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Natamycin Blocks Fungal Growth by Binding Specifically to Ergosterol without Permeabilizing the Membrane

2007; Elsevier BV; Volume: 283; Issue: 10 Linguagem: Inglês

10.1074/jbc.m707821200

1083-351X

Yvonne M. te Welscher, Hendrik H. ten Napel, Miriam Masià Balagué, Cleiton Martins Souza, Howard Riezman, Ben de Kruijff, Eefjan Breukink,

Fungal and yeast genetics research

Natamycin is a polyene antibiotic that is commonly used as an antifungal agent because of its broad spectrum of activity and the lack of development of resistance. Other polyene antibiotics, like nystatin and filipin are known to interact with sterols, with some specificity for ergosterol thereby causing leakage of essential components and cell death. The mode of action of natamycin is unknown and is investigated in this study using different in vitro and in vivo approaches. Isothermal titration calorimetry and direct binding studies revealed that natamycin binds specifically to ergosterol present in model membranes. Yeast sterol biosynthetic mutants revealed the importance of the double bonds in the B-ring of ergosterol for the natamycin-ergosterol interaction and the consecutive block of fungal growth. Surprisingly, in strong contrast to nystatin and filipin, natamycin did not change the permeability of the yeast plasma membrane under conditions that growth was blocked. Also, in ergosterol containing model membranes, natamycin did not cause a change in bilayer permeability. This demonstrates that natamycin acts via a novel mode of action and blocks fungal growth by binding specifically to ergosterol. Natamycin is a polyene antibiotic that is commonly used as an antifungal agent because of its broad spectrum of activity and the lack of development of resistance. Other polyene antibiotics, like nystatin and filipin are known to interact with sterols, with some specificity for ergosterol thereby causing leakage of essential components and cell death. The mode of action of natamycin is unknown and is investigated in this study using different in vitro and in vivo approaches. Isothermal titration calorimetry and direct binding studies revealed that natamycin binds specifically to ergosterol present in model membranes. Yeast sterol biosynthetic mutants revealed the importance of the double bonds in the B-ring of ergosterol for the natamycin-ergosterol interaction and the consecutive block of fungal growth. Surprisingly, in strong contrast to nystatin and filipin, natamycin did not change the permeability of the yeast plasma membrane under conditions that growth was blocked. Also, in ergosterol containing model membranes, natamycin did not cause a change in bilayer permeability. This demonstrates that natamycin acts via a novel mode of action and blocks fungal growth by binding specifically to ergosterol. Fungal infections have recently become a growing threat to human health, especially in persons whose immune systems are compromised (for example, by human immunodeficiency virus and cancer chemotherapy). Only a few effective antifungal agents are currently in use; these include the polyenes, the fluorocytes, and the azole derivatives. One important problem is the increase of drug resistance, particularly against azole antimyotics and fluorocytosine (1Ghannoum M.A. Rice L.B. Clin. Microbiol. Rev. 1999; 12: 501-517Crossref PubMed Google Scholar). Resistance against polyene antibiotics is still a rare event, which makes these antibiotics particularly interesting as antifungal agents. The polyene antibiotics have a ring structure in which a conjugated double bond system is located opposite to a number of hydroxyl functions. Often a mycosamine group is present in combination with a carboxyl moiety, rendering the molecule amphoteric (Fig. 1). In the past convincing evidence has been presented that several members of this class of antibiotics target sterols and in particular ergosterol, the abundant and main sterol of fungal membranes (2Kruijff de B. Demel R.A. Biochim. Biophys. Acta. 1974; 339: 57-70Crossref PubMed Scopus (566) Google Scholar, 3Bolard J. Biochim. Biophys. Acta. 1986; 864: 257-304Crossref PubMed Scopus (690) Google Scholar). Different types of polyene antibiotics were shown to have different modes of action despite that they share a common target. The larger polyenes like amphotericin B and nystatin form pores together with ergosterol in the plasma membrane that collapse vital ion gradients, thereby killing the cells. The smaller uncharged filipin also destroys the membrane barrier, but by a completely different mechanism. Filipin forms large complexes with sterols between the leaflets of the lipid bilayer, resulting in loss of the barrier function (2Kruijff de B. Demel R.A. Biochim. Biophys. Acta. 1974; 339: 57-70Crossref PubMed Scopus (566) Google Scholar). Natamycin (also called pimaricin) is a very effective member of the polyene antibiotic family with a large standing record of applications. It is produced by Streptomyces natalensis and used against fungal infections, but it is also widely utilized in the food industry to prevent mold contamination of cheese and other nonsterile foods (e.g. cured meats) (4Aparicio J.F. Colina A.J. Ceballos E. Martin J.F. J. Biol. Chem. 1999; 274: 10133-10139Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). Surprisingly, the mechanism of action of this antifungal agent is still unknown and it is even unknown whether it targets ergosterol in the fungal membrane. It is relatively small while it contains a tetraene compared with a pentaene in filipin, which is already considered as a small polyene antibiotic (Fig. 1). It contains a mycosamine group that renders it amphoteric, which is a feature that is also present in nystatin. Whereas natamycin has similar features of both filipin (small) and nystatin (amphoteric), it is difficult to predict its mechanism of action. We wanted to gain more insight into the mode of action of natamycin, which could in turn help to develop new or improved antifungal formulations or result in novel strategies to prevent fungal spoilage. To determine the interaction of natamycin with membranes in relation to its sterol composition, we tested in a comparative manner using filipin and nystatin as references, the interaction of natamycin with phosphatidylcholine model membranes of varying sterol composition using isothermal titration calorimetry (ITC) 2The abbreviations used are:ITCisothermal titration calorimetryDOPC1,2-dioleoyl-sn-glycero-3-phosphocholineCFDA-SE5-(and -6)-carboxyfluorescein diacetate, succinimidyl esterHPTS8-hydroxypyrene-1,3,6-trisulfonic acid trisodium saltDDAON,N-dimethyldodecylamine-N-oxideMICminimum inhibitory concentrationCFcarboxyfluoresceinLUVslarge unilamellar vesiclesMES4-morpholineethanesulfonic acid. and other binding studies. In addition, the ability of natamycin to permeabilize these model membranes was studied. isothermal titration calorimetry 1,2-dioleoyl-sn-glycero-3-phosphocholine 5-(and -6)-carboxyfluorescein diacetate, succinimidyl ester 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt N,N-dimethyldodecylamine-N-oxide minimum inhibitory concentration carboxyfluorescein large unilamellar vesicles 4-morpholineethanesulfonic acid. Parallel to the studies performed on model membranes, the effect of natamycin on yeast growth, the binding of the antibiotic with intact yeast cells, and the plasma membrane integrity were determined. These studies were performed using strains that carry specific mutations in the ergosterol biosynthetic pathway (ergΔ) or that were reprogrammed to contain cholesterol as the main sterol (5Heese-Peck A. Pichler H. Zanolari B. Watanabe R. Daum G. Riezman H. Mol. Biol. Cell. 2002; 13: 2664-2680Crossref PubMed Scopus (133) Google Scholar). We could demonstrate that, differently from any other polyene antibiotic of which the mode of action is known, natamycin blocks fungal growth by binding specifically to ergosterol, but without permeabilizing the membrane. Chemicals—1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) and cholesterol were purchased from Avanti Polar Lipids Inc. (Alabaster, AL). Ergosterol was purchased from Larodan AB (Sweden). DOPC or sterols were dissolved in chloroform to a stock concentration of 20 mm. The phospholipid concentration of DOPC was determined by phosphate analysis according to Rouser et al. (6Rouser G. Fkeischer S. Yamamoto A. Lipids. 1970; 5: 494-496Crossref PubMed Scopus (2880) Google Scholar). The polyene antibiotics nystatin and filipin were dissolved in Me2SO, whereas natamycin was dissolved in 85:15 Me2SO/H2O (v/v); all were obtained from Sigma. All antibiotic solutions were prepared freshly before the start of an experiment and the concentrations of the polyene antibiotics were determined by UV absorption on a PerkinElmer UV-visible spectrometer (Lambda 18). The molar extinction coefficients of the polyene antibiotics were determined in methanol to be 7.6 × 104 m-1 cm-1 (318 nm), 6.7 × 104 m-1 cm-1 (318 nm), and 8.5 × 104 m-1 cm-1 (356 nm) for natamycin, nystatin, and filipin, respectively. The molar extinction coefficient of ergosterol was measured in methanol to be 0.97 × 104 m-1 cm-1 (262 nm). The ionophore nigericin (dissolved in ethanol), ampicillin sodium salt, and the amino acids adenine, uracil, and l-tryptophan were obtained from Sigma. 5-(and -6)-Carboxyfluorescein diacetate, succinimidyl ester (CFDA-SE) (dissolved in Me2SO) and 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (HPTS) were both purchased from Invitrogen. N,N-Dimethyldodecylamine-N-oxide (DDAO) was bought from Fluka Biochimica (Buchs). All other chemicals used were of analytical or reagent grade. Strains and Growth Conditions—For all experiments, medium was inoculated directly from plates with colonies that were not older than 2 weeks. Unless otherwise mentioned, cells were grown overnight at 30 °C in rich medium (10 g/liter yeast extract, 20 g/liter Bacto-peptone, and 20 g/liter dextrose with 1 g/liter adenine, 2 g/liter uracil, and 1 g/liter tryptophan (YPUADT)) supplemented with 0.1 mg/ml ampicillin. For strains RH6611 and RH6613 SD medium was used (1.7 g/liter yeast nitrogen base without amino acids, 20 g/liter glucose, 2 mg/liter trace components, 5 g/liter ammonium sulfate) supplemented with vitamins and the appropriate amino acids minus histidine and leucine (SD-His-Leu). Yeast strains used in this study are listed with their relevant genotypes in Table 1 and the plasmids in Table 2.TABLE 1Strains used in this study The source of these strains is described in Ref. 5Heese-Peck A. Pichler H. Zanolari B. Watanabe R. Daum G. Riezman H. Mol. Biol. Cell. 2002; 13: 2664-2680Crossref PubMed Scopus (133) Google Scholar.StrainNameGenotypeWild typeRH448MATa his4 leu2 ura3 lys2 bar1Erg2ΔRH2897MATa erg2(end11)-1Δ::URA3 leu2 ura3 his4 lys2 bar1Erg2Δerg6ΔRH3616MATa erg2(end11)-1Δ::URA3 erg6Δ leu2 ura3 bar1Erg6ΔRH3622MATa erg6Δ::LEU2 leu2 ura3 his4 bar1Erg3ΔRH4213MATa erg3Δ::LEU2 leu2 ura3 his4 lys2 bar1Erg3Δerg6ΔRH5225MATa erg3Δ::LEU2 erg6Δ::LEU2 leu2 ura3 his4 lys2 bar1Erg2Δerg3ΔRH5228MATa erg2Δ (end11)-1Δ::URA3 erg3Δ::LEU2 leu2 ura3 his4 lys2 bar1Erg4Δerg5ΔRH5233MATa erg4Δ::URA3 erg5Δ::kanMX4 leu2 ura3 his4 lys2 bar1Wild typeRH6611MATa his3 ura3 leu2 (pRS423) (pRS425)CholesterolRH6613MATa erg5Δ::TRP1 erg6::TRP1 his3 ura3 leu2 trp1 (pRS423-DHCR7) (pRS425-DHCR24) Open table in a new tab TABLE 2Plasmids used in this studyPlasmidCharacteristicsRef.pRS423Multicopy vector containing GDP promoter and HIS330Mumberg D. Muller R. Funk M. Gene (Amst.). 1995; 156: 119-122Crossref PubMed Scopus (1603) Google ScholarpRS423-DHCR7pRS423 derivative vector containing DHCR7 gene–aC. M. Souza, H. Pichler, E. Leitner, X. Guan, M. R. Wenk, I. Tornare, and H. Riezman, submitted for publicationpRS425Multicopy vector containing GDP promoter and LEU230Mumberg D. Muller R. Funk M. Gene (Amst.). 1995; 156: 119-122Crossref PubMed Scopus (1603) Google ScholarpRS425-DHCR24pRS425 derivative vector containing DHCR24 gene–aC. M. Souza, H. Pichler, E. Leitner, X. Guan, M. R. Wenk, I. Tornare, and H. Riezman, submitted for publicationa C. M. Souza, H. Pichler, E. Leitner, X. Guan, M. R. Wenk, I. Tornare, and H. Riezman, submitted for publication Open table in a new tab MIC Value Determinations—Minimum inhibitory concentrations (MICs) were determined by diluting the polyene antibiotics in YPUADT (with 0.1 mg/ml ampicillin) to concentrations of 400, 350, 300, and 250 μm of which 100 μl was added to the first row of a 96-well suspension culture plate (U-form, Greiner Bio One). This was followed by a 1:1 dilution series in medium. Overnight cultures were diluted back to an A600 0.0001, of which 100 μl was added to the culture plate. The total volume per well was 200 μl. Strains RH6611 and RH6613 (in SD-His-Leu medium) were diluted to an A600 0.01, because they had a very slow growth rate. The MIC value was determined to be the lowest concentration of antibiotic, which inhibits the growth of the yeast strain and could be determined by eye on the 96-well plate after an incubation of 24 h at 30 °C. The experiments were performed in triplicate. Preparation of Large Unilamellar Vesicles (LUVs)—LUVs with a mean diameter of 200 nm were prepared using the following protocol. Aqueous phospholipid suspensions were prepared by premixing ergosterol or cholesterol with DOPC in the desired molar ratios as solutions in chloroform and evaporating the solvent in a stream of nitrogen, followed by drying the lipid film for 20 min under vacuum. Sterols were present in a range of 10 to 30 mol %. All following handlings were performed at 50 °C. The lipid film was hydrated and repeatedly vortexed until all lipid was removed from the walls of the test tube. Then a freeze-thaw cycle was repeated eight times using liquid nitrogen and a water bath. Subsequently, the lipid suspension was extruded 8 times through a polycarbonate membrane filter with a pore size of 0.2 μm (Whatman International). The size of the vesicles was determined after extrusion by using the Zetasizer 3000 (Malvern Instruments). The average of the size of the vesicles was 168 ± 3.7 nm for vesicles without sterols, 165 ± 1.2 nm for vesicles with 10% cholesterol, and 173 ± 8 nm for vesicles with 10% ergosterol. Thus no significant differences in size were observed. The resulting vesicle suspension was stored at 4 °C. The final phospholipid concentration was determined by phosphate analysis according to Rouser et al. (6Rouser G. Fkeischer S. Yamamoto A. Lipids. 1970; 5: 494-496Crossref PubMed Scopus (2880) Google Scholar). ITC Measurements—Titration experiments were carried out on a MCS titration calorimeter from Microcal Inc. LUVs were prepared as described above in 50 mm MES, 100 mm K2SO4, pH 6.0, or 10 mm HEPES, 100 mm NaCl, pH 7.0. Similar results were obtained with the different buffers. The vesicles were injected into a sample cell (volume = 1.345 ml) containing 50 μm antibiotic in the same buffer as used for the vesicle suspension. Because the polyene antibiotics are dissolved in Me2SO, an equal amount was added to the LUV suspension to compensate for any heat generated by dilution of this solvent. No more than 1% Me2SO was present. The solutions were degassed, before the start of the titration. The experiments consisted of 44 injections, 5 μl each, of a stock solution of vesicles at 25 °C (8 mm final phospholipid concentration). The results were analyzed using the ORIGIN software (version 2.9) provided by Microcal Inc. The interaction between the vesicles and the antibiotics was complex in that no clear saturation of this interaction was observed. Therefore the stoichiometry of the interaction could not be determined. An approximation of the binding constant was made using the ORIGIN software, where the value of integrated heat of the last injection was subtracted from all data and the model of one set of sites was fitted to the resulting data. Binding Assay Using Centrifugation of Model Membranes—Vesicles were prepared as described above in 10 mm MES/Tris, 15 mm K2SO4 at pH 7. The reduced ion strength facilitated the pelleting of the vesicles. The concentrations of antibiotics and vesicles were varied from 0 to 0.1 and 0.5 to 5 mm, respectively, unless indicated otherwise. Vesicles were incubated with the polyene antibiotics for 1 h in an Eppendorf incubator (22 °C, 650 rpm), with a maximum of 1% Me2SO present. To spin down the vesicles and the bound antibiotic, 1 ml of the mixture was centrifuged in a TLA 120.2 rotor in a Beckman Ultracentrifuge (TL-100) for 1.5 h at 100 krpm and 20 °C. The amount of antibiotic before centrifugation and in the supernatant and pellet was determined by UV absorption after 7 times dilution in methanol followed by centrifugation to remove any precipitated salts. The phospholipid concentrations were determined by phosphate analysis according to Rouser et al. (6Rouser G. Fkeischer S. Yamamoto A. Lipids. 1970; 5: 494-496Crossref PubMed Scopus (2880) Google Scholar). Under these conditions less than 10% of the phospholipids remained in the supernatant. The antibiotics were not pelleted in the absence of lipid below a concentration of 75, 34, and 30 μm, respectively, of natamycin, nystatin, and filipin. The binding isotherms of the interaction of natamycin with ergosterol could be described by the Langmuir adsorption model assuming that ergosterol was the only binding site for natamycin in the DOPC vesicles and that only the ergosterol in the outer leaflet of the bilayer could have an interaction with natamycin. The Langmuir adsorption model was applied to the data of the amount of natamycin bound to the vesicles versus the amount of free natamycin in the supernatant (7Langmuir I. J. Am. Chem. Soc. 1916; 38: 2221-2295Crossref Scopus (7969) Google Scholar). From using this model in Sigmaplot (10.0), the binding constant and the binding saturation of natamycin with ergosterol could be determined. Binding Assay Using Centrifugation of Intact Cells—Yeast were grown to the mid-logarithmic phase in 200 ml of YPUADT (with 0.1 mg/ml ampicillin) or SD medium. As a negative control the Escherichia coli strain DH5α was used that was grown to the logarithmic phase in 100 ml of Luria Broth (LB) medium at 37 °C. The cells were harvested by centrifugation at room temperature at 3600 × g for 10 min in a Sorvall RC 5B centrifuge (SLA 1500), washed two times in 100 ml of 10 mm MES/Tris, 15 mm K2SO4 at pH 7, and resuspended in a small volume of buffer. The A600 of the cell suspensions was determined and a series of 1-ml cell suspensions were prepared ranging from an A600 of 0 to 15. The cells were centrifuged at 3000 × g for 5 min at room temperature and resuspended in the same buffer containing 30 μm natamycin. As a control, cells were resuspended in buffer with no natamycin. The cells were incubated for 1 h in an Eppendorf incubator (900 rpm at room temperature) and spun down for 15 min at 3000 × g. The amount of natamycin in the supernatant was determined by UV absorption as described above (spectrum from 250 to 350 nm) and used to calculate the amount of natamycin bound to the yeast cells. Carboxyfluorescein Permeability Assay in Large Unilamellar Vesicles—Carboxyfluorescein (CF)-loaded vesicles were prepared as described above in 50 mm MES/KOH buffer at pH 7 (8Breukink E. van Kraaij C. Demel R.A. Siezen R.J. Kuipers O.P. Kruijff de B. Biochemistry. 1997; 36: 6968-6976Crossref PubMed Scopus (154) Google Scholar). To remove the untrapped CF, a Sephadex G-50 spin column equilibrated with 50 mm MES, 100 mm K2SO4 buffer at pH 7 was used. The CF-loaded vesicles were diluted in 1200 μl of 50 mm MES, 100 mm K2SO4 buffer at pH 7 followed by the addition of the antibiotic. The antibiotic-induced CF leakage from the vesicles was monitored by measuring the fluorescence intensity at 513 nm (excitation set at 430 nm) on a SLM AMINCO Spectrofluorometer (SPF-500). The detergent Triton X-100 was added at the end of the experiment to destroy the lipid vesicles and the resulting fluorescence was taken as the 100% leakage value. Proton Permeability Assay in Large Unilamellar Vesicles—Proton permeability was determined in an assay with HPTS-loaded vesicles as performed by van Kan et al. (9van Kan E.J. Demel R.A. Breukink E. der Bent van A. Kruijff de B. Biochemistry. 2002; 41: 7529-7539Crossref PubMed Scopus (49) Google Scholar). The assay is based on the strong pH dependence of the fluorescence of HPTS. Vesicles were prepared as described above in a 2 mm HPTS solution in 0.2 m NaH2PO4/Na2HPO4 buffer at pH 7. To create a lower pH at the outside and remove all the untrapped HPTS, a Sephadex G-25 spin column was used equilibrated with 10 mm MES, 0.2 m Na2SO4 buffer at pH 5.5. To determine the phospholipid concentration of the resulting vesicles the lipids were first extracted according to Bligh-Dyer (10Bligh E.G. Dyer W.J. Can J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (43133) Google Scholar) to exclude the phosphate from the buffer in the following phosphate analysis according to Rouser et al. (6Rouser G. Fkeischer S. Yamamoto A. Lipids. 1970; 5: 494-496Crossref PubMed Scopus (2880) Google Scholar). The effects of the polyene antibiotics on the proton permeability of the lipid vesicles was monitored by adding aliquots of antibiotic to 1200 μl of 10 mm MES and 0.2 m Na2SO4 buffer, pH 5.5, containing HPTS-loaded vesicles (35 μm phospholipid phosphorous). The fluorescence emission was detected at 508 nm (excitation at 450 nm) on a SLM AMINCO Spectrofluorometer (SPF-500). Differing from van Kan et al. (9van Kan E.J. Demel R.A. Breukink E. der Bent van A. Kruijff de B. Biochemistry. 2002; 41: 7529-7539Crossref PubMed Scopus (49) Google Scholar), the detergent DDAO was used instead of Triton X-100, because DDAO did not have any effect on the fluorescence of the probe where Triton X-100 did have an effect (not shown). DDAO was added at the end to destroy the lipid vesicles and the resulting fluorescence was taken as the 100% leakage value, whereas the blank without antibiotic was used as a reference for 0% leakage. Nigericin, a polyether ionophore known to collapse proton gradients, was used as a positive control (11Boyer M.J. Hedley D.W. Methods Cell Biol. 1994; 41: 135-148Crossref PubMed Scopus (48) Google Scholar). Proton Permeability Assay in Yeast—The assay was based on the loading of yeast cells with the probe CFDA-SE as described by Bracey et al. (12Bracey D. Holyoak C.D. Caron Nebe-von G. Coote P.J. J. Microbiol. Methods. 1998; 31: 113-125Crossref Scopus (62) Google Scholar, 13Bracey D. Holyoak C.D. Coote P.J. J. Appl. Microbiol. 1998; 85: 1056-1066Crossref PubMed Scopus (97) Google Scholar). CFDA-SE is a non-polar molecule that spontaneously penetrates cell membranes and is converted to the anionic pH-sensitive 5-(and-6)-carboxyfluorescein succinimidyl ester (CF-SE) by intracellular esterases (9van Kan E.J. Demel R.A. Breukink E. der Bent van A. Kruijff de B. Biochemistry. 2002; 41: 7529-7539Crossref PubMed Scopus (49) Google Scholar). Once the probe is internalized, amine reactive coupling of succinimidyl groups of CF-SE to aliphatic amines of intracellular proteins results in the formation of membrane-impermeable pH-sensitive probe conjugates. Wild type yeast cells from an overnight culture were diluted to an A600 of ∼0.8 and then centrifuged at 3000 × g for 3 min. The cells were washed and resuspended in an equal volume of 100 mm citric/phosphate buffer at pH 4 (100 mm citric acid, 50 mm NaH2PO4, and 50 mm KOH). CFDA-SE (100 μm) was added and the cells were incubated overnight while shaking at 37 °C. The viability of the cells was not significantly compromised by the loading conditions. Loaded cells were harvested (3000 × g for 3 min), washed, and resuspended in YPUADT buffered with 50 mm citric/phosphate (pH 4) to an A600 of 0.4. To recover from the stress imposed by the probe loading conditions, the cultures were left for 1 h at 30 °C with shaking. The effects of the polyene antibiotics on the proton permeability of the yeast cells were monitored by adding aliquots of antibiotic to 5 ml of culture and measuring the A600 and fluorescence at regular intervals. The A600 was determined on a Helios Epsilon UNICAM spectrometer and the fluorescence emission was detected at 525 nm (excitation at 495 nm) on a SLM AMINCO Spectrofluorometer (SPF-500). Sterol Specificity of Natamycin Binding to Membranes—To test whether sterols are required for membrane affinity of natamycin we used phosphatidylcholine model membranes containing ergosterol, the main fungal sterol or cholesterol, the main sterol in mammals. The interaction between natamycin and sterols in the model membrane was first studied using ITC. ITC measurements were performed where LUVs containing either no sterols, cholesterol, or ergosterol were titrated into a solution of natamycin (Fig. 2). Natamycin displayed no interaction with vesicles containing no sterols as the resulting heats were no different from the control (Fig. 2A). LUVs containing 10 mol % cholesterol produced only minor heat effects during the first injections, which indicates that natamycin displayed only a very small interaction with cholesterol containing vesicles (Fig. 2B). Interestingly, 10 mol % ergosterol containing vesicles displayed a significant amount of interaction with natamycin as evidenced by the consecutive heat effects (Fig. 2C). This titration curve differs from a normal titration curve as no clear saturation of the interaction was observed. The binding constant between natamycin and ergosterol was estimated to be 5.7 × 104 m-1 (see "Experimental Procedures"). Comparable large differences in effects between cholesterol and ergosterol were observed for sterol concentrations of 20 mol % (data not shown). Furthermore, the binding of natamycin to vesicles was studied by separating the bound from the free natamycin by centrifugation. Fig. 3A shows a representative graph of these results, from which can be concluded that ergosterol containing vesicles had a significant interaction with natamycin. In the absence of sterols or in the presence of cholesterol very little interaction with natamycin was observed consistent with the ITC experiments (Fig. 2). A similar sterol dependence of natamycin binding was observed when varying the concentrations of vesicles (data not shown). The binding constant was determined by the Langmuir adsorption model in SigmaPlot (10.0) to be 2.5 ± 1.0 × 104 m-1, which is in reasonable agreement with the binding constant determined in the ITC measurements. The binding saturation from the Langmuir adsorption model was determined at 72 ± 12 μm by extrapolating the data in SigmaPlot (10.0). By assuming that only the sterol in the external leaflet of the lipid vesicles could establish an interaction with the antibiotic, the sterol to antibiotic ratio was calculated to be ∼1:1. If all sterols would be available for the interaction, because of sterol flip-flop, the ratio would be 2:1. The affinity of natamycin for ergosterol containing vesicles was compared with that of filipin and nystatin to get insight into the relative strength of this interaction. Fig. 3B shows a representative graph of the results obtained with these antibiotics. Of the three polyene antibiotics filipin showed the highest affinity, followed by natamycin and nystatin. Sterol Specificity in the Antibiotic Action—To test if ergosterol is needed for natamycin to exert its antifungal activity in vivo, yeast strains carrying specific mutations in the ergosterol biosynthesis pathway (ergΔ) were used. Because of these mutations, the strains cannot synthesize ergosterol. However, they each accumulate a distinct set of sterols that, compared with ergosterol, have structural differences in the side chain and double bonds in the B or C ring (Fig. 4). The availability of these strains allows us to address the sterol specificity for polyenes, in relation to their inhibitory activity. The most prominent sterols present in the ergΔ mutants are tabulated in percentage of total sterol present, together with their MIC values for the polyene antibiotics natamycin, nystatin, and filipin in Table 3. The sterol composition of the erg strains given in Table 3 was taken from Heese-Peck et al. (5Heese-Peck A. Pichler H. Zanolari B. Watanabe R. Daum G. Riezman H. Mol. Biol. Cell. 2002; 13: 2664-2680Crossref PubMed Scopus (133) Google Scholar) and specifies the percentage of a listed sterol compared with the total sterol composition of a cell. The most sensitive erg strain is erg4Δerg5Δ, which has a MIC value of the wild type strain. The least sensitive toward natamycin was erg2Δerg6Δ, which contained mostly zymosterol. From the strain with the highest sensitivity toward the lowest, the most striking sterol structural feature that causes the loss of activity is the loss of double bonds in ring B. For example, the sterols in erg3Δ have one double bond at position C-7,8 and it is only 3 times less sensitive to natamycin compared with the wild type, whereas erg2Δerg6Δ has lost both double bonds at C-5,6 and C-7,8 and is 37 times less sensitive compared with the wild type. Variations in the C17 side chain of the sterols did not have very large effects on the sensitivity toward natamycin, which can be observed when comparing erg4Δerg5Δ with the wild type. The yeast strain sensitivities toward nystatin were similar compared with natamycin. Filipin sensitivity seemed not to be so dependent on the sterol structure. The results demonstrate that double bonds in the B ring of the sterols are very important for natamycin to inhibit the growth of yeast, whereas changes of the C17 side chain are of less importance.TABLE 3The minimum concentration of the polyene antibiotics needed to inhibit the growth of different ergΔ mutants The MIC values for natamycin (MICnatam), nystatin (MICnyst), and filipin (MICfilip) are given for the different ergΔ strains, together with the structure and percentage of the most abundant sterols in an ergΔ strain, as stated in Ref. 5Heese-Peck A. Pichler H. Zanolari B. Watanabe R. Daum G. Riezman H. Mol. Biol. Cell. 2002; 13: 2664-268