Ultrashort antibacterial and antifungal lipopeptidesArik Makovitzki, Dorit Avrahami, and Yechiel Shai*

Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot 76100, Israel

Edited by Michael Sela, The Weizmann Institute of Science, Rehovot, Israel, and approved August 28, 2006 (received for review July 20, 2006)

Host-defense cationic antimicrobial peptides (�12–50 aa long) playan essential protective role in the innate immune system of allorganisms. Lipopeptides, however, are produced only in bacteriaand fungi during cultivation, and they are composed of specificlipophilic moieties attached to anionic peptides (six to seven aminoacids). Here we report the following. (i) The attachment of analiphatic chain to otherwise inert, cationic D,L tetrapeptides en-dows them with potent activity against various microorganismsincluding antibiotic resistance strains. (ii) Cell specificity is deter-mined by the sequence of the short peptidic chain and the lengthof the aliphatic moiety. (iii) Despite the fact that the peptidic chainsare very short, their mode of action involves permeation anddisintegration of membranes, similar to that of many long antimi-crobial peptides. Besides adding important information on theparameters necessary for host-defense lipopeptides to kill micro-organisms, the simple composition of these lipopeptides and theirdiverse specificities should make them economically available,innate immunity-mimicking antimicrobial and antifungal com-pounds for various applications.

antimicrobial peptides � innate immunity � peptide–membraneinteraction � carpet model � lytic peptides

The increasing resistance of bacteria and fungi to availableantibiotics is a major concern worldwide, leading to enor-

mous efforts to develop new antibiotics with new modes ofaction. Two promising families of drugs that meet these criteriaare host-defense cationic antimicrobial peptides (AMPs) andlipopeptides. AMPs are produced by all species of life andrepresent key components of the innate immune system, pro-viding a fast acting weapon against invading pathogens includingbacteria, fungi, and yeast (1–4). Importantly, many AMPsphysically and rapidly permeate and destroy the cell membrane,causing damage hard to fix, in contrast to conventional antibi-otics, which act on specific targets such as enzymes or DNA.Therefore, microbial resistance may occur with lower probabilitythan that observed with available antibiotics (2). Structurally,AMPs are �12–50 aa long, carry a net positive charge of �2, andare composed of �50% hydrophobic amino acids. Many of themadopt an amphipathic structure only in membrane environ-ments, which is considered to be a prerequisite for their lyticactivity (5, 6). The diversity in AMPs’ lengths, sequences,structures, and spectra of activities is matched by a number ofmodels attempting to describe and explain their modes of action(7–10). There is a consensus, however, that one major step in theactivity of cationic AMPs is their initial binding to the negativelycharged lipopolysaccharide (LPS) of Gram-negative bacteria orto the lipoteichoic acid of Gram-positive bacteria. AMPs thentraverse into the inner phospholipid membrane [highly enrichedin phosphatidylglycine (PG)] and permeate it. In fungi, thepeptides bind to the negatively charged membrane phosphati-dylinositol (PI) and�or to the cell wall, which is composed ofpolybranched (1,3)-�-D-glucan (11–14). Many AMPs lyse mem-branes via the ‘‘carpet’’ mechanism, in which the peptide accu-mulates on the surface of the membrane until a thresholdconcentration has been reached; membrane permeation and�ordisintegration in a detergent-like manner (10, 15) follow. If longenough, AMPs can form toroidal-like pores (16).

Lipopeptides differ from AMPs in that they are produced onlyin bacteria and fungi during cultivation on various carbon sources(11, 12). They are composed of a specific lipophilic moietyattached to an anionic peptide (six to seven amino acids). Someof them destabilize the membrane by unknown mechanisms(17–19). Unfortunately, native lipopeptides belonging to thisgroup are non-cell-selective and therefore toxic to mammaliancells, too. Despite this toxicity, a member of this family, dapto-mycin, which is active only toward Gram-positive bacteria, wasrecently approved by the Food and Drug Administration (FDA)for the treatment of complicated skin and skin structure infec-tions (20).

Recently, we reported that the conjugation of aliphatic acidsto the N terminus of native or designed AMPs endowed themwith selective antimicrobial, antifungal, or both antimicrobialand antifungal activities (14, 21–23). These studies revealed thata threshold of hydrophobicity and a defined structure of peptidicmoiety are required for antimicrobial activity. Intriguingly, herewe report on a family of ultrashort lipopeptides with potentantimicrobial activity against a variety of microorganisms. Theyare composed of only four D,L amino acids conjugated toaliphatic acids with different chain lengths. The sequence of thepeptidic moiety and the length of the aliphatic acid determinetheir spectra of antimicrobial activity. Importantly, despite theirshort peptidic chain length, their mode of action involves thepermeation and disintegration of the membrane, similar to thatof many classical AMPs.

ResultsWe synthesized a series of lipopeptides composed of four L andD amino acids linked to aliphatic acids with different lengths.The sequence of the peptidic moiety was KXXK (X hererepresents L, A, G, K, or E). A peptide comprising the fourN-terminal amino acids of magainin served as a negative control.All of the peptides were amidated at their C terminus, and oneof their amino acids was replaced with the D-enantiomer. Table1 lists the lipopeptides investigated.

Biological Activity of the Lipopeptides. The lipopeptides wereassayed against representative Gram-positive and Gram-negative bacteria, yeast, and fungi, including gentamicin-resistant Acinetobacter baumannii, which are common in humaninfections, as well as against a highly diluted solution [4%(vol�vol)] of human erythrocytes. The antibiotics gentamicinand amphotericin B served as controls for bacteria and fungi,respectively, and gave the reported minimal inhibitory concen-trations (MICs). Importantly, the data shown in Table 1 indicatea repertoire of very short lipopeptides with potent antibacterialand antifungal activities. Interestingly, in contrast to many

Author contributions: Y.S. designed research; A.M. and D.A. performed research; and A.M.and Y.S. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS direct submission.

Abbreviations: AMP, antimicrobial peptide; PG, phosphatidylglycine; PI, phosphatidylino-sitol; MIC, minimal inhibitory concentration; SUV, small unilamellar vesicles; PC, phosphati-dylcholine; PE, phosphatidylethanolamine; hRBC, human RBC.

*To whom correspondence should be addressed. E-mail: yechiel.shai@weizmann.ac.il.

© 2006 by The National Academy of Sciences of the USA

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AMPs, in which increasing the hydrophobicity of the amino acidsincreases biological activity, our lipopeptides preserved potentbiological activity independently of the hydrophobicity of theuncharged amino acids (L, A, or G). However, the type of thehydrophobic amino acid determined the microorganism’s spec-ificity. For example, C16-KLLK was active only toward theGram-positive bacteria and fungi tested, whereas C16-KGGKwas active toward all types of cells tested. Furthermore, speci-ficity was also dictated by the length of the aliphatic acid, as canbe seen with KLLK and KAAK, each of which is attached tohexadecanoic, tetradecanoic, and dodecanoic acid.

The hemolytic activity of the lipopeptides against the highlydiluted solution of human erythrocytes is also shown in Table 1.Unexpectedly, the hexadecanoyl-linked peptide of the less hydro-phobic amino acid glycine was hemolytic, whereas the alanine-containing lipopeptide was practically nonhemolytic. Note, how-ever, that even the hemolytic lipopeptides were practically inactiveup to �20 �M.

Membrane Disruption Induced by the Lipopeptides. The lipopeptideswere investigated for their ability to induce calcein release fromsmall unilamellar vesicles (SUVs) composed of three types ofphospholipids, which mimic the membrane composition of fungi,bacteria, and mammalian cells. Lipopeptides at increasing con-centrations were added to a suspension of SUVs encapsulatedwith calcein, and membrane permeability was followed by mon-itoring the fluorescence recovery. The level of the maximumleakage reached as a function of peptide-to-lipid molar ratio isshown in Fig. 1 A, B, and C for phosphatidylcholine (PC)�phosphatidylethanolamine (PE)�PI�ergosterol (5:4:1:2, wt�wt�wt�wt), PE�PG (7:3, wt�wt), and PC�cholesterol (10:1 wt�wt),respectively. The data reveal a direct correlation between theactivity of the lipopeptides on these model membranes and thecorresponding microorganisms. The only exception is C16-KLLK, which was not active on Escherichia coli but stronglypermeated bacterial model membranes, the reason for which willbe discussed later.

Permeation of Bacterial and Fungal Cell Membranes. The extent ofthe membrane damage caused by the lipopeptides was investi-gated by using two independent methods, described below.Measuring the entrance of SYTOX green into the microorganism’s cells.The cationic dye SYTOX green (molecular mass of 900 Da)cannot enter into an intact cell unless its membrane is disrupted

by external compounds. The fluorescence of SYTOX greenincreases drastically when bound to intracellular nucleic acids.We found a marked enhancement in the fluorescence of E. coli,Staphylococcus aureus, and Candida albicans that were treatedwith the biologically active lipopeptides (e.g., C16-KGGK andC16-KAAK), whereas no activity was observed with these celltypes when treated with lipopeptides that were not bioactive onthese microorganisms (e.g., C12-KAAK and C16-GIGK) (Fig. 2).Overall, there was a direct correlation between the potency ofthe different lipopeptides in inducing the influx of SYTOX greeninto the cells and their corresponding biological activities onthose cells.Measuring the entrance of FITC. FITC (molecular mass of 389.4 Da),a green fluorescent probe, can also traverse only through thedamaged cytoplasmic membrane of a cell. When we treated C.albicans and E. coli ATCC 25922 with the different lipopeptidesin the presence of FITC, we observed that FITC could penetrateonly into cells that were treated with the lipopeptides that wereactive on them. More specifically, there was a marked fluores-cence signal within C. albicans when treated with the three activelipopeptides C16-KAAK, C16-KKKK, and C16-KLLK (Fig. 3Left), but not with the untreated control and the inactiveC12-KAAK and C16-GIGK (Fig. 3 Right). Similar results wereobtained with E. coli ATCC 25922 (data not shown). Theseresults further support a membranolytic mechanism of killing.

Visualization of Cell Damage by Using Transmission EM. C. albicans,S. aureus, and E. coli were treated with the different lipopeptidesfor 15 min at their MICs and visualized by using transmission EM(Fig. 4). The images reveal a direct correlation between thebiological function of the lipopeptides (Table 1) and their abilityto disrupt the cell membrane (Fig. 4). For example, C16-KAAK,C16-KLLK, and C16-KKKK lyse C. albicans, whereas C16-GIGK is not active. Similar patterns were observed with the twobacteria tested (data not shown). Note that C16-KLLK, which ispractically inactive toward E. coli, does not affect most of thebacteria, but some of the population has a ‘‘ghost-like’’ shape.

DiscussionThe repertoire of AMPs has dramatically increased during thepast decade (2). The list includes �800 natural peptides with alength ranging mostly from �12 to 100 aa (an online list can befound at www.bbcm.univ.trieste.it��tossi�antimic.html). Theiramino acid composition, amphipathic structure, net positive

Table 1. Antimicrobial MICs of the ultrashort lipopeptides (�M) and their hemolytic activity

Peptidedesignation

E.c.

P.a.27853

S.a.6538P

A.b.19606*

A.fu.26430

A.fl.9643

C.a.10231

C.n.MYA-422

Hemolysis, %

D21 25922At

20 �MAt

50 �M

C16-KLLK 100 100 100 3.12 50 6.25 12.5 6.25 1.5 9 45C14-KLLK 25 25 12.5 3.12 �100 6.25 12.5 6.25 2.3 13 50C12-KLLK 50 50 25 12.5 �100 25 100 12.5 3 0 0C16-KAAK 6.25 6.25 12.5 6.25 12.5 6.25 100 12.5 3 0 3C14-KAAK 50 50 50 12.5 100 25 100 50 6.25 0 0C12-KAAK �100 100 �100 100 �100 �100 �100 �100 50 0 0C16-KGGK 3 3 6.25 6.25 12.5 12.5 12.5 12.5 1.5 8 75C16-KKKK 6.25 6.25 3 6.25 12.5 25 100 25 1.5 0 6C16-KKEK 12.5 12.5 50 12.5 37 25 100 100 6.25 0 0C16-GIGK �100 �100 �100 �100 �100 �100 �100 �100 100 6 10Gentamicin 3.12 6.25 1.56 6.25 �100 — — — — — —Ampotericin B — — — — — 1.6 3.2 1.5 1.5 — —

The abbreviations used for genus and species names are as follows: E.c., Escherichia coli; P.a., Pseudomonas aeruginosa; S.a., Staphylococcus aureus II; A.b.,Acinetobacter baumannii; A.fu., Aspergillus fumigatus; A.fl., Aspergillus flavus; C.a., Candida albicans; C.n., Cryptococcus neoformans. The numbers that followthe abbreviated genus and species names correspond to American Type Culture Collection (ATCC) numbers. Amino acids in italics are the D-enantiomers.*Gentamicin-resistant.

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charge, and size were found to be important properties that allowthem to bind, insert, and destabilize the membrane of thepathogen via various mechanisms (2, 8, 10). Other properties ofAMPs, such as the ability to assemble in solution and inmembranes, also affect their propensity to selectively kill aparticular family, or several families, of microorganisms.

Our study demonstrates three intriguing findings. First, theattachment of an aliphatic chain to otherwise very short, inertcationic peptides can compensate for the length and hydropho-bicity of the peptidic chain and endow the resulting lipopeptideswith potent antimicrobial activity. Importantly, the MICs ofthese lipopeptides are similar or better than the values reportedfor many native AMPs, particularly while taking into accounttheir relatively low molecular mass (�600 Da). Second, thesequence of a peptidic chain as short as four amino acids alone,or the size of the attached aliphatic chain, is sufficient to controlthe specificity of the lipopeptides toward the different types ofcells. Importantly, most of the lipopeptides are practically nothemolytic against a highly diluted solution of human RBCs(hRBCs) at their MICs or at higher concentrations. Third, incontrast to most known AMPs or natural lipopeptides that areactive either on bacteria alone or fungi alone, several membersof this family of lipopeptides are highly potent against bothbacteria and fungi.

A major question asks how these lipopeptides exert theiractivity. Most AMPs studied so far adopt �-helical and�or�-sheet amphipathic structures that have been shown to beimportant for their function (5, 6, 9). Here, all of the peptidescontain only four amino acids, which makes it difficult for thepeptides to create a defined and stable amphipathic structure.Note also that neither the hydrophobicity of the peptidic chainnor the length of the aliphatic moiety directly correlates with thebiological function of the peptides. For example, the less hydro-phobic lipopeptide C16-KGGK is the most potent compound. Asecond example is C14-KLLK, which is active against Gram-negative bacteria, whereas the more hydrophobic analog C16-KLLK is not active.

Most natural antifungal lipopeptides are composed of six toseven amino acids, are cyclic (12), relatively hydrophobic, andcarry a net negative charge. They act via two major mechanisms:(i) inhibiting the synthesis of cell wall components such as

Fig. 1. Calcein release induced by the lipopeptides. The lipopeptides wereadded to SUVs with different phospholipid compositions (final concentrationof 2.5 �M) and encapsulated with calcein. (A) PC�cholesterol (10:1). (B) PC�PE�PI�ergosterol. (C) PE�PG. Designations are as follows: �, C16-KAAK; {,C14-KAAK; ‚, C12-KAAK; ■ , C16-KLLK; }, C14-KLLK; Œ, C12-KLLK; F, C16-KKKK; E, C16-KGGK; ✳, C16-GIGK; �, C16-KKEK.

Fig. 2. Influx of the vital dye SYTOX green into E. coli (A), S. aureus (B), andC. albicans (C) after the addition of the lipopeptides. The increase in fluores-cence was monitored with excitation set at 485 nm and emission at 520 nm.The fluorescence increase obtained by using 50 �M melittin was taken as100%. All readings were normalized by subtracting parasite scattering andthe basal fluorescence of the dye. Designations are as follows: �, C16-KAAK;{, C14-KAAK; ‚, C12-KAAK; ■ , C16-KLLK; }, C14-KLLK; Œ, C12-KLLK; F,C16-KKKK; E, C16-KGGK; ✳, C16-GIGK; �, C16-KKEK.

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(1,3)-�-D-glucan or chitin (11, 24) and (ii) lysing membranes,although the details in most cases are unknown (e.g., iturins,bacillomycin, and surfactin) (17–19). Daptomycin, for example,a lipopeptide recently approved by the Food and Drug Admin-istration, can depolarize the membrane of only Gram-positivebacteria and only in the presence of calcium ions (20, 25).

The following data suggest that, similar to those of most nativeAMPs, a major target of our lipopeptides is the membrane of thepathogen. Importantly, we found a direct correlation among theMICs of the lipopeptides on the different microorganisms (Table

1), their ability to disrupt and increase the permeability of thecytoplasmic membrane of these microorganisms (Figs. 2 and 3),as well as the corresponding model vesicles (Fig. 1), and theirability to damage the structures of the pathogens, as revealed bythe EM studies (Fig. 4). This is demonstrated in Fig. 5, whichshows in general a direct correlation between the activities of thelipopeptides on the selected microorganisms (Table 1) and thecorresponding model membranes (Fig. 1). A similar relationship(data not shown) was found between the activities shown inTable 1 and the data presented in Fig. 2. C12-KLLK (Fig. 5B)seems to be an exception because it is highly active on C. albicansbut could disrupt the fungal membrane only slightly. A secondexception is C16-KLLK, which is highly potent against allmicroorganisms except E. coli, although it strongly disrupts E.coli-mimicking model membranes (Fig. 1C). A possible expla-nation is that C16-KLLK, being the most hydrophobic lipopep-tide, forms aggregates that hardly traverse the LPS outer layerof E. coli, similar to what has been shown with a 12-merlipopeptide (26).

Note that the ultrashort lipopeptides comprise fatty acidsattached to short cationic peptides and therefore could mimicpartially alkylated ammonium salts, which constitute a broadclass of metabolites commonly occurring in nature. However,whereas the lipopeptides can be modified easily to acquiredifferent spectra of biological activities with no hemolytic ac-

Fig. 3. Lipopeptide-induced influx of FITC into C. albicans. Cells (2 � 107)were incubated with 50 �M lipopeptides at 30°C for 60 min and immobilizedon poly(L-lysine)-coated glass slides. FITC (500 �l of a diluted solution in 10 mMsodium phosphate buffer) was spread on the slides, which were then incu-bated at 30°C for 30 min. After washing, the slides were examined by confocallaser scanning microscopy to assess the affect of the lipopeptides on FITC influxinside the bacterial and yeast cells. Blank, cells with FITC.

Fig. 4. Electron micrographs of negatively stained C. albicans, E. coli, and S.aureus untreated or treated with the lipopeptides. The lipopeptide concen-trations used were their MICs.

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tivity against a highly diluted solution of hRBCs [4% (vol�vol)],alkylated ‘‘ammonium’’ salts are toxic to all types of cells (27).Furthermore, ammonium salts with a short alkyl chain, C12, arepractically inactive (minimal bactericidal activity at the millimo-lar range) (28), compared with our C12 lipopeptides, which arehighly active at the low micromolar range, depending on thesequence of the peptide (e.g., C12-KLLK).

It has been shown in the case of chemical surfactants that thelonger the lipophilic acyl chain, the more effective the micelliza-tion, because of an increase in intermolecular hydrophobicinteractions (29). For example, the micellar concentrations ofquaternary ammonium salts conjugated to alkyl groups are12,700, 1,790, and 210 �M for C12, C14, and C16 alkyl groups,respectively (30). Furthermore, previous studies (38) have alsoshown that the micellar concentration of hexadecanoic acidconjugated to an amino acid is in a micromolar range and shouldsignificantly decrease by increasing the peptidic chain. Thissuggests that the lipopeptides with long fatty acids exist, at leastpartially, as micelles at the concentrations at which they exertbiological function.

In summary, besides adding important information about theparameters required for host-defense-like lipopeptides to killmicroorganisms, we present a strategy that allows the creation ofa repertoire of a promising family of very short cationic peptides:although they cannot perturb the integrity of phospholipidmembranes alone, they are highly potent toward various micro-organisms when conjugated to aliphatic chains with differentlengths. Studies on their plausible mode of action support amembranolytic or detergent-like effect [probably via the carpetmechanism (10)] that, similar to many membrane-active AMPs,should make it difficult for the microorganisms to developresistance. The detergent-like properties of AMPs have been

discussed (31–34). Furthermore, the incorporation of D aminoacids should give these lipopeptides several advantages com-pared with their all L amino acid parental lipopeptides, such ascontrolled enzymatic degradation, which has been recentlyshown for 15-mer diastereomeric antibacterial peptides (35).Finally, their simple composition and diverse specificities shouldmake them economically viable antimicrobial and antifungalcompounds for many applications.

Materials and MethodsMaterials. Rink amide-4-methylbenzhydrylamine hydrochloridesalt (MBHA) resin, 4-methylbenzhydrylamine (BHA) resin, and9-fluorenylmethoxycarbonyl (Fmoc) amino acids were obtainedfrom Calbiochem Novabiochem (Darmstadt, Germany). Lauricacid (dodecanoic acid) was purchased from Sigma (St. Louis,MO). Other reagents used for peptide synthesis included trif-luoroacetic acid (TFA, Sigma), piperidine (Merck, Darmstadt,Germany), N,N-diisopropylethylamine (DIEA; Sigma), N-methylmorpholine (NMM; Fluka, Seelze, Germany), N-hydroxybenzotriazole hydrate (HOBT, Aldrich, St. Louis, MO),and 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HBTU) and dimethylformamide (DMF,peptide synthesis grade; Bio-Lab, St. Paul, MN). PC (from eggyolk), PE (from E. coli), PI (from bovine liver), egg PG, andergosterol were purchased from Sigma. Cholesterol (extra pure)was supplied by Merck. Calcein was purchased from MolecularProbes (Junction City, OR). All other reagents were of analyticalgrade. Buffers were prepared in double-distilled water. Ampho-tericin B and gentamicin were purchased from Sigma. RPMImedium 1640 was purchased from Biological Industries (BeitHaemek, Israel).

Peptide Synthesis, Acylation, and Purification. Peptides were synthe-sized by an Fmoc solid-phase method on Rink amide-4-methylbenzhydrylamine hydrochloride salt (MBHA) resin, by usingan Applied Biosystems (Foster City, CA) 433A automatic peptidesynthesizer. The lipophilic acid was attached to the N terminus ofa resin-bound peptide by standard Fmoc chemistry followed bypeptide cleavage from the resin and purification by RP-HPLC(�98%) (21). Lipopeptide composition was confirmed by electro-spray mass spectroscopy and amino acid analysis.

Antifungal Activity. The antifungal activity of the lipopeptides wasmeasured according to the conditions of National Committee forClinical Laboratory Standards document M27-A. The peptideswere examined in sterile 96-well plates [Nunc (Roskilde, Den-mark) F96 microtiter plates] in a final volume of 200 �l asfollows: 100 �l of a suspension containing fungi at a concentra-tion of 2 � 103 cfu�ml in culture medium (RPMI medium 1640and 0.165 M Mops, pH 7.4, with L-glutamine, without NaHCO3medium) was added to 100 �l of water containing the peptide inserial 2-fold dilutions. The fungi were incubated for 24 h forAspergillus fumigatus (ATCC 26430) and Aspergillus flavus(ATCC 9643) and 48–72 h for C. albicans (ATCC 10231) andCryptococcus neoformans (ATCC MYA-422) using a Binder(Tuttingen, Germany) KB115 incubator. Growth inhibition wasdetermined by measuring the absorbance at 620 nm in an El309microplate autoreader (Biotek Instruments, Luton, U.K.). An-tifungal activities are expressed as the MIC, the concentration atwhich no growth was observed.

Antibacterial Activity. The antibacterial activity of the lipopep-tides was examined in sterile 96-well plates (Nunc F96 microtiterplates) in a final volume of 100 �l as follows: aliquots (50 �l) ofa suspension containing bacteria at a concentration of 106 cfu�mlin culture medium were added to 50 �l of water containing thepeptide (prepared from a stock solution of 1 mg�ml peptide inwater) in serial 2-fold dilutions in LB. Inhibition of growth was

Fig. 5. Relationship between the relative activities of the different lipopep-tides against S. aureus (A) and C. albicans (B) and their ability to induce calceinrelease from the corresponding model membranes. The most active lipopep-tide in each assay was scored as 100. Designations are as follows: �, C16-KAAK;{, C14-KAAK; ‚, C12-KAAK; ■ , C16-KLLK; }, C14-KLLK; Œ, C12-KLLK; F,C16-KKKK; E, C16-KGGK; ✳, C16-GIGK; �, C16-KKEK.

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determined by measuring the absorbance at 492 nm with anEl309 microplate autoreader (Biotek Instruments) after anincubation of 18–20 h at 37°C. Antibacterial activities wereexpressed as the MIC, the concentration at which no growth wasobserved after 18–20 h of incubation. The bacteria used were E.coli ATCC 25922, E. coli ATCC D21, Pseudomonas aeruginosaATCC 27853, S. aureus ATCC 6538P, and gentamicin-resistantA. baumannii.

Hemolysis of hRBCs. The assay was done by using a protocoldescribed previously (36), with a final volume of 100 �l of PBSsolution containing the lipopeptides and hRBCs [final concen-tration of 4% (vol�vol)]. The release of hemoglobin was moni-tored by measuring the absorbance of the supernatant at 540 nm.Controls for 0% hemolysis (blank) and 100% hemolysis con-sisted of hRBCs suspended in PBS and 1% Triton X-100,respectively.

Preparation of Liposomes. SUVs were prepared by dissolving drylipids in chloroform�MeOH (2:1, vol�vol), evaporating thesolvent under a stream of nitrogen, lyophilizing overnight,resuspending in buffer (10 mg�ml), vortex mixing, and sonicat-ing. Lipid films were prepared from PC�PE�PI�ergosterol(5:4:1:2, wt�wt�wt�wt), PE�PG (7:3, wt�wt), and PC�cholesterol(10:1, wt�wt), which mimic the outer leaflets of the plasmamembranes of C. albicans (32), E. coli (33), and hRBCs (34),respectively.

Membrane Permeability Studies. Calcein release from vesicles induced bythe lipopeptides. Calcein (60 mM, self-quenching concentration)was entrapped in self-quenched concentrations in SUVs com-posed of PC�cholesterol (10:1, wt�wt), PE�PG (7:3, wt�wt), orPE�PC�PI�ergosterol (5:4:1:2, wt�wt�wt�wt) as described pre-viously (21). The buffer was 10 mM Hepes and 150 mM NaCl(pH 7.4). The nonencapsulated calcein was removed from theliposome suspension by gel filtration (35). Calcein release due tomembrane permeation induced by the lipopeptides was moni-tored by the fluorescence increase at room temperature (exci-tation wavelength of 485 nm and emission wavelength of 515nm). Complete dye release was obtained by using 0.1% TritonX-100.

SYTOX green uptake assay. C. albicans was grown in potato dextrosebroth, whereas E. coli and S. aureus were grown in LB at 37°C,washed, and suspended in PBS. Cells were suspended (2 � 107

cells per ml) in 10 mM sodium phosphate buffer (pH 7.4) andwere incubated with 1 �M SYTOX green for 15 min withagitation in the dark (37). After the addition of the peptides, theincrease in fluorescence, due to the binding of the dye tointracellular DNA, was monitored (excitation wavelength of 485nm and emission wavelength of 520 nm).FITC uptake assay. The assay was done as previously described (37).E. coli and S. aureus were grown in LB at 37°C, and C. albicanswas grown in potato dextrose broth. Cells were suspended (2 �107 cells per ml) in 10 mM sodium phosphate buffer (pH 7.4) andincubated with the lipopeptides for 60 min at 30°C. The micro-organisms were immobilized on poly(L-lysine)-coated glassslides by incubation at 30°C for 45 min. The slides were washedwith 10 mM sodium phosphate buffer, 6 �g�ml FITC (stocksolution is 10 mg�ml in acetone) in 10 mM sodium phosphatebuffer was spread on them, and they were incubated at 30°C for30 min. The slides were then washed with 10 mM sodiumphosphate buffer. Controls were run in the presence of peptidesolvents. After washes with sodium phosphate buffer (pH 7.4),the slides were examined by confocal microscopy with an exci-tation wavelength of 488 nm.EM. Samples containing C. albicans ATCC 10231, E. coli ATCC25922, and S. aureus ATCC 6538P (3 � 107 cfu�ml) wereincubated with 50 �M lipopeptides dissolved in PBS at theirMICs for 15 min and were centrifuged at 420 � g. Controls weremade in the presence of lipopeptides. The yeasts were fixed byincubation with 1% glutaraldehyde in PBS for 20 min. Thepellets were resuspended; a drop containing the fungi wasdeposited onto a carbon-coated grid and negatively stained with1% (wt�vol) uranyl acetate, and the bacteria were negativelystained with 2% (wt�vol) phosphotungstic acid. The grids wereexamined by using a JEOL (Tokyo, Japan) JEM 100B electronmicroscope.

We thank Vladimir Kiss for help with the fluorescence microscopy, andBatya Zarmi for technical assistance, and Yehuda Marikovsky for EMstudies. This work was supported in part by the Dr. Joseph Cohn MinervaCenter for Biomembrane Research. Y.S. is the incumbent of the HaroldS. and Harriet B. Brady Professorial Chair in Cancer Research.

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