Structure-based phylogeny of polyene macrolide antibiotic glycosyltransferases

Antibiotic glycosyltransferases (AGts) attach unusual deoxy-sugars to aglycons so antibiotics can exert function. It has been reported that polyene macrolide (PEM) AGts have different evolutionary origin when compared with other polyketide AGts, and our previous analysis have suggested that they could be results of horizontal gene transfer (HGT) from eukaryotes. In this paper, we compared the structures of PEM AGts with structures of eukaryotes and other AGts, and then built models of the representative PEM AGts and GT-1 glycosyltransferases. We also constructed the Neighbor-Joining (NJ) trees based on the normalized Root Mean Square (RMS) distance, the Bayesian tree guided by structural alignments, and carried out analysis on several key conserved residues in PEM AGts. The NJ tree showed a close relationship between PEM AGts and eukaryotic glycosyltransferases, and Bayesian tree further supported their affinity with UDP-glucuronosyltransferases (UGTs). Analysis on key conserved residues showed that PEM AGts may have similar interaction mechanism such as in the formation of hydrogen bonds as eukaryotic glycosyltransferases. Using structure-based phylogenetic approaches, this study further supported that PEM AGts were the result of HGT between prokaryotes and eukaryotes.     

Biosynthesis of the polyene macrolide antibiotic nystatin in Streptomyces noursei

The polyene macrolide antibiotic nystatin, produced commercially by the bacterium Streptomyces noursei, is an important antifungal agent used in human therapy for treatment of certain types of mycoses. Early studies on nystatin biosynthesis in S. noursei provided important information regarding the precursors utilised in nystatin biosynthesis and factors affecting antibiotic yield. New insights into the enzymology of nystatin synthesis became available after the gene cluster governing nystatin biosynthesis in S. noursei was cloned and analysed. Six large polyketide synthase proteins were implicated in the formation of the nystatin macrolactone ring, while other enzymes, such as P450 monooxygenases and glycosyltransferase, were assumed responsible for ring decoration. The latter data, supported by analysis of the polyene mixture synthesised by the nystatin producer, helped elucidate the complete nystatin biosynthetic pathway. This information has proved useful for engineered biosynthesis of novel nystatin analogues, suggesting a plausible route for the generation of potentially safer and more efficient antifungal drugs.     

Stimulation of ion release from liposomes by the polyene antibiotic, filipin.

The effect of the polyene antibiotic, filipin, upon release of the ions Ca²⁺, Sr²⁺, SO₄^2? and phosphate out of phospholipid and phospholipid-cholesterol liposomal vesicles was studied. The addition of filipin at concentrations stoichiometrically comparable to the cholesterol concentration in the liposomes, resulted in 2–10 × stimulation of the rate of release of all of these ions. The filipin mediated stimulation of release of ions from liposomes was dependent upon the presence of cholesterol. The relative effectiveness of filipin increased when the mole percent of cholesterol incorporated into the liposomes increased from 10 to 50% and when the molar filipin:cholesterol ratio increased from 0.2 to 1.0. It has been previously shown that there is a 1:1 stoichiometry of interaction between filipin and cholesterol [Biochem. Biophys. Acta339, 57 (1974)]. The present studies suggest that this 1:1 stoichiometric interaction may also be responsible for the increased release of entrapped ions.A possible mechanism of action of polyene antibiotics is discussed which suggests that the rearrangement of membrane constituents occurring upon interaction of filipin with cholesterol is the basis for the enhancement of ion release. This would imply that the ion specificity observed upon interaction of polyene antibiotics with membranes would not only be determined by the polyene antibiotic itself, but also by the intrinsic properties of the membrane.     

A photophysical study of the polyene antibiotic filipin. Self-aggregation and filipin--ergosterol interaction

Filipin, a macrolide polyene antibiotic, is known to interact selectively with ergosterol, a constituent of fungi membranes. In this work, the fluorescence resonance energy transfer (FRET) between a fluorescent analog of ergosterol, dehydroergosterol (DHE), and filipin was measured in small unilamellar vesicles of dipalmitoylphosphatidylcholine at 25 degrees C. The time-resolved FRET results were rationalized in the framework of the mean concentration model, and were complemented with steady-state fluorescence intensity, anisotropy and absorption measurements. The results point to the formation of both DHE--filipin aggregates (evidence from static quenching of DHE fluorescence by filipin) and filipin--filipin aggregates (evidence from: (i) the FRET acceptor concentration distributions; (ii) spectral changes of filipin absorption in the vesicles, the excitonic interaction suggesting a stack arrangement; (iii) filipin fluorescence self-quenching), even in presence of DHE and low antibiotic mole fractions (<1 mol%). These results point out that apparently contradictory biochemical models for the action of filipin (some based on the presence of sterols, others not) can be equally valid. Moreover, since results (ii) and (iii) are also observed when a sterol is present, both models of action can actually coexist in membranes with a low sterol content.     

Interaction of the polyene antibiotic filipin with model and natural membranes containing plant sterols

The interaction of the polyene antibiotic, filipin, with individual or mixed plant sterols (stigmasterol, sitosterol, campesterol and 24-methylpollinastanol) incorporated into large unilamellar vesicles (LUV) of soybean phosphatidylcholine (PC) as well as the filipin interaction with purified membrane fractions from maize roots containing these sterols was investigated by ultraviolet (UV) absorption and and circular dichroism (CD) spectroscopy. With both types of membrane preparation, dramatic changes in the UV absorption and CD spectra of the antibiotic were evidenced. When LUV containing stigmasterol, sitosterol and/or campesterol were incubated with low filipin concentrations (i.e., for filipin/sterol molar ratios (rst) lower than 1), CD signal characteristic of the formation of filipin-sterol complexes were observed. At higher rst values, the filipin-sterol interaction was shown to be in competition with a filipin-phospholipid interaction. With 24-methylpollinastanol-containing LUV, the filipin-phospholipid interaction was detected even at rst values lower than 1, which suggests a lower affinity of filipin for this sterol and emphasizes the structural differences between delta 5-sterols and 9 beta,19-cyclopropylsterols. With sterol-free soybean PC LUV, a filipin-phospholipid interaction could also be evidenced. With maize root cell membranes containing either delta 5-sterols or 9 beta,19-cyclopropylsterols, CD spectra similar to those obtained in the presence of LUV having these sterols as components were observed. Thus, the protein component of the membranes does not appear to be an important feature.     

Solution NMR structure of five representative glycosylated polyene macrolide antibiotics with a sterol-dependent antifungal activity.

Glycosylated polyene macrolide antibiotics, as nystatins and amphotericins, are amphiphilic structures known to exert antifungal activity by disrupting the fungal cell membrane, leading to leakage of cellular materials, and cell death. This membrane disruption is strongly influenced by the presence and the exact nature of the membrane sterols. The solution structures of five representative glycosylated members, three tetraenes (pimaricin, nystatin A1 and rimocidin) and two heptaenes (candidin and vacidin A) have been calculated using geometric restraints derived from 1H-NMR data and random searches of their conformational space. Despite a different apparent structural order, the NMR solutions structure indicate that the hydroxyl groups all clustered on one side of the rod-shaped structures, and the glycosyl moieties are structurally conserved both in their conformation and their apparent order. The molecular structures afford an understanding of their selective interaction with the membrane sterols and the design of new polyene macrolides with improved activities.     

多烯大环内酯类抗生素生物合成基因簇中调控因子的研究进展

多烯大环内酯类抗生素具有良好的抗真菌活性,广泛应用于医疗卫生、食品加工和农业生产领域。随着高通量测序技术和生物信息学技术的发展,越来越多的链霉菌抗生素生物合成基因簇被发现和鉴定,调控因子作为生物合成基因簇中的重要组成部分,在庞大复杂的调控网络中起着至关重要的作用。本文总结了链霉菌中重要的调控因子类型,综述了多烯大环内酯类抗生素生物合成基因簇中调控因子的生物学功能、结合位点、作用机制等研究进展,并展望了后续研究工作。     

Sterol and fatty acid composition of polyene macrolide antibiotic resistant Torulopsis glabrata.

Successive reculturing of Torulopsis glabrata on media containing increasing concentration of the polyene macrolide antibiotics nystalin or lucensomycin resulted in the segregation of cultures resistant to these antibiotics. Isolates resistant to lucensomycin showed good resistance to nystatin, and vice versa. Analysis of the sterols and fatty acids of sensitive and polyene resistant T. glabrata revealed that compositional changes occurred in both classes of lipids upon acquistion of resistance. The sterol composition of nystatin and lucensomycin resistant cultures possessed reduced amounts of, or no ergosterol (the major sterol of the sensitive parent culture), and increased amounts of sterols which were biogenetically more primitive than ergosterol. Resistant cultures in which ergosterol was absent possessed a fatty acid composition that did not differ significantly from the parent sensitive culture grown under identical conditions. Resistant cultures containing significantly reduced amounts of ergosterol were found to possess altered fatty acid compositions. Generally it was observed that these latter cultures possessed fatty acids containing shorter and more saturated chains. These results are considered to indicate that alteration in both lipid and sterol composition is involved in determination of culture resistance to polyene macrolides.     

Distribution of intramembranous particles and filipin-sterol complexes in mouse sperm membranes: polyene antibiotic filipin treatment

The distribution of intramembranous particles (IMPs) and membrane filipin-sterol complexes (FSC) was examined ultrastructurally in mouse spermatozoa from the male reproductive tract and ejaculates. IMPs were qualitatively analyzed on freeze-fracture replicas of glutaraldehyde-fixed tissue, while membrane FSC were quantitatively analyzed on replicas of filipin-treated cells. The distribution pattern of IMPs of mouse spermatozoa was fundamentally similar to that of other mammalian spermatozoa. 1) In the head, the plasma membrane had a heterogeneous population density, e.g., few IMPs on the acrosomal region, particularly few on the marginal segment, and somewhat regularly arranged IMPs on the postacrosomal region. The acrosomal membrane had many IMPs in hexagonal arrays. The nuclear membrane had many IMPs on the P-face, few IMPs on the variegated E-face, and an intense population density on the P-face of the basal plate. 2) In the neck, the plasma membrane had many IMPs with square arrangements of small IMPs in some areas on the P-face; the redundant nuclear membrane had a few IMPs on both P- and E-faces. 3) In the tail, the plasma membrane had diagonal rows of IMPs in some areas amongst larger IMPs on the middle piece, while it had "zippers" composed of IMPs running parallel to the axis on the principal piece. The distribution of sperm membrane FSC may be summarized as follows: 1) In the head, the acrosomal plasma membrane, which was heavily labeled with filipin, had much more FSC in the equatorial segment than in the marginal segment throughout the study. The postacrosomal plasma membrane generally had no FSC, but some sperm in ejaculates were slightly positive to filipin. The acrosomal membranes (both outer and inner) had no FSC. The nuclear membrane in the main part of the head had less FSC in vas deferens and ejaculated sperm than in the epididymal sperm. The nuclear membrane on the basal plate had no FSC. 2) In the neck, the plasma membrane had little FSC. The redundant nuclear envelope had scattered FSC with a higher incidence in the epididymal sperm than in those from the vas deferens and ejaculates. The membrane scroll, which was elongated from the extreme caudal end of the redundant nuclear envelope, had abundant FSC in the vas deferens and ejaculated sperm. 3) The tail plasma membrane (both middle and principal piece), which was weakly labeled with filipin, had less FSC in sperm from the vas deferens and ejaculates than in those from the epididymis. The limiting membrane covering the mitochondria had no FSC.     

Molecular modelling of membrane activity of amphotericin B, a polyene macrolide antifungal antibiotic

Amphotericin B (AmB) is a well known polyene macrolide antibiotic used to treat systemic fungal infections. Despite its toxicity AmB is still regarded as a life-saving drug. The lack of adequate knowledge of the AmB mechanism of action is a serious obstacle to efficient development of new less toxic derivatives. Complementary to various experimental approaches, computational chemistry methods were used to study AmB mechanism of action. A programme lasting for a decade, that was run by our group covered studies of: i) molecular properties of AmB and its membrane targets, ii) structure and properties of AmB membrane channels, and iii) interaction of AmB with the membrane.     

Characterization of an endophytic whorl-forming Streptomyces from Catharanthus roseus stems producing polyene macrolide antibiotic

An endophytic whorl-forming Streptomyces sp. designated as TS3RO having antifungal activity against a large number of fungal pathogens, including Sclerotinia sclerotiorum, Rhizoctonia solani, Colletotrichum gloeosporioides, Cryphonectria parasitica, Fusarium oxysporum, Pyrenophora tritici-repentis, Epidermophyton floccosum, and Trichophyton rubrum, was isolated from surface-sterilized Catharanthus roseus stems. Preliminary identification showed that Streptomyces cinnamoneus subsp. sparsus was its closest related species. However, strain TS3RO could readily be distinguished from this species using a combination of phenotypic properties, 16S rDNA sequence similarity, and phylogenetic analyses. Thus, the whorl-forming Streptomyces sp. strain TS3RO is likely a new subspecies within the Streptomyces cinnamoneus group. Direct bioautography on a thin-layer chromatography plate with Cladosporium cucumerinum was conducted throughout the purification steps for bioassay-guided isolation of the active antifungal compounds from the crude extract. Structural elucidation of the isolated bioactive compound was obtained via LC-MS spectrometry, UV-visible spectra, and nuclear magnetic resonance data. It revealed that fungichromin, a known methylpentaene macrolide antibiotic, was the main antifungal component of TS3RO strain, as shown by thin-layer chromatography bioautography. This is the first report of an endophytic whorl-forming Streptomyces isolated from the medically important plant Catharanthus roseus.     

The effect of fetal bovine serum on polyene macrolide antibiotic cytotoxicity and antifungal activity

Summary The relationships between fetal bovine serum (FBS) concentration and polyene macrolide antibiotic cytotoxicity to animal cells and to fungi were evaluated. The toxicity of amphotericin B (AB) and its derivative, amphotericin B methyl ester (AME), toward KB cells was found to be directly related to fetal bovine serum concentration. At higher FBS levels, increased concentrations of AB and AME were required to reduce 72-hr KB viable cell numbers to 50% of control values. Similarly, polyene macrolide antibiotic levels required to inhibit the growth ofSaccharomyces cerevisiae to 50% of controls, and for obtaining minimum fungicidal concentrations (MFC), were greater when higher levels of FBS were used. In addition, AME was less toxic than AB toward KB cells grown in media containing 2, 5, 10, 15 or 20% FBS, whereas the antifungal activities of AB and AME were similar. AME was also capable of eliminatingCandida albicans, Saccharomyces cerevisiae, Aspergillus niger orFusarium moniliforme from KB cultures at antibiotic levels which exhibited less cell toxicity than did the concentrations of AB required for a similar response. These findings indicate that AME may be a potentially useful antifungal antibiotic for tissue culture systems. Portions of this paper were presented at the 25th Annual Meeting of the Tissue Culture Association at Miami, Florida, 1974. This investigation was supported in part by contract NIH 69-2161, NIH grant no. AI-02095 and NIH training grant no. GM 507 from the National Institute of General Medical Sciences.     

Effects and mechanisms of action of polyene macrolide antibiotic nystatin on Babesia gibsoni in vitro

Nystatin is a membrane-active polyene macrolide antibiotic and a channel-forming ionophore. Nystatin exhibits in vitro activity against Babesia gibsoni infecting normal canine erythrocytes containing low potassium (LK) and high sodium concentrations, i.e., LK erythrocytes. The calculated IC(50) value of nystatin against B. gibsoni infecting LK erythrocytes was 31.96 μg/ml. The anti-babesial activity of nystatin disappeared when B. gibsoni in LK erythrocytes were incubated in culture media containing high potassium concentrations (HK). Moreover, when the parasites were harbored in canine HK erythrocytes, which contained high potassium and low sodium concentrations as a result of high Na-K-ATPase activity, the in vitro anti-babesial activities of nystatin also disappeared, apparently due to protection by HK erythrocytes. This suggested that nystatin could show in vitro anti-babesial activity against B. gibsoni by its ionophorous activity, the same as other ionophores such as valinomycin. Subsequently, the effects of nystatin on the host cells were observed. Nystatin could not modify the intracellular concentrations of potassium, sodium, adenosine triphosphate, or glucose in either LK or HK erythrocytes, although it caused weak hemolysis in HK erythrocytes. In addition, nystatin did not affect the survival of canine peripheral polymorphonuclear leukocytes. In conclusion, nystatin destroyed B. gibsoni by ionophorous activity but did not affect either canine erythrocytes or leukocytes in vitro.     

Chemistry and biology of the polyene macrolide antibiotics

[This corrects the article on p. 166 in vol. 37, PMID: 4578757.].     

Fluorescence study of the macrolide pentaene antibiotic filipin in aqueous solution and in a model system of membranes.

The polyene antibiotic filipin (a pentaene) has been studied using photophysical techniques. The polyene self-aggregates in water with a critical micellar concentration of 2 microM. Two approaches were used to evaluate the aggregate dimensions: (a) a lower limit of 10 nm for the aggregate radius was obtained from energy transfer experiments; (b) a formula for rationalizing the turbidity spectrum was derived, and from its application a spherical shape of radius about 50 nm was deduced. The low value for the fluorescence anisotropy of the aggregate (r = 0.02) is compatible with a very loose structure, i.e. the chromophore has very efficient depolarization dynamics that is not controlled by the aggregate size. The Stern-Volmer plot of aggregated filipin fluorescence quenching by iodide is non-linear, presenting a downward curvature. A model was used for the interpretation of these data, along with a study of the quenching in transient state; it was concluded that all the components of the decay are affected by the quencher, i.e. the aggregate has a very open structure with respect to the iodide ion. The partition constants of the polyene, Kp, between a model system of membranes (small unilamellar vesicles of dipalmitoylglycerophosphocholine) and the aqueous phase were determined from anisotropy measurements; the values obtained were Kp (gel phase) = (3.4 +/- 0.8) x 10(3) and Kp (liquid crystal phase) = (7.7 +/- 2.2) x 10(2). The observation that the polyene incorporation is efficient is at variance with the belief that the presence of sterols are essential for the interaction of polyene antibiotics with membranes [for review see Bolard, J. (1986) Biochim. Biophys. Acta 864, 257-304].     

Coupled transepithelial sodium and potassium transport across isolated frog skin: Effect of ouabain,amiloride and the polyene antibiotic filipin

Summary Addition of the polyene antibiotic filipin (50 m) to the outside bathing solution (OBS) of the isolated frog skin resulted in a highly significant active outward transport of K⁺ because filipinper se increases the nonspecific Na⁺ and K⁺ permeability of the outward facing membrane. The K⁺ transport was calculated from the chemically determined changes in K⁺ concentrations in the solution bathing the two sides of the skin. The active transepithelial K⁺ transport required the presence of Na⁺ in the OBS, but not in the inside bathing solution (IBS), and it was inhibited by the Na⁺, K⁺-ATPase inhibitor ouabain. The addition of Ba⁺⁺ to the IBS in the presence of filipin in the OBS resulted in an activation of the transepithelial K⁺ transport and in an inhibition of the active Na⁺ transport. This is in agreement with the notion that Ba⁺⁺ decreases the passive K⁺ permeability of the inward facing membrane. In the presence of amiloride (which blocks the specific Na permeability of the outward facing membrane) and Ba⁺⁺ there was a good correlation between the active Na⁺ and K⁺ transport. It is concluded that the active transepithelial K⁺ transport is carried out by a coupled electrogenic Na–K pump, and it is suggested that the pump ratio (Na/K) is 1.5.