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We report on the synthesis, biological function, and a plausible mode of action of a new group of lipopeptides with potent antifungal and antibacterial activities. These lipopeptides are derived from positively charged peptides containing d- and l-amino acids (diastereomers) that are palmitoylated (PA) at their N terminus. The peptides investigated have the sequence K4X7W, where X designates Gly, Ala, Val, or Leu (designated d-X peptides). The data revealed that PA-d-G and PA-d-A gained potent antibacterial and antifungal activity despite the fact that both parental peptides were completely devoid of any activity toward microorganisms and model phospholipid membranes. In contrast, PA-d-L lost the potent antibacterial activity of the parental peptide but gained and preserved partial antifungal activity. Interestingly, both d-V and its palmitoylated analog were inactive toward bacteria, and only the palmitoylated peptide was highly potent toward yeast. Both PA-d-L and PA-d-V lipopeptides were also endowed with hemolytic activity. Mode of action studies were performed by using tryptophan fluorescence and attenuated total reflectance Fourier transform infrared and circular dichroism spectroscopy as well as transmembrane depolarization assays with bacteria and fungi. The data suggest that the lipopeptides act by increasing the permeability of the cell membrane and that differences in their potency and target specificity are the result of differences in their oligomeric state and ability to dissociate and insert into the cytoplasmic membrane. These results provide insight regarding a new approach of modulating hydrophobicity and the self-assembly of non-membrane interacting peptides in order to endow them with both antibacterial and antifungal activities urgently needed to combat bacterial and fungal infections. We report on the synthesis, biological function, and a plausible mode of action of a new group of lipopeptides with potent antifungal and antibacterial activities. These lipopeptides are derived from positively charged peptides containing d- and l-amino acids (diastereomers) that are palmitoylated (PA) at their N terminus. The peptides investigated have the sequence K4X7W, where X designates Gly, Ala, Val, or Leu (designated d-X peptides). The data revealed that PA-d-G and PA-d-A gained potent antibacterial and antifungal activity despite the fact that both parental peptides were completely devoid of any activity toward microorganisms and model phospholipid membranes. In contrast, PA-d-L lost the potent antibacterial activity of the parental peptide but gained and preserved partial antifungal activity. Interestingly, both d-V and its palmitoylated analog were inactive toward bacteria, and only the palmitoylated peptide was highly potent toward yeast. Both PA-d-L and PA-d-V lipopeptides were also endowed with hemolytic activity. Mode of action studies were performed by using tryptophan fluorescence and attenuated total reflectance Fourier transform infrared and circular dichroism spectroscopy as well as transmembrane depolarization assays with bacteria and fungi. The data suggest that the lipopeptides act by increasing the permeability of the cell membrane and that differences in their potency and target specificity are the result of differences in their oligomeric state and ability to dissociate and insert into the cytoplasmic membrane. These results provide insight regarding a new approach of modulating hydrophobicity and the self-assembly of non-membrane interacting peptides in order to endow them with both antibacterial and antifungal activities urgently needed to combat bacterial and fungal infections. Together with the growing number of individuals with impaired host defenses, invasive mycoses have emerged as major causes of morbidity and mortality in the last decade (1Groll A.H. Shah P.M. Mentzel C. Schneider M. Just-Nuebling G. Huebner K. J. Infect. 1996; 33: 23-32Abstract Full Text PDF PubMed Scopus (686) Google Scholar, 2Minamoto G.Y. Rosenberg A.S. Med. Clin. N. Am. 1997; 81: 381-409Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 3Walsh T.J. Hiemenz J.W. Anaissie E. Infect. Dis. Clin. North Am. 1996; 10: 365-400Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 4Ellis M. Richardson M. de Pauw B. Hosp. Med. 2000; 61: 605-609Crossref PubMed Scopus (30) Google Scholar, 5Odds F.C. Brown A.J. Gow N.A. Trends Microbiol. 2003; 11: 272-279Abstract Full Text Full Text PDF PubMed Scopus (896) Google Scholar). Although the spectrum of fungal pathogens has changed, the vast majority of invasive fungal infections are still due to Aspergillus and Candida species (4Ellis M. Richardson M. de Pauw B. Hosp. Med. 2000; 61: 605-609Crossref PubMed Scopus (30) Google Scholar, 5Odds F.C. Brown A.J. Gow N.A. Trends Microbiol. 2003; 11: 272-279Abstract Full Text Full Text PDF PubMed Scopus (896) Google Scholar, 6Denning D.W. J. Antimicrob. Chemother. 1991; 28: 1-16Crossref PubMed Google Scholar). Because of their eukaryotic nature fungal cells have only a restricted set of unique targets. This makes it difficult to selectively target fungal cells. Two major families have been used for more than two decades to combat fungi; these include azoles, which inhibit sterol formation, and polyenes, which bind to mature membrane sterols. However, the development of fluconazole resistance among different pathogenic strains and the high toxicity of amphotericin B (7Alexander B.D. Perfect J.R. Drugs. 1997; 54: 657-678Crossref PubMed Scopus (143) Google Scholar, 8Mukherjee P.K. Chandra J. Kuhn D.M. Ghannoum M.A. Infect. Immun. 2003; 71: 4333-4340Crossref PubMed Scopus (418) Google Scholar, 9Kontoyiannis D.P. Mantadakis E. Samonis G. J. Hosp. Infect. 2003; 53: 243-258Abstract Full Text PDF PubMed Scopus (103) Google Scholar) have prompted the studies of new antifungal agents with new modes of actions. The investigation of antimicrobial peptides, from a wide range of biological sources, and their synthetic derivatives is a novel inroad to new antifungal agents. Antimicrobial peptides are gene-encoded and are part of the innate immunity to the microbial invasion of microorganisms of all types. The most studied group are short (<40 amino acids), positively charged, linear peptides whose possible mechanisms have been reviewed in detail (10Bechinger B. Biochim. Biophys. Acta. 1999; 1462: 157-183Crossref PubMed Scopus (439) Google Scholar, 11Blondelle S.E. Perez-Paya E. Houghten R.A. Antimicrob. Agents Chemother. 1996; 40: 1067-1071Crossref PubMed Google Scholar, 12Dathe M. Wieprecht T. Biochim. Biophys. Acta. 1999; 1462: 71-87Crossref PubMed Scopus (636) Google Scholar, 13Tossi A. Sandri L. Giangaspero A. Biopolymers. 2000; 55: 4-30Crossref PubMed Scopus (1056) Google Scholar, 14Oren Z. Shai Y. Biopolymers. 1998; 47: 451-463Crossref PubMed Scopus (766) Google Scholar, 15Hwang P.M. Vogel H.J. Biochem. Cell Biol. 1998; 76: 235-246Crossref PubMed Scopus (293) Google Scholar, 16Zasloff M. Nature. 2002; 415: 389-395Crossref PubMed Scopus (6852) Google Scholar). It is believed that most of these peptides are targeted to biological membranes, increase their permeability, and kill the cells. However, other additional mechanisms were proposed for some of them (17Hancock R.E. Rozek A. FEMS Microbiol. Lett. 2002; 206: 143-149Crossref PubMed Google Scholar). Studies that investigated the mode of action of native antimicrobial peptides that are also endowed with antifungal activity are limited and included, for example, LL-37 and dermaseptines (18Oren Z. Lerman J.C. Gudmundsson G.H. Agerberth B. Shai Y. Biochem. J. 1999; 341: 501-513Crossref PubMed Scopus (493) Google Scholar, 19Ghosh J.K. Shaool D. Guillaud P. Ciceron L. Mazier D. Kustanovich I. Shai Y. Mor A. J. Biol. Chem. 1997; 272: 31609-31616Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 20Strahilevitz J. Mor A. Nicolas P. Shai Y. Biochemistry. 1994; 33: 10951-10960Crossref PubMed Scopus (122) Google Scholar). These studies showed that they self-associate in solution and reach the membrane as oligomers (18Oren Z. Lerman J.C. Gudmundsson G.H. Agerberth B. Shai Y. Biochem. J. 1999; 341: 501-513Crossref PubMed Scopus (493) Google Scholar, 19Ghosh J.K. Shaool D. Guillaud P. Ciceron L. Mazier D. Kustanovich I. Shai Y. Mor A. J. Biol. Chem. 1997; 272: 31609-31616Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 20Strahilevitz J. Mor A. Nicolas P. Shai Y. Biochemistry. 1994; 33: 10951-10960Crossref PubMed Scopus (122) Google Scholar). Previously, we reported on a new approach for increasing the hydrophobicity and self-assembly in a solution of antibacterial peptides that are not active on fungi (21Avrahami D. Shai Y. Biochemistry. 2002; 41: 2254-2263Crossref PubMed Scopus (165) Google Scholar). We showed that peptide oligomerization in solution is crucial for the peptide to endow antifungal activity. In another study we examined the effect of multiple substitutions of different amino acids regarding the structure, binding, and biological function of positively charged diastereomers (include both d- and l-amino acids) of linear lytic peptides (22Avrahami D. Oren Z. Shai Y. Biochemistry. 2001; 40: 12591-12603Crossref PubMed Scopus (72) Google Scholar). For this purpose, a group of diastereomeric peptides with the sequence K4X7W were synthesized, where X designates one of the aliphatic amino acids, Gly, Ala, Val, or Leu. The results revealed that the peptides containing Ala and Gly are not active against microorganisms, concomitant with their inability to bind membranes. This is in agreement with the notion that a threshold of hydrophobicity along with a defined structure is for antimicrobial activity. we to this of diastereomers and investigated their biological function and mode of we that the most potent antibacterial and antifungal lipopeptides are derived from the peptides containing Ala and Studies on the function and structure of the peptides with their with bacteria, and model on their mode of these studies that a for the hydrophobicity and structure of the to a for antimicrobial activity. was from used for peptide include and and used attenuated total reflectance circular high and were from and were from other were of were in B and were from was from was from and were by a on using peptide The was to the N of a peptide using of the from the N of the peptide with a solution of in the in was to the used for the of amino The peptides were from the by and by high on a or the The peptides were to by The of the lipopeptides by increase in hydrophobicity to the of the spectroscopy was used to their and amino was used to the of the antifungal activity of the peptides and their was to the of for The peptides were examined in in a of as of a containing fungi at a of in with was to of containing the peptide in The fungi were for for fungi or for Candida and at using a was by the at in a activity is as the the at which was The fungi used were Aspergillus Aspergillus Aspergillus C. and C. antibacterial activity of the peptides was examined in in a of as of a containing bacteria at a of in were to of containing the peptide in in from a solution of peptide in of was by the at with a of at activities were as the the at which was of The bacteria used were and of with were with by for at and in in were to of a solution of the in to reach a of The was with for at The were at for The of was by the of the at for and of in and of were by as Y. D. A. J. Biol. Chem. Full Text PDF PubMed Google Scholar). were in The were a of and The were in the with and the were in a the The were using a were from a of and a used to the major of the of C. B. G. A. M. K. C. G. J. Cell Biol. 1999; PubMed Scopus Google Scholar). of the lipopeptides were with a The were with a cell with a of spectrum was in of at of at a range of to The lipopeptides were at a of in and in the of K. Biochemistry. PubMed Scopus Google Scholar, N. Biochemistry. PubMed Scopus Google Scholar) were where is the at and for and to and at are to and K. Biochemistry. PubMed Scopus Google Scholar). In in and which results in the of transmembrane was by using a fluorescence A.S. J.R. Biochemistry. PubMed Scopus Google Scholar) for bacteria and The the membrane to the cell transmembrane in a of the membrane a of the transmembrane that was by increase in fluorescence due to of the were performed in in a of as of the cell and the were to of containing the peptide in depolarization was by increase in the fluorescence of on the C. was performed with yeast. cells were at for at in a at with for in with and The cells were in and to of were with by fluorescence a was the of the the membrane. in E. of the bacteria E. were by the J. J. Biochem. 2000; PubMed Scopus Google Scholar). were at with to the from were by and with were in the containing of at with the cells were by and in of at with the were by The were to of in a containing The cells were with by fluorescence a was the of the into the bacteria membrane. were with a with a and with the as has been in detail in other studies (21Avrahami D. Shai Y. Biochemistry. 2002; 41: 2254-2263Crossref PubMed Scopus (165) Google Scholar). The was by of the to of The was due to the of the of to the the was by of the of with the we the using were to the of the in the These were used as for with and of the were a agreement the of all and the was The of different structure were by the of to a structure by the of the of the of were N. Biochemistry. PubMed Scopus Google Scholar, Scholar) as where is the of a to the at the of and and are the of as and the membrane as these and are and The with the as the of a membrane and are used to the order by the where is the the of the of and the from to were reported in the for Biophys. 1997; PubMed Scopus Google Scholar). We used the of N. Biochemistry. PubMed Scopus Google Scholar, N.A. PubMed Scopus Google Scholar) and Brown L. A. J. Biol. PubMed Scopus (122) Google Scholar) for order were from the and modes using the N. Biochemistry. PubMed Scopus Google Scholar). the of the peptides we in the tryptophan fluorescence in and to peptide was to or containing were on a with the set at using a and in the range of In these studies were used to D. Biochemistry. PubMed Scopus Google Scholar) and the was high of peptide diastereomeric peptides were to the sequence Gly, Ala, Val, or were to a structure in their as revealed by the and Lett. PubMed Scopus Google Scholar). acids at X Ala, Val, or or at X was in peptide to as peptides were at their and at their N to a linear of the sequence of the peptides and their and and in a new activity was against strains of A. and E. and two strains of B. the of the peptides against the The data revealed that both d-V and its palmitoylated analog were devoid of antibacterial activity. However, in to was highly active toward all bacteria but its palmitoylated analog was also devoid of antibacterial activity. both and were devoid of antibacterial their palmitoylated PA-d-G and PA-d-A were highly active toward all the bacteria of the peptides against in a new We the antifungal activity of the peptides against pathogenic and fungi. The C. C. A. A. and A. The results are in The data revealed that in all the antifungal activity. to the antibacterial the lipopeptides PA-d-G and PA-d-A were the most active toward all the fungi and the PA-d-V and are only of the peptides against and in a new The hemolytic activity of the lipopeptides against a highly solution of is in The data revealed that PA-d-G and which were highly active against bacteria, and fungi showed hemolytic activity. In contrast, PA-d-V and which were devoid of antibacterial activity and only and were highly We the fluorescence spectrum of the tryptophan in We that the of the in PA-d-V was of the which causes Because tryptophan is along the peptide we that the as in as for the (22Avrahami D. Oren Z. Shai Y. Biochemistry. 2001; 40: 12591-12603Crossref PubMed Scopus (72) Google Scholar). The data also revealed a in the of which that the peptide is in a more This the peptide with the by on as with the all l-amino (21Avrahami D. Shai Y. Biochemistry. 2002; 41: 2254-2263Crossref PubMed Scopus (165) Google or of the of in which the peptide is to the In contrast, was for PA-d-G and which that these lipopeptides are or the the is to the of the lipopeptides in solution or in the of of was used in all was was of was used in all was in a new we the effect of the on the structure of the peptides in as by spectroscopy to of the lipopeptides showed that only PA-d-L a well defined structure, which is peptide a structure in solution it that the are one against another and the the solution it is into a that by of the diastereomeric peptides were to in solution (22Avrahami D. Oren Z. Shai Y. Biochemistry. 2001; 40: 12591-12603Crossref PubMed Scopus (72) Google Scholar). the in fluorescence in the revealed that the peptide is in a These data the the of The structure of the lipopeptides was in by using We with to it possible to from a structure, the of the structure to a than the Biophys. J. 1991; Full Text PDF PubMed Scopus Google Scholar). The of the lipopeptides are in derivatives with were to the of the in the and are in the in We used these as for with the and of the The of the different to the was to the from and M. Biochem. Biol. PubMed Scopus Google Scholar). The data that the major of PA-d-V is at to the that of PA-d-L has two at and the of both and In contrast, both PA-d-G and PA-d-A are spectroscopy in these structure of the lipopeptides in in a new of the lipopeptides in were at a of in containing of The was performed as The are as spectroscopy was used to the of the membrane. The and the of are to the of a of the dichroism of infrared the order and of the membrane to the However, the of the was than were only on from the The data revealed that the are in a that the used were well N. Biochemistry. PubMed Scopus Google Scholar, Biochim. Biophys. Acta. PubMed Scopus Google Scholar). The effect of the lipopeptides on the order by the of phospholipid and The data that the effect on the order was with PA-d-V the of all the other lipopeptides into the membrane the order to a they are on the C. the ability of the lipopeptides to the cell we the of the transmembrane in in the of the lipopeptides by using a the lipopeptides were to the membrane of C. to a high Interestingly, these results well with the antifungal activity of the different peptides against C. However, is a the and the of peptides to of the transmembrane This due to differences in the used for the antifungal and the depolarization These results suggest that the target of the lipopeptides is the membrane of the cells. of E. the cytoplasmic membrane of bacteria is charged with the membrane of we performed depolarization assays using of the bacteria E. of the depolarization activity is in B. the lipopeptides are highly active in the membrane of the E. However, a the antibacterial activity and the to the cytoplasmic membrane was only with PA-d-G and The that both PA-d-V and PA-d-L are highly active on but not on bacteria and that both oligomers that they are not active on E. they are not to the cell into the cytoplasmic target membrane. The most result in this study is the that the of to the N of positively charged short peptides, which are toward microorganisms endowed them with a spectrum of potent antibacterial and antifungal activity and with a hemolytic activity against the highly solution of In contrast, the to a potent antimicrobial peptide its antibacterial activity but both its antifungal and hemolytic activities. the parental d-V peptide was inactive toward bacteria, and its palmitoylated was active only toward the two strains of and studies with peptides have the of hydrophobicity and structure for their biological function K. Biochim. Biophys. Acta. 1999; 1462: PubMed Scopus Google Scholar, Y. Biopolymers. 2002; PubMed Scopus Google Scholar). all parental peptides were derived from a and the number of at the However, they in their We that to the N of the inactive peptides for the hydrophobicity of the that the antibacterial activity of a peptide of which antibacterial activity A. J. J. Antimicrob. Chemother. 2003; PubMed Scopus Google or endowed antibacterial peptides with antifungal activity (21Avrahami D. Shai Y. Biochemistry. 2002; 41: 2254-2263Crossref PubMed Scopus (165) Google Scholar). The and the on the mode of action of these new we examined their oligomeric state and their ability to increase the membrane permeability as well as their structure and to membranes. studies showed that the parental peptides in their ability to bind and phospholipid membranes, in their structure in the and in the oligomeric state in solution and (22Avrahami D. Oren Z. Shai Y. Biochemistry. 2001; 40: 12591-12603Crossref PubMed Scopus (72) Google Scholar). These studies that and peptides are toward both and charged phospholipid and are in In contrast, both d-V and peptides and both of phospholipid membranes. However, d-V oligomers in and membranes, is In all the parental peptides were also to in all the palmitoylated peptides are to studies showed that the of to amino is in the range and by increasing the M. J. Am. Scopus Google Scholar). However, the structure and the of the in solution are different for the spectroscopy revealed a defined structure in solution only for PA-d-L the of this peptide is more than that in solution This the with the by the the of of the the notion that in to the by the the of PA-d-V is also In contrast, the of the in PA-d-G and PA-d-A that their is not and not the This in agreement with the that these two parental peptides bind only to (22Avrahami D. Oren Z. Shai Y. Biochemistry. 2001; 40: 12591-12603Crossref PubMed Scopus (72) Google Scholar). The of on studies that the peptide oligomerization is a of antimicrobial peptides that act on which are in the membrane of eukaryotic from fungi to (18Oren Z. Lerman J.C. Gudmundsson G.H. Agerberth B. Shai Y. Biochem. J. 1999; 341: 501-513Crossref PubMed Scopus (493) Google Scholar, 19Ghosh J.K. Shaool D. Guillaud P. Ciceron L. Mazier D. Kustanovich I. Shai Y. Mor A. J. Biol. Chem. 1997; 272: 31609-31616Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, I. M. L. Mor A. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). Because of peptides have both antifungal and hemolytic activities. However, oligomerization has been to for antibacterial and antifungal it was not crucial for hemolytic activity of antimicrobial peptides highly hemolytic N. Oren Z. Shai Y. Biochemistry. 2002; 41: PubMed Scopus Google Scholar). all lipopeptides oligomers in However, in two of the and Leu the the in PA-d-V is and in is the of these two oligomers is more difficult than the of the lipopeptides containing Gly and Ala they to the target to for antimicrobial activity pathogens bacteria, and fungi are in to the by which to reach the cytoplasmic phospholipid possible target of the as in the they to the cell as the peptides the cell of the the only is the membrane. the the of the the is its ability to the cell to reach the membrane. This result in biological activity. and this for bacteria a R.E. Infect. Immun. PubMed Google Scholar). the most active and with the spectrum of activity are PA-d-G and However, both are also the hemolytic This we into the charged the cytoplasmic membrane of The positively charged peptides are to the However, the has to membranes, it makes it more difficult for these two peptides to into the cell phospholipid membrane with the more The the of the to other antimicrobial peptides, the target of the lipopeptides is the membrane. This is by the of a the antifungal activity of the lipopeptides against C. and their ability to the membrane of the In we the cell of E. all the lipopeptides high activity which with the of the We have also performed studies by using model phospholipid membranes. These studies the of the to the effect of the lipopeptides on the and the structure of the lipopeptides the phospholipid membranes. The data revealed that lipopeptides and a and effect on the order as well as the of of the This is despite differences in their However, PA-d-V was it the order to a and fluorescence from its This is the peptide is and the fluorescence is This was by spectroscopy which showed that PA-d-V in the PA-d-L a and PA-d-G and PA-d-A that native lipopeptides and they a spectrum of and activities K. A. G. Biochem. Biophys. PubMed Scopus Google Scholar, J. Microbiol. 61: PubMed Scopus Google Scholar, D. M. J. G. 1997; PubMed Scopus Google Scholar, J. Antimicrob. Chemother. 2003; PubMed Scopus (30) Google Scholar, D.M. Antimicrob. Agents Chemother. 2003; 47: PubMed Scopus Google Scholar) L. Infect. Dis. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). However, with the lipopeptides most of them have a unique to a short peptide amino acids) and are A.J. T.J. Antimicrob. 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Avrahami et al. (Mon,) studied this question.