Effects of antimycotic azoles on growth and sterol biosynthesis of Leishmania promastigotes

Promastigotes of 36 World Health Organization reference (and other) strains of 6 species and 10 subspecies of Leishmania were cultured in the presence of 3 antimycotic azole drugs (ketoconazole, itraconazole, fluconazole) and their population growth determined. A representative of each subspecies was also analyzed for its sterol composition. For all strains the order of azole drug activity with respect to both growth and sterol biosynthesis inhibition was itraconazole greater than or equal to ketoconazole greater than fluconazole. The inhibitory actions of the three azole drugs were greater on L. donovani and L. braziliensis subspecies and on L. mexicana amazonensis than on L. aethiopica, L. major, L. tropica and L. mexicana mexicana. The nature of the changes in sterol composition caused by the drugs was the same for all strains. The normal, major endogenous sterols of the promastigotes (5-dehydroepisterol and ergosterol) were reduced in amount to 1-2% of the total free sterols and were replaced by endogenous 14 alpha-methyl sterols and exogenous cholesterol. The changes occurred rapidly, were drug concentration dependent and coincided with growth inhibition. Six strains of those Leishmania species less sensitive to the azole drugs could be subcultured indefinitely at reduced growth rates in the presence of a ketoconazole concentration causing the same extraordinary alterations in sterol composition. This suggested that the bulk membrane functions of sterols in leishmanias can be served by 14 alpha-methyl sterols and cholesterol, albeit imperfectly, while traces of 14 alpha-desmethyl sterols are needed for uncharacterized metabolic functions.     

Effects of ketoconazole on growth and sterol biosynthesis of Leishmania mexicana promastigotes in culture

Ketoconazole, a clinically effective antimycotic agent active in vitro against the amastigote stage of Leishmania mexicana Walter Reed 227 in human monocyte-derived macrophages, was found to inhibit growth and impair sterol biosynthesis of the cultured promastigote stage by approx. 50% at a concentration of approx. 10(-8)M. Sterol biosynthesis was interfered with at the level of the removal of the 14 alpha-methyl group of lanosterol, as judged by changes in the distribution of [2-14C]mevalonate radioactivity among desmethyl sterol and methyl sterol thin-layer chromatography fractions, by the loss of 4-desmethyl sterols (mainly 5-dehydroepisterol), and by the accumulation of 14 alpha-methyl sterols. The growth inhibition and sterol changes were evident in promastigotes cultured in a cholesterol-rich medium and in a cholesterol-poor medium, even though promastigotes incorporated cholesterol. The mechanism of action of ketoconazole against promastigotes may be that postulated for Candida albicans: interference with membrane permeability secondary to loss of desmethyl sterols and accumulation of 14 alpha-methyl sterols.     

Biosynthesis and secretion of acid phosphatase by Leishmania donovani promastigotes

Metabolic labeling and immunoprecipitation experiments demonstrated that soluble acid phosphatase (EC 3.1.3.2) was rapidly synthesized and released into culture medium by Leishmania donovani promastigotes. The kinetics of release indicated a constitutive secretory process (t 1/2 = 45 min). Moreover, acid phosphatase was the major secretory protein. The extracellular enzyme is composed of two heterodisperse bands of approximately 110 and 130 kDa in sodium dodecyl sulphate-polyacrylamide gels. It is synthesized as two intracellular precursors of 92.5 and 107 kDa which acquire the heterodisperse form characteristic of the mature extracellular enzyme during biosynthesis. Labeling in the presence of tunicamycin altered the electrophoretic mobility of the acid phosphatase, indicating the presence of several N-linked oligosaccharides on the mature enzyme. However, tunicamycin did not block secretion of the enzyme or its processing to the heterodisperse form. The biosynthetic effect of tunicamycin was mimicked by N-glycosidase F treatment of acid phosphatase immunoprecipitates. In contrast to tunicamycin, labeling in the presence of monensin inhibited processing of the phosphatase to its heterodisperse form. This indicates that Golgi processing, probably glycosylation, is responsible for the heterodispersity of the mature enzyme in sodium dodecyl sulphate-polyacrylamide gels. As with tunicamycin, monensin treatment did not prevent secretion of the acid phosphatase. These cumulative results demonstrate that release of this enzyme by L. donovani promastigotes occurs via a secretory pathway.     

Effects of oxygen concentration on the intermediary metabolism of Leishmania major promastigotes

Leishmania major promastigotes grown in late log phase were incubated with glucose as sole exogenous carbon source in the presence of 5% CO2 and the amounts of glucose consumed and of the major products formed--succinate, pyruvate, alanine, acetate, glycerol, and D-lactate--were measured as a function of pO2. Glucose consumption increased as pO2 was lowered to 6% (a positive Pasteur effect) and then declined to the same level at 95% N2 as at 95% O2. The production of D-lactate and of glycerol increased as pO2 dropped from 95%, reaching a maximum at about 2% O2. Succinate production, however, increased dramatically when pO2 was reduced to 6% and remained at that level with further reduction of pO2. The amount of succinate produced relative to the amount of glucose carbon consumed suggests utilization of an endogenous carbon source. Acetate production did not change between 95% O2 and 6% O2 and then declined with decreasing pO2. These observations suggest the presence of two sensors, one with a high and one with a low affinity for oxygen. When glycerol or alanine were the only exogenous sources of carbon, the primary products released were acetate and succinate. Acetate production from alanine declined slightly as pO2 was reduced to 2%, and then dropped markedly when pO2 was reduced to 0%. Acetate production from glycerol increased over 4-fold when the pO2 was reduced from 95% to 4%, and then declined with further reduction in pO2. No succinate was formed from either substrate until complete anaerobiosis. This pattern of response, while differing from that when glucose was sole exogenous carbon source, is also consistent with the regulation of metabolism by a high and a low affinity O2 sensor. Cells from cultures in early stationary phase, before the appearance of metacyclic forms, consumed glucose at about the same rate as log phase promastigotes, but did not show a Pasteur effect. Stationary cells also consumed glycerol at the same rate as did log phase promastigotes, but consumed alanine at a much lower rate. Reduction of pO2 affected product formation from each of these substrates differently than for log phase promastigotes, demonstrating the sensitivity of several pathways of intermediary metabolism to regulation by pO2 during the transition from log to stationary phase.     

Golgi-mediated post-translational processing of secretory acid phosphatase by Leishmania donovani promastigotes

Monensin, an inhibitor of Golgi function, was used to investigate the role of this cell compartment in the glycosylation of Leishmania donovani promastigote secretory acid phosphatase (EC 3.1.3.2). Monensin-treated cells demonstrated morphological changes in the Golgi complex and secreted enzyme with an altered electrophoretic mobility: two discrete bands of approximately 95 and 110 kDa were found, as compared to the heterodisperse nature of the enzyme from untreated controls. Chemical deglycosylation by mild acid hydrolysis resulted in a similar effect on the electrophoretic mobility of purified extracellular enzyme. Acid phosphatase was also treated with N-glycosidase F (EC 3.5.1.52) to remove N-linked oligosaccharides. The altered lectin-binding properties of the enzyme after these two treatments demonstrated that an unusual type of galactose-containing acid-labile carbohydrate was present in secretory acid phosphatase in addition to the N-linked oligosaccharides. Further, experiments with 32P-labelled enzyme indicated that phosphodiester bonds were the structural component responsible for the sensitivity of this carbohydrate to mild acid hydrolysis. Cumulatively, these results demonstrated that a novel form of Golgi-mediated posttranslational modification had occurred to the secretory acid phosphatase presumably by the addition of an acid-labile phosphoglycan.     

Effects of hypoxia and acute osmotic stress on intermediary metabolism in Leishmania promastigotes.

This study further explores the effects of hypoxia and acute osmotic stress on intermediary metabolism of Leishmania major and Leishmania donovani. Late log phase promastigotes were washed and incubated with glucose as the sole exogenous carbon source, and rates of glucose consumption and product formation were measured as a function of osmotic strength (610, 305, and 167 mOsm kg ¹) and pO₂ (95, 10, and 0% O₂) in the presence of 5% CO₂. Very mild hypoxia dramatically altered flux through the pathways of intermediary metabolism and increased the rates of production of the major metabolites, thus confirming the presence of a low-affinity O₂ sensor which was active under all osmolalities tested. The data also require that as pO₂ is lowered towards anoxia an endogenous carbohydrate source is mobilized. Under aerobic conditions, acute hypo-osmotic stress had little effect on product formation, whereas acute hyperosmotic stress altered metabolism in a manner similar to mild hypoxia, with the exception of decreasing the rates of acetate and succinate production. It was also shown in L. donovani promastigotes that the effects of anoxia and hyperosmolality were not additive. Thus, separate sensors with partially overlapping actions are involved in the metabolic responses to hypoxia and hyperosmolality. There was no apparent species-specificity for the responses to pO₂ and osmotic stress. Uncoupling with carbonyl cyanide p-trifluoromethoxyphenylhy-drazone caused changes in metabolite flux patterns which differed from the changes caused by either hypoxia or acute osmotic stress, while rotenone and calcium ionophore A23187 had no significant effects. The identity of the sensors responsive to pO₂and osmolality, and the mechanisms by which they regulate flux through the pathways of intermediary metabolism, require further study.     

Effects of dietary supplementation with eicosapentaenoic acid or gamma-linolenic acid on neutrophil phospholipid fatty acid composition and activation responses

Previous data that alimentation with fish oil rich in eicosapentaenoic acid (EPA; 20: 5n-3) or vegetable oil rich in gamma-linolenic acid (GLA; 18: 3n-6) can reduce symptoms of inflammatory skin disorders lead us to determine the effects of dietary supplements of oils rich in EPA or GLA on guinea pig (GP) neutrophil (PMN) membrane potential (), secretion, and Superoxide (O ₂ ^– ) responses. Weanling GPs were initially fed diets supplemented with olive oil (<0.1% EPA; <0.1% GLA) for 2 weeks, followed by a crossover by two sets of animals to diets supplemented with fish oil (19% EPA) or borage oil (25% GLA). At 4-week intervals, 12% sterile casein-elicited peritoneal neutrophils (PMN) were assessed for membrane polyunsaturated fatty acid (PUFA) profiles and FMLP-, LTB ₄ ^s- , and PMA-stimulated changes, changes in flow cytometrically measured forward scatter (FWD-SC) (shape change), 90 scatter (90-SC) in cytochalasin B-pretreated-PMN (secretion response), and Superoxide responses. GP incorporated EPA and GLA (as the elongation product, dihomo-GLA or DGLA) into their PMN phospholipids by 4 weeks. The peritoneal PMN of all groups demonstrated broad resting FWD-SC and poor activation-related FWD-SC increases, suggesting in vivo activation. While secretion was comparable in the three groups in response to FMLP, there was a trend toward inhibition of LTB₄-stimulated 90-SC loss in both fish and borage oil groups. This was significant only with borage oil (21.7±2.1 vs 15.3±1.2% loss of baseline 90-SC, olive vs borage;P=0.03). PMN from borage- and fish oil-fed GPs showed a progressively lower O ₂ ^– response to FMLP than the olive oil group (73.9±3.9 and 42.9±6.8% of olive oil response for borage and fish oils, respectively;P< 0.005 andP<0.01, respectively, at 12 weeks), while PMA-stimulated O ₂ ^– was inhibited only in the fish oil-fed group and only at 12 weeks (62.0±2.7% of control;P<0.025). We conclude that dietary supplementation with oils rich in PUFAs can modify PMN activation responses.This work was supported in part by Research Grants AM-30679, AR 39040, and P30-AM 35747-02 from the U.S. Public Health Service.     

Effect of protein kinase inhibitors on the growth, morphology, and infectivity of Leishmania promastigotes

The effect of two protein kinase inhibitors, staurosporine and H-7, on the growth, morphology and infectivity of Leishmania major and Leishmania amazonensis promastigotes was examined. Incubation with H-7 (600 μM) for up to one hour had no effect on parasite growth, morphology or infectivity. Staurosporine, however, was cytotoxic for promastigotes and incubation for 1, 5 or 15 minutes with 10 μM inhibitor killed 19, 34 and 59 %, respectively, of the parasites. Longer incubations, up to one hour, at this concentration did not increase parasite killing. However, treatment with 25 μM staurosporine for one hour was highly toxic, only 4 % of the promastigotes surviving after 72 h. Lower concentrations of staurosporine, 0.25 and 2.5 μM, had only minor effects on parasite growth. Incubation of either L. major or L. amazonensis with staurosporine (10 μM for 10 minutes) caused marked morphological changes in the size and appearance of the flagellar pocket, and/or cytoplasm of the viable parasites. Treated parasites were still capable of infecting mouse peritoneal macrophages and causing disease in BALB/c mice, though the treated parasites were less virulent than control promastigotes. These results indicate that staurosporine, while inhibiting promastigote growth, does not prevent differentiation to amastigotes and amastigote replication. Received: 26 June 1996 / Accepted: 20 August 1996     

Detection of characteristic distributions of phospholipid head groups and fatty acids on neurite surface by time-of-flight secondary ion mass spectrometry

Neurons have a large surface because of their long and thin neurites. This surface is composed of a lipid bilayer. Lipids have not been actively investigated so far because of some technical difficulties, although evidence from cell biology is emerging that lipids contain valuable information about their roles in the central nervous system. Recent progress in techniques, e.g., mass spectrometry, opens a new epoch of lipid research. We show herein the characteristic localization of phospholipid components in neurites by means of time-of-flight secondary ion mass spectrometry. We used explant cultures of mouse superior cervical ganglia, which are widely used by neurite investigation research. In a positive-ion detection mode, phospholipid head group molecules were predominantly detected. The ions of m/z 206.1 [phosphocholine, a common component of phosphatidylcholine (PC) and sphingomyelin (SM)] were evenly distributed throughout the neurites, whereas the ions of m/z 224.1, 246.1 (glycerophosphocholine, a part of PC, but not SM) showed relatively strong intensity on neurites adjacent to soma. In a negative-ion detection mode, fatty acids such as oleic and palmitic acids were mainly detected, showing high intensity on neurites adjacent to soma. Our results suggest that lipid components on the neuritic surface show characteristic distributions depending on neurite region.     

Characterization of RNA from Leishmania tropica and Leishmania d.donovani promastigotes

RNA has been prepared from promastigotes of Leishmania tropica and Leishmania d.donovani using three different methods. Extraction by hot phenol/isothiocyanate gave the best quantitative and qualitative results. The analysis of total RNA on methyl mercuric agarose gels shows that the large rRNA species is nicked: it is composed of a 630 and a 560 kDa molecule. The small rRNA species has a molecular weight of 800,000. Poly(A+) RNA can be translated in a rabbit reticulocyte lysate system. The newly synthesized products comprise high molecular weight proteins and show different patterns using RNA from L. tropica or from L. d. donovani promastigotes.     

Cytotoxicity of ester and ether lysophospholipids on Leishmania donovani promastigotes

The cytotoxic activity of four ester lysophospholipids, three ether lysophospholipids, and two radylglycerols on Leishmania donovani promastigotes was determined by measuring the inhibition of cell growth. The 1-acyl lysophospholipids reduced cell growth to 50% of controls at concentrations of 6.4-10.9 microM. In contrast, 1-O-alkenyl-sn-glycero-3-phosphoethanolamine, 1-O-hexadecyl-sn-glycero-3-phosphocholine, and 1-O-hexadecyl-sn-glycerol already showed a 50% inhibition of growth at concentrations between 2.1 and 2.8 microM. Moreover, the unnatural alkyl lysophospholipid analogue 1-O-octadecyl-2-methoxy-sn-glycero-3-phosphocholine was even 10-fold more toxic. Incubations of L. donovani promastigotes with radioactively labelled ether lysophospholipids revealed a rapid uptake of these compounds and their incorporation into cellular lipids at a non-toxic concentration of 1.0 microM. An accumulation of the lysophospholipids in the cell due to insufficient metabolism may be the cause of its cytotoxic effect. The sensitivity of L. donovani cells towards ether lysophospholipids was found to be similar to that reported for tumor cells.     

Effects of polyunsaturated fatty acids on interleukin-2-dependent T cell growth

Cellular membranes, in addition to serving as structural constituents of cells, also provide precursors for a number of chemical messengers involved in intracellular signal transduction. This includes the eicosanoids (prostaglandins and leukotrienes) and diacylglycerol, and activator of protein kinase C (PKC). Changes induced in the fatty acid profile of lymphocytes can influence vital metabolic processes in cells. Such changes, independent of the function of fatty acids as prostaglandin and leukotriene precursors, can alter the development and regulation of immune responses. In this report we study the effects of the polyunsaturated fatty acids (PUFA) on proliferation and signal transduction in the interleukin-2 (IL-2)-dependent murine T cell line CTL.L-2. Culture of CTL.L-2 cells in the presence of specific PUFA resulted in their incorporation into the cellular phospholipids. IL-2-induced proliferation of CTL.L-2 cells was markedly suppressed in a dose-dependent fashion by incubation in media supplemented with dihomogammalinolenic acid (an n-6 PUFA) slightly inhibited proliferation, while eicosapentaenoic acid (an n-3 PUFA) had no effect. Neither indomethacin (a cyclooxygenase inhibitor) nor nordihydroguaiaretic acid (NDGA, a lipoxygenase inhibitor) reversed the effect of DGLA. In contrast, phorbol 12-myristate 13-acetate (a phorbol ester and activator of PKC), blocked, in a dose-dependent manner, the antiproliferative effect of DGLA. This study presents evidence that PUFA alter signal transduction in cells in a manner which is separate from their function as eicosanoid precursors. The botanical lipid-derived DGLA has a potent suppressive effect on IL-2-driven T cell proliferation and may alter signal transduction by modification of second messenger or PKC activity.     

Mechanism of action of amphotericin B on Leishmania donovani promastigotes

The growth of Leishmania donovani promastigotes in a liquid medium was completely inhibited by amphotericin B at a concentration of 0.3 microgram ml-1 (0.3 microM). Continuous release of small molecules that absorb at 260 nm and 280 nm was observed after contact with the drug. Uptake of [U-14C]glucose was inhibited in cells treated with the drug. An immediate release of isotopic glucose and its metabolites from preloaded cells could be demonstrated after incubation with amphotericin B (0.4 microM). Inhibition of respiration by the drug was a comparatively slower process. All the above effects could be effectively prevented in the presence of either cholesterol or ergosterol. The primary site of action of amphotericin B on L. donovani promastigote cells appears to be membrane sterols that result in a loss of the permeability barrier to small metabolites. An interesting biochemical similarity, thus, emerges between flagellated protozoa and fungi.     

Effects of fatty acids on growth of Bordetella pertussis in defined medium.

The effects of saturated and unsaturated fatty acids on the growth of Bordetella pertussis strain 114 in defined medium were tested. Individual fatty acids were found to be either inhibitory or stimulatory to growth, depending on concentration. Myristic (C14), pentadecanoic (C15), and palmitic (C16) acids were the most inhibitory saturated fatty acids tested. B. pertussis 114 was extremely sensitive to the unsaturated fatty acids oleic (C18; cis-9), elaidic (C18; trans-9) and petroselinic (C18; cis-6).     

Rapid uptake and esterification of arachidonic acid and other fatty acids by microfilariae of Brugia malayi

We have examined the differential incorporation and esterification of exogenous fatty acids by microfilariae of the human filarial parasite Brugia malayi. Microfilariae incubated with 2 nM [3H]arachidonic acid over 1 h rapidly took up this fatty acid. Palmitic, oleic and linoleic acids were also incorporated by parasites. In contrast to these other fatty acids, little incorporated arachidonic acid remained as free fatty acid within microfilariae. Arachidonate was rapidly esterified into phospholipids, with 66% of incorporated arachidonate esterified into phospholipids at 1 min. Esterification of other fatty acids into phospholipids was quantitatively lesser and occurred into phosphatidylcholine and phosphatidylethanolamine. Arachidonate was preferentially esterified into phosphatidylinositol, which constituted only 10% of the total parasite phospholipid pool, and into phosphatidylcholine. By 1 min these two phospholipid classes, respectively, comprised 53% and 43% of [3H]arachidonyl-phospholipids. Neither the microfilarial incorporation of arachidonate nor its esterification into parasite phospholipids could be saturated by noncytotoxic concentrations of up to 600 microM. Microfilariae, which in vivo are exposed to arachidonate in blood, can rapidly, avidly and with high capacity incorporate exogenous arachidonate and esterify it preferentially into specific classes of phospholipids, including phosphatidylinositol. Like many mammalian cells, these phylogenetically distinct metazoan parasites possess efficient means for utilizing host-derived arachidonic acid.     

Inhibition of interferon gamma-induced macrophage microbicidal activity against Leishmania major by liposomes: inhibition is dependent upon composition of phospholipid headgroups and fatty acids

Multilamellar liposomes of phosphatidylcholine and phosphatidylserine at a 7:3 molar ratio significantly inhibited activation of murine resident peritoneal macrophages by recombinant murine interferon-gamma for cytotoxicity against amastigotes of the protozoan parasite Leishmania major; other macrophage effector functions, such as particle phagocytosis or tumoricidal activity, were unaffected. This inhibition was not due to direct toxic effects of liposomes against parasite or macrophage, was fully reversible, and was directed at one or more early events in macrophage-LK interactions which ultimately induce microbicidal activity. Liposomes containing some natural phospholipids (phosphatidylserine, phosphatidylethanolamine, phosphatidic acid or diphosphatidyl glycerol), but not phosphatidylcholine, phosphatidylglycerol, or several synthetic saturated phospholipids, prevented the induction of macrophage microbicidal activity. Inhibition by liposomes of various composition was not related to the efficiency with which these vesicles were ingested by macrophages. Inhibitory activity was directly influenced by changes in the phospholipid head group, as well as by the number of unsaturated bonds in phospholipid fatty acids: for a given phospholipid in liposomes, inhibition was directly related to the number of unsaturated bonds among the fatty acids. These data support a role for phospholipids in postbinding regulation of macrophage activation and add to our understanding of how liposome delivery systems can be designed to avoid potential microbicidal suppressive effects.     

Isoenzyme and ultrastructural characterization of Leishmania tropica axenic amastigotes and promastigotes

Leishmania tropica is one of the main etiological agents of cutaneous leishmaniasis in Iran. For ultrastructural and isoenzyme study, axenic amastigotes were cultured in a brain–heart infusion medium containing 20 % fetal calf serum, pH?4.5, and incubated at 37 °C in 5 % CO₂. Different stages of L. tropica revealed the same isoenzyme profiles after comparing four enzyme systems including phosphoglucomutase, 6-phosphogluconate dehydrogenase, malate dehydrogenase, and nucleoside hydrolase II. Different isoenzyme patterns for glucose-phosphate isomerase, glucose-6-phosphate dehydrogenase, nucleoside hydrolase I, and malic enzyme enzymic systems were seen; thus, these isoenzyme systems among the eight systems studied were more efficient in characterizing L. tropica amastigotes. The structure of the axenic amastigotes was essentially similar to that of the promastigotes except for some important characteristics including the flagellum, flagellar pocket, paraxial rod, and the subpellicular microtubules.     

Effects of short-chain fatty acids on in vitro bacterial growth of Bacteroides fragilis and Escherichia coli

Short-chain fatty acids (SCFA) are produced in the large bowel of nonruminant mammals by bacterial anaerobic fermentation. The aim of the present study was to investigate the effects of SCFA on the in vitro growth of Bacteroides fragilis and Escherichia coli. B. fragilis and E. coli isolated from fresh human clinical samples and a reference strain for each species were incubated in a meat infusion broth with increasing amounts of SCFA and grown under anaerobic conditions at a temperature of 37 degrees C. Bacterial growth was estimated by spectrophotometry. Rate of growth was calculated from the logarithmic growth phase. SCFA, in concentrations normally found in the human colon, had a significant (p<0.01) inhibitory effect of the in vitro growth rate for E. coli, while they were without effect on the in vitro growth rate of B. fragilis. It may be concluded that under in vitro conditions SCFA had growth-inhibitory effects on E. coli, while they had no effect on B. fragilis.