, for example PKDL and DD8, as well as on species such as Leishmania donovani S1, Leishmania donovani infantum, Leishmania tropica, Leishmania amazonensis, and Leishmania mexicana. However, freshly transformed promastigotes appeared to be more susceptible than the ones subcultured. The strong leishmanicidal activity of SA-PC liposomes was also demonstrated toward intracellular L. donovani amastigotes. The SA-bearing vesicles could effectively inhibit the growth and multiplication of the parasites within the macrophages. The cytolytic activity of these liposomes on leishmanial parasites and low toxicity on host macrophages may, thus, find application in the therapy of leishmaniasis.     

Transplasma membrane electron transport in Leishmania donovani promastigotes

Leishmania donovani promastigotes are capable of reducing certain electron acceptors with redox potential at pH 7 down to -125 mV; outside the plasma membrane promastigotes can reduce ferricyanide. Ferricyanide has been used as an artificial electron acceptor probe for studying the mechanism of transplasma membrane electron transport. Transmembrane ferricyanide reduction by L. donovani promastigotes was not inhibited by such mitochondrial inhibitors as antimycin A or cyanide, but it responded to inhibitors of glycolysis. Transmembrane ferricyanide reduction by Leishmania appears to involve a plasma membrane electron transport chain dissimilar to that of hepatocyte cells. As with other cells, transmembrane electron transport is associated with proton release, which may be involved in internal pH regulation. The Leishmania transmembrane redox system differs from that of mammalian cells in being 4-fold less sensitive to chloroquine and 12-fold more sensitive to niclosamide. Sensitivities to these drugs suggest that transplasma membrane electron transport and associated proton pumping may be targets for the drugs used against leishmaniasis.     

Protective immunity against the protozoan Leishmania chagasi is induced by subclinical cutaneous infection with virulent but not avirulent organisms

Protective immunity against Leishmania major is provided by s.c. immunization with a low dose of L. major promastigotes or with dihydrofolate-thymidylate synthase gene locus (DHFR-TS) gene knockout L. major organisms. Whether these vaccine strategies will protect against infection with other Leishmania species that elicit distinct immune responses and clinical syndromes is not known. Therefore, we investigated protective immunity to Leishmania chagasi, a cause of visceral leishmaniasis. In contrast to L. major, a high dose s.c. inoculum of L. chagasi promastigotes was required to elicit protective immunity. Splenocytes from mice immunized with a high dose produced significantly greater amounts of IFN-gamma and lower TGF-beta than mice immunized with a low dose of promastigotes. The development of protective immunity did not require the presence of NK cells. Protection was not afforded by s.c. immunization with either attenuated L. chagasi or with L. major promastigotes, and s.c. L. chagasi did not protect against infection with L. major. Subcutaneous immunization with DHFR-TS gene knockouts derived from L. chagasi, L. donovani, or L. major did not protect against L. chagasi infection. We conclude that s.c. inoculation of high doses of live L. chagasi causes a subclinical infection that elicits protective immune responses in susceptible mice. However, L. chagasi that have been attenuated either by long-term passage or during the raising of recombinant gene knockout organisms do not elicit protective immunity, either because they fail to establish a subclinical infection or because they no longer express critical antigenic epitopes.     

Leishmania donovani: immunochemical localization and secretory mechanism of soluble acid phosphatase

Monoclonal antibodies specific for the soluble, secreted acid phosphatase (EC 3.1.3.2) of Leishmania donovani were used to investigate the localization of this enzyme in extracellular promastigotes and intracellular amastigotes. Indirect immunofluorescence showed a weak general staining in the promastigote cytoplasm, together with strong fluorescence in the flagellar reservoir. Immunofluorescence studies on U937 cells infected in vitro with L. donovani showed that the pathogenic amastigote stage also produced soluble acid phosphatase. Metabolic labeling experiments using promastigotes indicated that the intracellular enzyme was soluble prior to secretion and no evidence was found for the association of secretory acid phosphatase with cell membranes after protein synthesis. The rapid release of acid phosphatase from the flagellar reservoir was energy dependent and may be coupled to beating of the flagellum. The results demonstrated that acid phosphatase was secreted into the flagellar reservoir by Leishmania promastigotes using a conventional constitutive secretory mechanism, and subsequently released from the reservoir into the extracellular medium.     

A proteogenomic approach to map the proteome of an unsequenced pathogen - Leishmania donovani

Visceral leishmaniasis or kala azar is the most severe form of leishmaniasis and is caused by the protozoan parasite Leishmania donovani. There is no published report on L. donovani genome sequence available till date, although the genome sequences of three related Leishmania species are already available. Thus, we took a proteogenomic approach to identify proteins from two different life stages of L. donovani. From our analysis of the promastigote (insect) and amastigote (human) stages of L. donovani, we identified a total of 22,322 unique peptides from a homology-based search against proteins from three Leishmania species. These peptides were assigned to 3711 proteins in L. infantum, 3287 proteins in L. major, and 2433 proteins in L. braziliensis. Of the 3711 L. donovani proteins that were identified, the expression of 1387 proteins was detectable in both life stages of the parasite, while 901 and 1423 proteins were identified only in promastigotes and amastigotes life stages, respectively. In addition, we also identified 13 N-terminally and one C-terminally extended proteins based on the proteomic data search against the six-frame translated genome of the three related Leishmania species. Here, we report results from proteomic profiling of L. donovani, an organism with an unsequenced genome.     

Inhibition of Leishmania donovani promastigote DNA topoisomerase I and human monocyte DNA topoisomerases I and II by antimonial drugs and classical antitopoisomerase agents

We have compared the inhibitor sensitivities of DNA topoisomerase I (TOPI) from Leishmania donovani promastigotes and TOPs I and II of human monocytes using pentavalent and trivalent antimonials (SbV, SbIII) and classical TOP inhibitors. Bis-benzimidazoles (Hoechst-33258 and -33342) were potent inhibitors of both parasite and human TOPI, but Hoechst-33342 was markedly less cytotoxic to promastigotes than to monocytes in vitro. Leishmania donovani was also considerably less sensitive than monocytes to camptothecin, both at enzyme and cellular levels. Sodium stibogluconate (SSG) was the only antimonial to inhibit TOPI, exhibiting a significant (P < 0.05) 3-fold greater potency against the L. donovani enzyme but showed low cytotoxicities against intact promastigotes. The SbV meglumine antimoniate failed to inhibit TOPI and showed negligible cytotoxicities, whereas SbIII drugs were lethal to parasites and monocytes yet poor inhibitors of TOPI. Monocyte TOPII was inhibited by bis-benzimidazoles and insensitive to antimonials and camptothecin. The disparity between the high leishmanicidal activity and low anti-TOPI potency of SbIII indicates that in vivo targeting of L. donovani TOPI by the reductive pathway of antimonial activation is improbable. Nevertheless, the potent direct inhibition of TOPI by SSG and the differential interactions of camptothecin with L. donovani and human TOPI support the possibility of developing parasite-specific derivatives.     

Anti-leishmanial activity of neolignans from Virola species and synthetic analogues

Surinamensin, a neolignan isolated from Virola surinamensis, 3,4,5-trimethoxy-8-[2',6'-dimethoxy-4'-(E)-propenylphenoxy]-phenylpropane, a neolignan isolated from Virola pavonis, and 25 of its synthetic analogues or correlated substances with ether linkages and their corresponding C-8 sulphur and nitrogen analogues, were tested for activity against Leishmania donovani amastigotes and promastigotes in vitro. Some were active against L. donovani promastigotes at 30 microM but inactive against intracellular amastigotes. The natural neolignan from V. pavonis was active against promastigotes at 100 microM. The highest selective activity was found in those compounds with sulphur bridges. The beta-ketosulfide (3,4-dimethoxy)-8-(4'-methylthiophenoxy)-propiophenone produced 42% inhibition of L. donovani amastigotes in the liver of BALB/c mice at 100 mg/kg given once daily for five consecutive days (P>0.05).     

Nutrient broth for the cultivation of Leishmania

Nutrient broth containing fetal calf serum was used successfully in isolating Leishmania donovani from animals and cryopreserving Leishmania major, Leishmania donovani, and Leishmania adleri. It also supported heavy growth of promastigotes of laboratory strains of L. donovani, L. major, L. adleri, and uncharacterized reptilian Leishmania-like flagellates.     

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.     

Biphenylquinuclidines as inhibitors of squalene synthase and growth of parasitic protozoa

In this paper we describe the preparation of some biphenylquinuclidine derivatives and their evaluation as inhibitors of squalene synthase in order to explore their potential in the treatment of the parasitic diseases leishmaniasis and Chagas disease. The compounds were screened against recombinant Leishmania major squalene synthase and against Leishmania mexicana promastigotes, Leishmania donovani intracellular amastigotes and Trypanosoma cruzi intracellular amastigotes. Compounds that inhibited the enzyme, also reduced the levels of steroids and caused growth inhibition of L. mexicana promastigotes. However there was a lower correlation between inhibition of the enzyme and growth inhibition of the intracellular parasites, possibly due to delivery problems. Some compounds also showed growth inhibition of T. brucei rhodesiense trypomastigotes, although in this case alternative modes of action other than inhibition of SQS are probably involved.     

Carbocyclic inosine as a potent anti-leishmanial agent: the metabolism and selective cytotoxic effects of carbocyclic inosine in promastigotes of Leishmania tropica and Leishmania donovani

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.