Energy-dependent efflux from Leishmania promastigotes of substrates of the mammalian multidrug resistance pumps.

We demonstrated the existence of three transport activities in promastigotes of Leishmania braziliensis, Leishmania guyanensis, and Leishmania mexicana. The first activity, an energy-dependent efflux of pirarubicin, was observed in all Leishmania species and inhibited by verapamil, by 2-[4-(diphenylmethyl)-1-piperazinyl]ethyl-5-(trans-4,6-dimethyl-1, 3,2-dioxaphosphorinan-2-yl)-2,6-dimethyl-4-(3-nitrophenyl)-3-py ridinecarboxylate P oxide (PAK104P) and by the phenothiazine derivatives: thioridazine, prochlorperazine, trifluoperazine, chlorpromazine and trifluoropromazine. The second activity, an energy-dependent efflux of calcein acetoxymethylester, was observed in all Leishmania species and inhibited by PAK104P and the same phenothiazine derivatives, but not by verapamil. The third activity, an energy-dependent efflux of calcein, was clearly detected in L. braziliensis and guyanensis and inhibited only by prochlorperazine and trifluoperazine. The fact that prochlorperazine and trifluoperazine inhibited the energy-dependent efflux of the three substrates suggests that these activities are mediated by the same transport system. It is noteworthy that the transport system identified in this study shares several properties with the mammalian multidrug resistance pump, MRP1. Pirarubicin, calcein acetoxymethylester and calcein are well known substrates of the MRP. Furthermore, the three types of inhibitors are also inhibitors of the MRP function.

Antiproliferative effect of illimaquinone on Leishmania mexicana.

Illimaquinone, a sponge metabolite that disrupts the Golgi complex in mammalian cells, stopped proliferation and induced morphological and ultrastructural changes in promastigotes of L. mexicana. Radioactive labeling of proteins demonstrates an increased excretion function and diminution of membrane acid phosphatase activity, due probably to the vesiculation of the Golgi complex and alteration of the cell protein sorting mechanism. The result indicated that illimaquinone could be useful for the study of intracellular traffic in Trypanosomatidae.

Characterization of mitochondrial electron-transfer in Leishmania mexicana.

Some general features of the respiratory chain and respiratory control were characterized in coupled mitochondrial preparations from Leishmania mexicana promastigotes. O2 uptake was sensitive to the electron-transfer inhibitors rotenone, flavone, malonate, 4,4,4-trifluoro-1-(2-thienyl) 1.3 butanedione (TTFA), antimycin A, 2n-nonyl-4-hydroxyquinoline-N-oxide (HQNO), myxothiazol, cyanide and azide. A high concentration of rotenone (60 microM) was required to inhibit O2 uptake effectively. Difference spectra revealed the presence of cytochromes (a + a3), b and c. Respiratory control was stimulated 2-fold by ADP with different exogenous oxidizable substrates. Calculated ADP/O ratios were consistent with the notion that ascorbate/N,N,N',N'-tetramethylphenylenediamine (TMPD)-linked and FAD-linked respiration proceeds, respectively, with one third and two thirds of the ATP producing capacity of NADH-linked respiration. State 3 was suppressed by the ATP synthase inhibitors oligomycin and aurovertin and by the adenine nucleotide translocator inhibitors atractyloside and carboxy atractyloside. The protonophore carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) provoked state 3u respiration. The mitochondrial preparation was capable of Ca2+ uptake and Ca2+ stimulated respiration. Data obtained suggests strongly that mitochondrial complexes I, II, III and IV are present in a major pathway of electron-transfer and that oxidative phosphorylation might proceed with high bioenergetic efficiency.

Naturally azole-resistant Leishmania braziliensis promastigotes are rendered susceptible in the presence of terbinafine: comparative study with azole-susceptible Leishmania mexicana promastigotes.

Leishmania braziliensis (isolate 2903) was naturally resistant to ketoconazole or the bis-triazole D0870, inhibitors of sterol C-14 demethylase, which produced only moderate effects on the proliferation of promastigotes at 10 microM. In contrast, Leishmania mexicana (isolate NR) was extremely susceptible to the azoles, as complete growth arrest and cell lysis were induced by incubation of the parasites with 0.05 microM concentrations of the drugs for 72 h. The opposite response was observed with terbinafine, an inhibitor of squalene epoxidase: L. braziliensis 2903 was three times more susceptible to the drug than L. mexicana NR (MICs of 5 and 15 microM, respectively). However, when the L. braziliensis stock was grown in the presence of 1 microM terbinafine, which by itself produced only marginal (< 10%) effects on growth, it became highly susceptible to the azoles, with an MIC of 0.03 microM. Analysis of cellular free sterols by high-resolution capillary gas chromatography coupled to mass spectrometry showed that 14-methyl sterols can support normal growth of L. braziliensis 2903 but not of L. mexicana NR. On the other hand, the higher susceptibility of the L. braziliensis isolate to terbinafine was correlated with a massive accumulation of squalene in the presence of the allylamine while no significant effects on L. mexicana sterol composition were observed at drug concentrations up to 1 microM. Thus, the > 300-fold increase in the susceptibility of L. braziliensis promastigotes to azoles in the presence of terbinafine was attributed to the combined effect of squalene and the methylated sterol precursors on the physical properties of the cell's membranes, leading to the loss of cell viability. Combination therapy with azoles and terbinafine in the treatment of human L. braziliensis infections deserves further study.

Amphotericin B kills unicellular leishmanias by forming aqueous pores permeable to small cations and anions.

The polyene antibiotic amphotericin B (AmB) is known to form two types of ionic channels across sterol-containing liposomes, depending on its concentration and time after mixing (Cohen, 1992). In the present study, it is shown that AmB only kills unicellular Leishmania promastigotes (LPs) when aqueous pores permeable to small cations and anions are formed. Changes of membrane potential across ergosterol-containing liposomes and LPs were followed by fluorescence changes of 3,3' dipropylthiadicarbocyanine (DiSC3(5)). In KCl-loaded liposomes suspended in an iso-osmotic sucrose solution, low AmB concentrations ( NO3 > Cl > I > Br > acetate (SO2-4 being impermeable). Cell killing by AmB was followed by fluorescence changes of the DNA-binding compound ethidium bromide (EB). At low concentrations (/=0.1 microM), a salt influx via the aqueous pores formed by the antibiotic was followed by osmotic changes leading to cell lysis. This last stage is supported by electron microscopy observations of the changes of parasite morphology immediately upon addition of AmB, which indicated that the typical elongated promastigote cell forms became rounded and the flagella swells and round up. The present work is the first demonstration of the in vitro sensitivity of Leishmania promastigotes to osmotic lysis by AmB.

Ouabain-sensitive Na+,K(+)-ATPase in the plasma membrane of Leishmania mexicana.

The mechanism responsible for the regulation of intracellular Na+ and K+ concentrations in trypanosomatids is unknown. In higher eukaryotes a ouabain-sensitive Na+,K(+)-ATPase located in the plasma membrane is the main mechanism for the regulation of the intracellular concentrations of Na+ and K+, while in trypanosomatids there are conflicting evidences about the existence of this type of ATPase. By the use of a highly enriched plasma membrane fraction, we showed that an ouabain-sensitive Na+,K(+)-ATPase is present in L. mexicana. The affinity of the enzyme for Na+ and K+ is similar to that reported for the mammalian Na+,K(+)-ATPase, showing also the same kinetic parameters regarding the relative concentration of those cations that give the optimal activity. Vanadate (10 microM) fully inhibits the ATPase activity, suggesting that the enzyme belongs to the P-type family of ionic pumps. The enzyme is sensitive to ouabain and other cardiac glycosides. These cardiac glycosides do not show any appreciable effect on the higher Mg(2+)-ATPase activity present in the same preparation. By the use of [3H]ouabain, we also show in this report that the binding of the inhibitor to the enzyme was specific. Taken together, these results demonstrate that an ouabain-sensitive Na+,K(+)-ATPase is present in the plasma membrane of Leishmania mexicana. Therefore, this Na+,K(+)-ATPase should participate in the intracellular regulation of these cations in Leishmania.

The role of anions in pH regulation of Leishmania major promastigotes.

The pH regulation of Leishmania major promastigotes was studied as a function of the ionic composition of the medium and in response to acid and alkali load. Intracellular pH (pHi) was monitored by on-line ratio fluorescence using the fluorescence-dependent pH indicator 2',7'-bis-(carboxyethyl)-5,6-carboxyfluorescein (BCECF). In Cl(-)-based medium (pH 7.4, 30 degrees C), the steady state pHi was maintained at 6.75 +/- 0.01. Only a minor (< or = 0.07 +/- 0.02 unit) decrease in steady state pHi was observed when parasites were treated with H(+)-ATPase inhibitors such as vanadate, N-ethylmaleimide, or bafilomycin. After treatment with the impermeant anion transport blocker DIDS, or in the presence of the reduced analog H2DIDS, pHi decreased by > or = 0.2 unit. In gluconate-based medium, however, pHi gradually decreased to 6.53 +/- 0.05 and showed a swift but time-dependent recovery (alkalinization) when Cl- or other halides or nitrate were restored to the medium. That recovery was also inhibited by pretreating cells with DIDS or exposing them to H2DIDS. The findings provide evidence for Cl- transport mechanisms that support a pHi regulatory process which is operative in acidic-neutral cytoplasmic milieu. Under alkali load induced by weak base treatment, parasites undergo a rapid alkalinization which was followed first by a fast but limited acidification and subsequently by a slower but more robust acidification (recovery) to reach a pHi of 6.85 +/- 0.05. The recovery of pHi was markedly reduced in the presence of H2DIDS and/or in the absence of Cl- in the medium. Based on these results and on the fact that the natural parasite environment is both alkaline and rich in HCO3-/CO3(2-) ions, we propose (Cl-)o-(HCO3-)i or (Cl-)o-(OH-)i exchange as the major mechanism of regulatory cell acidification which is operative upon cell alkalinization. The possibility that similar pH regulatory mechanisms are operative in Leishmania promastigotes in both acidic and alkaline conditions is considered. The putative pH regulatory mechanisms might serve as potential targets for therapeutic intervention.

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 heterogeneity of Leishmania cell-surface antigens.

A comparative study of the radioiodinated promastigote cell-surface antigens of Leishmania mexicana and L. major was carried out under reduced and nonreduced conditions by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by autoradiography. Under reduced conditions, the cell surface of L. mexicana promastigotes showed three iodinated polypeptides with molecular weights of 65,000, 50,000 and 27,000 daltons, whereas L. major promastigotes displayed a single polypeptide of 63,000 daltons. Under nonreduced conditions, the radioiodinated cell-surface component of L. major shifted to a mol.wt. of 51,000 daltons, whereas only one of the three components of L. mexicana (mol.wt., 65,000 daltons) underwent a large shift (to 59,000 daltons). The different immunochemical nature of the L. mexicana cell-surface antigens was demonstrated by using different anti-Leishmania sera. The rabbit anti-promastigote serum immunoprecipitated mainly the 50,000- and 27,000-dalton L. mexicana cell-surface polypeptides, whereas the rabbit anti-amastigote serum as well as a serum from a patient with cutaneous leishmaniasis immunoprecipitated almost exclusively the 65,000-dalton polypeptide. Immunoblot studies using a rabbit antibody against the L. major deglycosylated major surface antigen gp63 confirmed the differences in nature of the 65,000- and 50,000-dalton cell-surface antigens of L. mexicana. The results obtained are discussed in the light of the differences in antigenic cell-surface expression among Leishmania isolates and their consequences in the development of a differential diagnosis of leishmaniasis.

Plasma membrane and cytoskeletal constituents in Leishmania mexicana.

The presence and the localization of actin, spectrin and ankyrin are studied by immunofluorescence and immunoblotting in Leishmania mexicana promastigotes growing in vitro. These proteins, amphitropic in nature, coexist both in soluble and insoluble forms. Our results demonstrate that the Triton insoluble form of these proteins constitutes beside tubulin the cytoskeletal scaffold of promastigotes in close association with the plasma membrane, the axoneme and the basal body of the parasite.

The effect of tunicamycin on Leishmania brasiliensis. Glycosylation and the cell surface components.

Culture conditions of Leishmania cells were developed to allow the study of the effect of tunicamycin (TM) on glycosylation and on the cell surface components. Leishmania incorporate [14C]-mannose and [35S]-methionine in vitro. The incorporation of [14C]-mannose is linear for 150 min and is inhibited by TM (2 micrograms/ml) in a time dependent effect which reaches a plateau of 45% inhibition at 36 h. Under the same experimental conditions [35S]-methionine incorporation into protein is slightly affected. This is reflected by an almost identical polypeptide pattern for TM treated and non-treated cells when analyzed on SDS-PAGE. On the contrary, strong differences were detected on the labeled compounds analyzed on SDS-PAGE followed by autoradiography when the precursor used was [14C]-mannose. A shift in the electrophoretic mobility of most of the glycopeptides synthesized in the presence of TM was observed, which is also reflected in the structure of the main Leishmania cell surface components. The findings are discussed in the light of biological implications.

The effect of tunicamycin on Leishmania braziliensis cell growth, cell morphology and ultrastructure.

The effect of tunicamycin (TM) on Leishmania braziliensis promastigotes in culture has been studied. TM at different concentrations (2, 4, 6 micrograms/ml) inhibits promastigote growth as the mean generation time of control cells, 36 hr, is changed to 41, 46 and 55 hr, respectively. Cells remain viable after long exposure to 2 micrograms/ml of TM and can be cultured in the presence of the drug for several generations. Under these conditions cells tend to round up and many "ruffle"-like structures appear at the parasite cell surface. At the ultrastructural level, cell coat disappears and the rough endoplasmic reticulum appears distended. Other structures remain unaltered by the drug treatment. The changes in cell morphology are discussed in relation to changes in cell surface morphology. The possible use of these TM-transformed cells as experimental systems for host-parasite studies is also considered.

Production and secretion of Leishmania braziliensis proteins.

The kinetics of secretion of proteins by Leishmania braziliensis was followed by incorporation of [3H]leucine into macromolecules produced by the cells which are released into the growth medium. About 10% of the total protein synthesized by actively growing cells is secreted. Cycloheximide (100 microgram/ml) and puromycin (0.5 mM) inhibited the incorporation of labelled leucine by 85 and 99%, respectively. The secreted proteins do not seem to result from cell lysis since, first, the kinetics of production are linear and, secondly, less than 1% of thymidine or uridine incorporated by the cells is found in the medium. Cells grown with [3H]leucine and then transferred to fresh medium show two phases of secretion. During the first six hours, it is slow and reaches a plateau. The release increases about ten-fold during the next six hours. An analysis of the secreted material showed that following precipitation with methanol and sodium acetate, three isotopically labelled peaks were eluted from Sephadex G-120-150. The first of these, containing 50% of the radioactivity, did not react with anti-leishmanial serum, while the last two did. Since the last two fractions could be labelled with [3H]glucosamine as well as [3H]leucine it is suggested that they are glycoprotein in nature and are similar to the products released by other species of Leishmania.