Amphotericin B-Induced Carbohydrate Changes on the Trypanosoma cruzi Surface Membrane

ABSTRACT Changes in the cell surface carbohydrates of Trypanosoma cruzi epimastigotes induced by Amphotericin B (AmB) were assessed by chemical methods and by agglutination assay employing a panel of highly purified lectins of various sugar specificities, Escherichia coli K12 with mannose-sensitive fimbriae was also used as an agglutination probe. Amphotericin B caused a decrease in the total carbohydrate content of all glycoconjugate fractions isolated. Exposure to AmB strongly affected the mannose/galactose ratio (1:5) in the CHCI3/methanol/H2O soluble fraction. These sugars in 1.4:1 ratio were the major hexose components of control cells. The decrease in the mannose content (48 to 15%) after AmB treatment agrees with the marked decrease in the T. cruzi cell surface receptors for fimbriated E. coli K12. Also, an increase in the galactose content (74%) as compared with control cells (34%) is in agreement with the peanut agglutinin and Euonymus europaeus lectins agglutination results. Differences in the cell surface carbohydrates induced by AmB could be associated with alterations in the membrane structure and organization.     

Amphotericin B-induced damage of Trypanosoma cruzi epimastigotes

Amphotericin B (AmB) autoxidation resulted in oxygen consumption, superoxide anion formation and production of thiobarbituric acid (TBA)-reactive material (malondialdehyde). Malondialdehyde formation increased after incubation of the drug with ascorbate-ADP-FeCl3. Growth of Trypanosoma cruzi epimastigotes in the presence of AmB induced a decrease in the free fatty acid content of the cells (57% in control cells vs. 7% in AmB-treated cells), and in the proportion of unsaturated fatty acids as well as cell killing. No changes were detected on sterol content. No evidence was found for lipid peroxidation as a mechanism of cell injury by this antibiotic.     

The cell surface of Phytomonas davidi

Carbohydrates were located on the surface of Phytomonas davidi using ultrastructural cytochemistry, and agglutination induced by lectins which bind to residues of mannose, N-acetylglucosamine, galactose, N-acetylgalactosamine, fucose and sialic acid. The surface charge of the cells was analysed by the binding of cationic particles (colloidal iron and cationized ferritin) to the cell surface and by cell electrophoretic mobility (EPM). Based on observations of binding of cationic particles to the cell surface; a decrease in the binding of these particles to the cell surface; a decrease in the mean EPM of the cells after their incubation in the presence of neuraminidase; and detection of N-acetylneuraminic acid by paper and gas-liquid chromatography, it was concluded that sialic acid residues are exposed on the surface of P. davidi. These residues may be glycolipids or are masked on the cell surface since only after brief trypsinization were the cells agglutinated by the lectin from Limulus polyphemus.     

Coflocculation of Escherichia coli and Schizosaccharomyces pombe

Several yeasts, such as Candida utilis, Dekkera bruxellensis, Hanseniaspora guilliermondii, Kloeckera apiculata, Saccharomyces cerevisiae and Schizosaccharomyces pombe, were found to coaggregate with Escherichia coli, but S. pombe showed much less coflocculation than the other yeasts (Peng et al. 2001)). S. pombe is known to have galactose-rich cell walls and we investigated whether this might be responsible for its different behavior by studying the wild-type TP4-1D, with a mannose to galactose ratio of 1 to 1.2, and the glycosylation mutant gms1delta (Man:Gal=1:0). The wild-type induced very low levels of coflocculation (3%) while gms1delta induced a remarkable amount of coflocculation (48%). Coflocculation of the mutant was inhibited by mannose but not affected by galactose or glucose. The S. cerevisiae mnn2 mutant, with a mannan structure similar to gms1delta, also showed a high degree of coflocculation (40%). However, S. cerevisiae mutant mnn9, with a mature core similar to S. pombe, showed decreased coflocculation (21.3%). Both these S. cerevisae mutants were sensitive to mannose inhibition. Coflocculation of E. coli and gms1delta also could be inhibited by gms1delta mannan and plant lectins, such as HHA, GNA and NPA, specific to either alpha-1-3- or alpha-1-6-linked mannosyl units. From these results we conclude that the E. coli lectins may have specificity for alpha-1-6- and alpha-1-3-linked mannose residues either in the outer chain or in the core of S. pombe, but in wild-type strains these mannose residues are shielded by galactose residues.     

A preliminary investigation on the chemical composition of the cell surface of five enteropathogenic Escherichia coli serotypes.

The cell surfaces of five enteropathogenic Escherichia coli serotypes (O111:H2; O111:H12; O125:H9; O119:H6; O26:H11) were assayed by chemical methods, lectin agglutination tests and spectroscopy associated to transmission electron microscopy. Results of lectin agglutination assays showed that all strains reacted with mannosebinding lectins. Strains belonging to serotype O125:H9 also agglutinated with lectins which recognize galactose and Nacetylgalactosamine residues. The bacterial cells were treated with 0.01M phosphate buffered saline (pH 7.0) at 100 degree C for 2 hr and the extracts were submitted to precipitation and fractionated by Cetavlon. Phosphate, total sugar and protein contents were determined. Gas liquid chomatography-mass spectrometry analysis of alditol acetates showed the presence of galactose, mannose, fucose, glucose and traces of ribose. Spectroscopic analysis of intact cells showed the presence of a capsule-like structure which was not totally preserved after extraction. Some cells were still surrounded by an amorphous capsular-like material after polysaccharide extraction.     

Changes in cell-surface carbohydrates of Trypanosoma cruzi during metacyclogenesis under chemically defined conditions.

Highly purified lectins with specificities for receptor molecules containing sialic acid, N-acetylglucosamine (D-GlcNAc), N-acetylgalactosamine (D-GalNAc), galactose (D-Gal), mannose-like residues (D-Man) or L-fucose (L-Fuc), were used to determine changes in cell-surface carbohydrates of the protozoal parasite Trypanosoma cruzi during metacyclogenesis under chemically defined conditions. Of the D-GalNAc-binding lectins, BS-I selectively agglutinated metacyclic trypomastigotes, MPL was selective for replicating epimastigotes, whereas SBA strongly agglutinated all developmental stages of T. cruzi. WGA (sialic acid and/or D-GlcNAc specific) was also reactive with differentiating epimastigotes and metacyclic trypomastigotes but displayed a higher reactivity with replicating epimastigote forms. A progressive decrease in agglutinating activity was observed for jacaline (specific for D-Gal) during the metacyclogenesis process; conversely, a progressive increase in affinity was observed for RCA-I (D-Gal-specific), although the reactivity of other D-Gal-specific lectins (PNA and AxP) was strong at all developmental stages. All developmental stages of T. cruzi were agglutinated by Con A and Lens culinaris lectins (specific for D-Man-like residues); however, they were unreactive with the L-fucose-binding lectins from Lotus tetragonolobos and Ulex europaeus. These agglutination assays were further confirmed by binding studies using 125I-labelled lectins. Neuraminidase activity was detected in supernatants of cell-free differentiation medium using the PNA hemagglutination test with human A erythrocytes. The most pronounced differences in lectin agglutination activity were observed between replicating and differentiating epimastigotes, suggesting that changes in the composition of accessible cell-surface carbohydrates precede the morphological transformation of epimastigotes into metacyclic trypomastigotes.     

Cell Surface Carbohydrates in Tritrichomonas foetus1

The cell surface of Tritrichomonas foetus was characterized by using 18 highly purified lectins with specificities for N-acetyl glucosamine, N-acetyl galactosamine, galactose, mannose, and sialic acid. The specificity of the lectin-induced cell agglutination was verified by inhibition of the agglutination with the specific sugars. By using cytochemical techniques associated with electron microscopy, carbohydrates were detected on the cell surface of T. foetus. The following techniques were used: periodic acid-thiosemicarbazide-silver proteinate, concanavalin A-horseradish peroxidase, and ruthenium red. Anionic sites were detected on the cell surface of the protozoan at pH's 1.8 and 7.2 with the use of colloidal iron hydroxide and cationized ferritin particles, respectively. The binding of colloidal iron particles, as well as the agglutination induced by the lectin from Limulus polyphemus, indicated the presence of sialic acid on the cell surface of T. foetus.     

Amphotericin B mediates killing in Cryptococcus neoformans through the induction of a strong oxidative burst

We studied the effects of Amphotericin B (AmB) on Cryptococcus neoformans using different viability methods (CFUs enumeration, XTT assay and propidium iodide permeability). After 1h of incubation, there were no viable colonies when the cells were exposed to AmB concentrations ≥ 1 mg/L. In the same conditions, the cells did not become permeable to propidium iodide, a phenomenon that was not observed until 3h of incubation. When viability was measured in parallel using XTT assay, a result consistent with the CFUs was obtained, although we also observed a paradoxical effect in which at high AmB concentrations, a higher XTT reduction was measured than at intermediate AmB concentrations. This paradoxical effect was not observed after 3h of incubation with AmB, and lack of XTT reduction was observed at AmB concentrations higher than 1mg/L. When stained with dihydrofluorescein, AmB induced a strong intracellular oxidative burst. Consistent with oxidative damage, AmB induced protein carbonylation. Our results indicate that in C. neoformans, Amphotericin B causes intracellular damage mediated through the production of free radicals before damage on the cell membrane, measured by propidium iodide uptake.     

The quantitative determination of metabolites of 6-mercaptopurine in biological materials. VI. Evidence for posttranscriptional modification of 6-thioguanosine residues in RNA from L5178Y cells treated with 6-mercaptopurine

Cell surface glycoconjugates of epimastigotes of Trypanosoma cruzi have been isolated and analyzed to give their amino acid and carbohydrate compositions. Those which have been investigated are a complex of three closely associated glycoproteins, GP24, GP31, GP37, and a lipopeptidophosphoglycan. The GP24-GP31-GP37 complex has an unusual amino acid composition with very low levels of hydrophobic amino acids, it contains 56% (w/w) carbohydrate, with mannose, galactose and glucosamine (presumably N-acetyl) being present in approximately equal quantities. The lipopeptidophosphoglycan also has low levels of hydrophobic amino acids and contains equal levels of mannose and galactose together with lesser amounts of (N-acetyl) glucosamine. The glycoconjugates are contrasted and compared with two other previously characterised cell surface glycoproteins (GP25 and GP72) from T. cruzi.     

Membrane effects of the polyene antibiotic amphotericin B and of some of its derivatives on lymphocytes

Amphotericin B (AmB) exhibits immunomodulating properties in mice.In vitro studies on lymphocytes, in relation with these properties, are reported here with AmB and two of its derivatives: the N-Fructosyl (N-Fru AmB) and the N-thiopropionyl (AmBSH) derivatives. Interactions of these molecules with thymocytes, a sensitive cell type, demonstrated that the extent of binding is not a toxicity parameter. In contrast, membrane fludity changes have been observed and appeared to be related to toxicity.Experiments performed with normal B lymphocytes have shown that Amphotericin B derivatives were more potent polyclonal B cell activators than the parent compound. To go further in the understanding of these events, we have investigated in a B cell line WEHI 231, the changes in intracellular Ca²⁺ and membrane potential induced by AmB and AmBSH. The two polyenes were shown to induce membrane depolarization but no intracellular Ca²⁺ increase.     

Alteration of membrane permeability by amphotericin B

Amphotericin B (AmB) increased unidirectional Na transport and net transcellular sodium movements across the skin of the frog, Rana pipiens, when added to the solution bathing the corium side, but not from the outer epidermal surface. The AmB response was prevented with pretreatment with amiloride, ouabain and mucosal sodium substitution. Alteration in pH markedly reduced the permeability changes induced by AmB. AmB did not interfere with the increase in sodium transport induced by antidiuretic hormone. The present study demonstrates that AmB interacts with the skin of the frog, Rana pipiens, from the corium side specifically increasing transepithelial sodium transport. The increase in transport apparently occurs through the existing sodium pathway.     

Purification and characterization of three galactose specific lectins from mulberry seeds (Morus sp.).

Three lectins were extracted and purified from mulberry seeds by gel filtration of 100% ammonium sulfate saturated crude protein extract followed by ion-exchange chromatography on DEAE and CM-cellulose. The lectins were found to be homogeneous as judged by polyacrylamide disc gel electrophoresis. The molecular masses of the lectins as determined by gel filtration were 175 000 for MSL-1, 120 000 for MSL-2 and 89 500 for MSL-3. MSL-1 is dimer in nature, with the two monomers held together by disulfide bond(s), while MSL-2 and MSL-3 contain four nonidentical subunits that are held together by nonionic hydrophobic interactions. The lectins agglutinated rat red blood cells and this agglutination was inhibited specifically by galactose, methyl-alpha-d-galactopyranoside, methyl-beta-d-galactopyranoside, lactose and raffinose. The lectins MSL-1, MSL-2 and MSL-3 contained 5.7, 5.4 and 4.5% neutral sugars, respectively, and the sugar composition of the lectins was glucose and mannose for MSL-1 and galactose for both MSL-2 and MSL-3. The lectins exhibited strong cytotoxic effect in brine shrimp lethality bioassay.     

Features of the modulating effect of complexes of ribosome-inactivating proteins type II with sugars on respiratory burst of neturophils, caused by a chemotactic peptide

It was shown that agents inducing phagocytosis (zymosan, lectins) cause changes in the number of receptors responsible for fast neutrophil reaction (chemotaxis or respiratory burst) or inhibit the binding of the agonist to its receptor. Among lectins are ribosome-inactivating proteins of type II ricin and agglutinin ricin, which penetrate the cell by binding to mannose and galactose receptors. It was shown that ribosome-inactivating proteins of type II can exhibit the properties of the antagonist of the receptor N-formylmethionylleucylphenylalanine. Ricin is more effective in modulating the respiratory burst induced by the chemotactic peptide than agglutinin ricin. The modulating effect of ribosome-inactivating proteins of type II on neutrophils is likely to be mediated by their interaction with galactose rather than mannose receptors. Presumably, the affinity of ribosome-inactivating proteins to galactose receptors increases with increasing amount of saccharides bound to the protein molecule. The modulating effect of ribosome-inactivating proteins of type II on the respiratory burst of neutrophils induced the chemotactic peptide is due to the structural peculiarities of these proteins.     

Red cell aging results in a change of cell surface carbohydrate epitopes allowing for recognition by galactose-specific receptors of rat liver macrophages

Binding and uptake studies of in vitro aged or senescent rat erythrocytes by isolated rat liver macrophages suggest recognition by galactose-specific receptors on the cell surface of the macrophages. We analyzed carbohydrates exposed on old erythrocytes by plant lectins in an agglutination assay in comparison with freshly isolated untreated erythrocytes. Rat erythrocytes aged in vitro by storage are agglutinated by a panel of lectins that do not react with freshly isolated erythrocytes. Specificity of agglutination was shown by inhibition with monosaccharides. Antibodies eluted from senescent rat erythrocytes agglutinate in vitro aged as well as senescent rat erythrocytes, but not freshly isolated cells nor human erythrocytes. Galactose-specific lectins isolated from rat liver give similar results; they also agglutinate normal human erythrocytes. Agglutination by the liver lectin is inhibitable by galactose and N-acetylgalactosamine but not by N-acetylglucosamine or mannose. Furthermore, rat liver macrophages devoid of galactose-specific receptors show markedly reduced binding of senescent rat erythrocytes. We conclude that recognition of old rat erythrocytes is mediated by two systems: old erythrocytes expose different terminal sugar residues or a different arrangement of glycans when compared to young erythrocytes, rendering them recognizable by liver lectins and by autoantibodies.     

Changes to the galactose/mannose ratio in galactomannans during coffee bean (<Emphasis Type="Italic">Coffea arabica</Emphasis> L.) development: implications for in vivo modification of galactomannan synthesis

Endosperm was isolated from Arabica Caturra coffee beans 11, 15, 21, 26, 31 and 37 weeks after flowering, and the chemical composition and relative solubility of its component polysaccharides determined at each growth stage. Chemical analysis of the total mannan content of the cell wall material was done after solubilisation of galactomannans by alkaline extraction of the cell wall material followed by enzymatic digestion of the alkali-insoluble residue with a mixture of endo-mannanse and endo-glucanase. Eleven weeks after flowering, galactomannans accounted for approximately 10% of the polysaccharides but were highly substituted, with galactose/mannose ratios between 1:2 and 1:7. As the bean matured, galactomannan became the predominant polysaccharide, until 31 weeks after flowering it accounted for approximately 50% of the polysaccharides. However, it was less substituted, possessing galactose/mannose ratios between 1:7 and 1:40. Early in bean growth, up to 50% of the cell wall polysaccharides were extractable but as the galactomannan content of the bean increased there was a reduction in the extractability of all polysaccharides. The decrease in the galactose/mannose ratio of the galactomannans commenced between 21 and 26 weeks after flowering and was in synchrony with a rise in the concentration of free galactose in the beans. The results indicated that the degree of substitution of the galactomannans in coffee beans is developmentally regulated and may result, in part, from the modification of a primary synthetic product by the action of an alpha-galactosidase.     

Two-step impact of Amphotericin B (AmB) on lipid membranes: ESR experiment and computer simulations

Abstract

In this study, the electron spin resonance (ESR) method was used to examine the effect of Amphotericin B (AmB) molecules on the fluidity of model membranes made of dipalmitoylphosphatidylcholine (DPPC). The changes occurring under increased AmB concentrations in the spectroscopic parameters of spin probes placed in liposomes were determined. Three probes were used, penetrating the membrane at different depths which allowed the changes in its fluidity to be found in the transverse section. A computer model of the surface layer of membrane, with AmB admixture, was developed and subjected to computer simulation. The effect of changing concentration of the admixture on the binding energy in the system of dipoles representing the surface of the membrane was examined. The ESR studies showed that the process of accumulation of AmB molecules in the membrane has two stages, marked by local maxima in the ESR spectra. The first appears for concentrations of ca. 0.25–0.5% and the second appears for ca. 2.5–3% AmB of its molar ratio to DPPC. The computer simulations permitted reconstructing the two-stage mechanism of interaction between the molecules and the membrane. They demonstrated that, at low concentrations, the AmB molecules position themselves flat on the membrane surface. After the threshold concentration is exceeded, they re-orientate to a vertical position. This process leads to the perforation of the membrane.     

Mannose-binding plant lectins: different structural scaffolds for a common sugar-recognition process

Mannose-specific lectins are widely distributed in higher plants and are believed to play a role in recognition of high-mannose type glycans of foreign micro-organisms or plant predators. Structural studies have demonstrated that the mannose-binding specificity of lectins is mediated by distinct structural scaffolds. The mannose/glucose-specific legume (e.g., Con A, pea lectin) exhibit the canonical twelve-stranded beta-sandwich structure. In contrast to legume lectins that interact with both mannose and glucose, the monocot mannose-binding lectins (e.g., the Galanthus nivalis agglutinin or GNA from bulbs) react exclusively with mannose and mannose-containing N-glycans. These lectins possess a beta-prism structure. More recently, an increasing number of mannose-specific lectins structurally related to jacalin (e.g., the lectins from the Jerusalem artichoke, banana or rice), which also exhibit a beta-prism organization, were characterized. Jacalin itself was re-defined as a polyspecific lectin which, in addition to galactose, also interacts with mannose and mannose-containing glycans. Finally the B-chain of the type II RIP of iris, which has the same beta-prism structure as all other members of the ricin-B family, interacts specifically with mannose and galactose. This structural diversity associated with the specific recognition of high-mannose type glycans highlights the importance of mannose-specific lectins as recognition molecules in higher plants.     

Lectins in fish skin: do they play a role in host-monogenean interactions?

Mucus samples from rainbow trout skin with or without infections by Gyrodactylus derjavini were tested for the presence of lectins reacting with mannose, galactose and lactose. The samples inhibited the binding of biotinylated lectins (from Canavalia ensiformis, Artocarpus integrifolia and Erythrina corallodendron, respectively) to microtitre plates with covalently bound carbohydrates (mannopyranoside, galactopyranoside and lactose, respectively). However, the inhibition of C. ensiformis and A. integrifolia lectins was slightly greater when mucus from infected (but recovering) fish was used, suggesting an increase of mannose and galactose binding lectins in fish skin exposed to parasites. As mannose, galactose and lactose are present on the glycocalyx of Gyrodactylus derjavini, it is suggested that lectins could play a dual role in interactions between fish hosts and their monogenean parasites. Thus, recognition between parasite and host and also host responses towards parasite infections could both, at least partly, involve carbohydrate-lectin binding.     

Organization of polyene antibiotic amphotericin B at the argon-water interface

Amphotericin B (AmB) is a life-saving antibiotic, used to treat deep-seated mycotic infections. Both the therapeutic and toxic side effects of AmB are directly dependent on its molecular organization. Organization of AmB was studied in monocomponent monomolecular layers formed at the argon-water interface, by means of polarized and non-polarized electronic absorption spectroscopy and analyzed in terms of the exciton splitting theory. The results provide direct indication that AmB forms spontaneously dimers that can be assembled into molecular structures characterized by homogeneous orientational distribution in the monolayer, interpreted as cylindrical pores. The structures are not stable at surface pressures higher than 20 mN/m and therefore dimers are concluded as abundant molecular organization forms of AmB in biomembranes. Possibility of stabilization of the cylindrical structures, at higher surface pressures, by other molecules, e.g. sterols, is also discussed.     

Molecular aspects of the interaction between amphotericin B and a phospholipid bilayer: molecular dynamics studies

Amphotericin B (AmB) is a polyene macrolide antibiotic used to treat systemic fungal infections. The molecular mechanism of AmB action is still only partly characterized. AmB interacts with cell-membrane components and forms membrane channels that eventually lead to cell death. The interaction between AmB and the membrane surface can be regarded as the first (presumably crucial) step on the way to channel formation. In this study molecular dynamics simulations were performed for an AmB–lipid bilayer model in order to characterize the molecular aspects of AmB–membrane interactions. The system studied contained a box of 200 dimyristoylphosphatidylcholine (DMPC) molecules, a single AmB molecule placed on the surface of the lipid bilayer and 8,065 water molecules. Two molecular dynamics simulations (NVT ensemble), each lasting 1 ns, were performed for the model studied. Two different programs, CHARMM and NAMD2, were used in order to test simulation conditions. The analysis of MD trajectories brought interesting information concerning interactions between polar groups of AmB and both DMPC and water molecules. Our studies show that AmB preferentially took a vertical position, perpendicular to the membrane surface, with no propensity to enter the membrane. Our finding may suggest that a single AmB molecule entering the membrane is very unlikely.Figure The figure presents the whole structure of the system simulated—starting point. AmB is presented as a space-filling model, DMPC molecules—green sticks, water molecules—red sticks