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

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 in medium yeast 20 and 20 with adenine, 2 uracil, and with strains and medium was used yeast without amino 20 2 with and the amino acids and Yeast strains used in this study are with in and the in used in this study The of these strains is in A. Pichler H. Zanolari B. Watanabe R. Daum G. Riezman H. Mol. Biol. Cell. 2002; 13: 2664-2680Crossref PubMed Scopus (133) Google in a new used in this containing and R. PubMed Scopus Google containing H. E. R. and H. for containing and R. PubMed Scopus Google containing H. E. R. and H. for H. E. R. and H. for in a new inhibitory concentrations were determined by the polyene antibiotics in to concentrations of and of which was to the of a This was by a in were to an of which was to the The was Strains and were to an because they a very growth The was determined to be the concentration of which the growth of the yeast and could be determined by on the an of The were performed in of with a of were prepared using the phospholipid were prepared by ergosterol or cholesterol with DOPC in the molar as solutions in chloroform and the in a of by the lipid for 20 under were present in a of to All were performed The lipid was and all lipid was from the of the a was using and a the lipid was a membrane with a of The of the vesicles was determined by using the The of the of the vesicles was for vesicles without sterols, for vesicles with and for vesicles with ergosterol. in were The resulting was The 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). were on a titration from Inc. were prepared as in or were obtained with the different The vesicles were into a cell containing antibiotic in the as used for the the polyene antibiotics are dissolved in Me2SO, an was to the to for any by of this more than was The solutions were before the start of the The of of a stock of vesicles phospholipid The were using the by Inc. The interaction between the vesicles and the antibiotics was in that of this interaction was the of the interaction could not be determined. of the binding was using the the of of the was from all and the model of of was to the resulting of were prepared as in The ion the of the The concentrations of antibiotics and vesicles were from to and to were with the polyene antibiotics for in an with a of To the vesicles and the of the was in a in a for and 20 The of antibiotic before and in the and was determined by UV absorption in methanol by to any 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). these conditions than of the in the The antibiotics were not in the of lipid a concentration of and of natamycin, nystatin, and The binding of the interaction of natamycin with ergosterol could be by the model that ergosterol was the binding for natamycin in the DOPC vesicles and that the ergosterol in the of the bilayer could have an interaction with The model was to the of the of natamycin to the vesicles the of natamycin in the J. Chem. Scopus Google Scholar). using this model in the binding and the binding of natamycin with ergosterol could be determined. of were grown to the in of or a the was used that was grown to the in of medium The cells were by × for in a in of and in a small of The of the cell was determined and a of cell were prepared from an of to The cells were × for and in the containing a cells were in with The cells were for in an and for × The of natamycin in the was determined by UV absorption as from to nm) and used to the of natamycin to the yeast cells. in vesicles were prepared as in E. Demel R.A. de B. PubMed Scopus Google Scholar). To the a with was The vesicles were in of by the of the The leakage from the vesicles was by the nm) on a The was the of the experiment to the lipid vesicles and the resulting was as the leakage in permeability was determined in an with vesicles as performed by et al. Demel R.A. E. A. de B. 2002; PubMed Scopus Google Scholar). The is on the strong of the of were prepared as in a 2 in To a the and all the a was used with To determine the phospholipid concentration of the resulting vesicles the were according to J. PubMed Scopus Google to the phosphate from the in the phosphate analysis according to Rouser et al. (6Rouser G. Fkeischer S. Yamamoto A. Lipids. 1970; 5: 494-496Crossref PubMed Scopus (2880) Google Scholar). The of the polyene antibiotics on the permeability of the lipid vesicles was by of antibiotic to of and containing vesicles phospholipid The was nm) on a from et al. Demel R.A. E. A. de B. 2002; PubMed Scopus Google the was used of because did not have any effect on the of the did have an effect was the to the lipid vesicles and the resulting was as the leakage whereas the without antibiotic was used as a for a ionophore known to collapse gradients, was used as a Biol. PubMed Scopus Google Scholar). in was on the of yeast cells with the as by et al. G. J. Microbiol. Scopus Google Scholar, J. Microbiol. PubMed Scopus Google Scholar). is a molecule that cell membranes and is to the succinimidyl ester by Demel R.A. E. A. de B. 2002; PubMed Scopus Google Scholar). the is of succinimidyl of to of in the of yeast cells from an overnight were to an of and × for The cells were and in an of and was and the cells were overnight while The of the cells was not compromised by the cells were × for and in with to an of To from the by the the were for with The of the polyene antibiotics on the permeability of the yeast cells were by of antibiotic to of and the and The was determined on a spectrometer and the was nm) on a of Natamycin to whether sterols are for membrane of natamycin we used phosphatidylcholine model membranes containing ergosterol, the main fungal sterol or the main sterol in The interaction between natamycin and sterols in the model membrane was using were performed containing sterols, or ergosterol were into a of natamycin (Fig. Natamycin interaction with vesicles containing sterols as the resulting were different from the (Fig. containing cholesterol produced the which that natamycin a very small interaction with cholesterol containing vesicles (Fig. ergosterol containing vesicles a of interaction with natamycin as by the consecutive (Fig. This titration from a titration as of the interaction was The binding between natamycin and ergosterol was to be × 104 m-1 large in between cholesterol and ergosterol were for sterol concentrations of 20 not the binding of natamycin to vesicles was by the from the natamycin by a of these from which be that ergosterol containing vesicles a interaction with In the of sterols or in the of cholesterol very interaction with natamycin was with the (Fig. similar sterol of natamycin binding was varying the concentrations of vesicles not The binding was determined by the model in to be × 104 which is in with the binding determined in the The binding from the model was determined by the in that the sterol in the of the lipid vesicles could an interaction with the the sterol to antibiotic was to be all sterols be for the because of sterol the be The of natamycin for ergosterol containing vesicles was compared with that of filipin and nystatin to insight into the of this a of the obtained with these the polyene antibiotics filipin the by natamycin and nystatin. in the ergosterol is for natamycin to its antifungal activity in yeast strains specific mutations in the ergosterol pathway (ergΔ) were of these the strains ergosterol. they a of sterols that, compared with ergosterol, have in the and double bonds in the B or ring (Fig. The of these strains to the sterol specificity for polyenes, in relation to inhibitory The sterols present in the mutants are in of sterol together with for the polyene antibiotics natamycin, nystatin, and filipin in The sterol composition of the strains in was from 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 and the of a sterol compared with the sterol composition of a The is which has a of the The natamycin was which the with the the the sterol feature that the loss of activity is the loss of double bonds in ring B. example, the sterols in have double bond and it is to natamycin compared with the whereas has both double bonds and and is compared with the in the of the sterols did not have very large on the natamycin, which be with the The yeast nystatin were similar compared with Filipin not to be on the sterol The demonstrate that double bonds in the B ring of the sterols are very important for natamycin to the growth of whereas of the are of minimum concentration of the polyene antibiotics to the growth of different mutants The for natamycin nystatin and filipin are for the different together with the structure and of the abundant sterols in an as in A. Pichler H. Zanolari B. Watanabe R. Daum G. Riezman H. Mol. Biol. Cell. 2002; 13: 2664-2680Crossref PubMed Scopus (133) Google The were determined in in a new a yeast was that is to ergosterol or its but was to This to the strong of natamycin for ergosterol cholesterol as in the model membrane The of growth are shown in and that the cholesterol was natamycin compared with the This demonstrates that also in vivo natamycin has a strong specificity for ergosterol the in of ergosterol and the importance of the double bonds of the B-ring for interaction with natamycin is with the of the the effect on the yeast strains as natamycin, whereas filipin is specific as it was as effective in killing the cholesterol as the minimum concentration of the polyene antibiotics to the growth of strains and The for natamycin nystatin and filipin are for the different together with the sterol structure and of the abundant sterols in the as H. E. R. and H. for The were determined in in a new To determine whether the of growth was to the of binding of natamycin to these yeast a binding with the different strains was All the strains were tested and in an E. was as a because it contains sterols in the plasma membrane. strains are in The of binding of natamycin was for the and together with The of binding was for the the E. whereas the of binding of the yeast The relation of the of binding of natamycin to the is in a cell to an of The an relation between the of natamycin to the of a particular that the in natamycin are directly to the in binding of natamycin to the yeast cells. In addition, binding studies with vesicles from lipid of plasma yeast membrane were performed and in a similar binding as compared with intact yeast cells not of on in binding as as the that is a specific interaction of natamycin with ergosterol, which to an of cell growth. To the interaction of natamycin with ergosterol to in membrane different leakage were Natamycin did not any carboxyfluorescein leakage from DOPC vesicles containing ergosterol in contrast to filipin, which did cause carboxyfluorescein leakage not nystatin, which is known to form also did not cause carboxyfluorescein from the the pores by this antibiotic are small to of this similar could be the for we an on leakage of that be small to This makes of a which has a and a Demel R.A. E. A. de B. 2002; PubMed Scopus Google Scholar). of the effect of polyene antibiotics on ergosterol containing vesicles is in was by of the vesicles to the and the in the was to the vesicles and the its was used as a and in an of the the model membrane. filipin and nystatin both in leakage of the membrane natamycin did not result in leakage this more analysis of the effect of the antibiotics on leakage in model membranes is in The that in strong contrast to filipin and nystatin, natamycin did not any leakage in ergosterol containing vesicles even very concentrations (Fig. This that natamycin not via a of the membrane barrier and has a completely different mode of action compared with filipin or nystatin. To similar could be in a leakage in yeast was of on and Growth in the from the in vitro leakage to an in vivo yeast cells were with the The effect of the polyene antibiotics the on the yeast is in The of the yeast was different (Fig. In the of the yeast cells a that in natamycin was in was nystatin was to the yeast cells an in was to the of pores in the plasma membrane. the of a increase of the was which that the yeast cells to the ion the plasma membrane. that with the conditions used to study the of growth was by both natamycin and nystatin the in mode of action between these polyene In natamycin not yeast cells by permeabilizing the plasma membrane. In this study we have that natamycin yeast by specifically binding to ergosterol but without permeabilizing the plasma membrane. This novel mechanism natamycin from other polyene antibiotics We of these as a in this The and direct binding studies in both model and yeast membrane systems that natamycin binds with an of specifically to ergosterol with a of or whether the sterol is for interaction in the or in both leaflets of the membrane. This is in with the before for other polyene B. A. Demel R.A. Biochim. Biophys. Acta. 1974; 339: PubMed Scopus Google Scholar). the of the binding and the unknown of the natamycin-ergosterol a more of the binding is not from the model system and the yeast mutants a of the the sterol structure for the binding to natamycin, in the double bonds of the B-ring in large in especially the of The of the sterol molecule together with natamycin is to this The of ring B in ergosterol from the of this ring in which is in The in ergosterol with an in a which is in cholesterol a (Fig. A. E. Biophys. J. PubMed Scopus Google Scholar). Natamycin has a that a very J. 2002; PubMed Scopus Google Scholar). it is very that the of the B-ring in ergosterol result in a more amphotericin similar were the was of importance for the interaction of this antibiotic with sterols in model membranes, whereas the double bond was not essential S. R. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The sterol specificity of natamycin in model and was more with nystatin than to This also be from the that are as The of binding for filipin in the experiment was ergosterol cholesterol sterol to the of and × 104 respectively. Filipin did not to be as on sterol structure the of sterols as the to different membranes did not with J. J. J. Biophys. J. PubMed Scopus Google Scholar, J. Biochim. Biophys. Acta. PubMed Scopus Google Scholar, E. M.A. J. Chem. PubMed Scopus Google Scholar). The binding of nystatin more similar to natamycin and the is compared with natamycin, to × 104 We have shown that the interaction between natamycin and ergosterol to an of yeast growth and cell this is not via a of the membrane as is by nystatin. The structure of the natamycin-ergosterol is but that it is similar to for the in mode of action. One is that the of natamycin and ergosterol be to even an ion as small as a the could be small to the the mode of action of natamycin not it In this it is that for the polyene antibiotics that are known to permeabilize the also other modes of have been as of membrane J. S. A. G. PubMed Scopus Google Scholar, J. G. J. 1986; PubMed Scopus Google Scholar, J. R. Biochim. Biophys. Acta. PubMed Scopus Google Scholar). The mode of action of natamycin be to an important function of ergosterol in the yeast cells. example, sterols are known to have an effect on the it is that they in specific in membranes and they are also known to be in and Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, Biochim. Biophys. Acta. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar). Natamycin these important by binding to ergosterol that the sterol its We R. and J. The for and with