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A Novel Linear Amphipathic β-Sheet Cationic Antimicrobial Peptide with Enhanced Selectivity for Bacterial Lipids*

Open AccessPublished:July 27, 2001DOI:https://doi.org/10.1074/jbc.M102865200
      All known naturally occurring linear cationic peptides adopt an amphipathic α-helical conformation upon binding to lipids as an initial step in the induction of cell leakage. We designed an 18-residue peptide, (KIGAKI)3-NH2, that has no amphipathic character as an α-helix but can form a highly amphipathic β-sheet. When bound to lipids, (KIGAKI)3-NH2 did indeed form a β-sheet structure as evidenced by Fourier transform infrared and circular dichroism spectroscopy. The antimicrobial activity of this peptide was compared with that of (KIAGKIA)3-NH2, and it was better than that of GMASKAGAIAGKIAKVALKAL-NH2(PGLa) and (KLAGLAK)3-NH2, all of which form amphipathic α-helices when bound to membranes. (KIGAKI)3-NH2 was much less effective at inducing leakage in lipid vesicles composed of mixtures of the acidic lipid, phosphatidylglycerol, and the neutral lipid, phosphatidylcholine, as compared with the other peptides. However, when phosphatidylethanolamine replaced phosphatidylcholine, the lytic potency of PGLa and the α-helical model peptides was reduced, whereas that of (KIGAKI)3-NH2 was improved. Fluorescence experiments using analogs containing a single tryptophan residue showed significant differences between (KIGAKI)3-NH2 and the α-helical peptides in their interactions with lipid vesicles. Because the data suggest enhanced selectivity between bacterial and mammalian lipids, linear amphipathic β-sheet peptides such as (KIGAKI)3-NH2 warrant further investigation as potential antimicrobial agents.
      PGLa
      GMASKAGAIAGKIAKVALKAL-NH2
      KIAGKIA
      (KIAGKIA)3-NH2
      KLAGLAK
      (KLAGLAK)3-NH2
      KIGAKI
      (KIGAKI)3-NH2
      CD
      circular dichroism
      FTIR
      Fourier transform infrared
      LUV
      large unilamellar vesicles
      POPC
      1-palmitoyl-2-oleoylphosphatidylcholine
      POPE
      1-palmitoyl-2-oleoylphosphatidylethanolamine
      POPG
      1-palmitoyl-2-oleoylphosphatidylglycerol
      DPG
      diphosphatidylglycerol
      TFE
      trifluoroethanol
      MIC
      minimum inhibitory concentration
      PIPES
      1,4-piperazinediethanesulfonic acid
      DiPoPE
      1,2-dipalmitoleoylphosphatidylethanolamine
      PE
      phosphatidylethanolamine
      T H
      bilayer-to-hexagonal phase transition temperature
      W-KIAGKIA
      KIAGKIAKWAGKIAKIAGKIA-NH2
      W-KIGAKI
      KIGAKIKWGAKIKIGAKI-NH2
      W-KLAGLAK
      KLAGLAKKWAGLAKKLAGLAK-NH2
      A diverse collection of host defense peptides discovered in a wide range of species shares the common characteristics of a net positive charge and the ability to form amphipathic structures. Many of these peptides appear to exert their protective effect by permeabilizing the membranes of target organisms. The efficacy of these peptides results from their ability to disrupt prokaryotic membranes at concentrations that are not harmful to host membranes (
      • Maloy W.L.
      • Kari U.P.
      ).
      Frog skin is a particularly rich source of defense peptides, including magainins (
      • Zasloff M.
      ) and PGLa1 (
      • Hoffmann W.
      • Richter K.
      • Kreil G.
      ,
      • Soravia E.
      • Martini G.
      • Zasloff M.
      ). These linear cationic peptides containing 21–23 amino acid residues demonstrate broad antimicrobial activity; however, relatively high concentrations are necessary to kill most target organisms. It is possible to enhance antimicrobial activity through simple modifications of the native peptides. For instance, substituting Ala for Glu-19 in magainin 2-amide substantially increases antimicrobial activity (
      • Cuervo J.H.
      • Rodriguez B.
      • Houghten R.A.
      ,
      • Hirsh D.J.
      • Hammer J.
      • Maloy W.L.
      • Blazyk J.
      • Schaefer J.
      ).
      Conformational studies of magainins revealed a significant increase in α-helical content in nonpolar solvents or upon binding to lipid bilayers (
      • Chen H.-C.
      • Brown J.H.
      • Morell J.L.
      • Huang C.M.
      ). Subsequently, a large body of experimental evidence has accumulated to support the notion that these peptides are mostly α-helical when bound to membranes (
      • Williams R.W.
      • Starman R.
      • Taylor K.M.
      • Gable K.
      • Beeler T.
      • Zasloff M.
      • Covell D.
      ,
      • Jackson M.
      • Mantsch H.H.
      • Spencer J.H.
      ,
      • Bechinger B.
      • Zasloff M.
      • Opella S.J.
      ,
      • Ludtke S.J.
      • He K.
      • Wu Y.
      • Huang H.W.
      ,
      • Matsuzaki K.
      • Murase O.
      • Tokuda H.
      • Funakoshi S.
      • Fujii N.
      • Miyajima K.
      ,
      • Wieprecht T.
      • Dathe M.
      • Schümann M.
      • Krause E.
      • Beyermann M.
      • Bienert M.
      ).
      PGLa (see Table I) possesses greater Gram-positive antimicrobial activity than magainin 2 (
      • Maloy W.L.
      • Kari U.P.
      ). PGLa is largely α-helical when bound to lipid bilayers (
      • Bechinger B.
      • Zasloff M.
      • Opella S.J.
      ) and appears to form pores in membranes (
      • Wieprecht T.
      • Apostolov O.
      • Beyermann M.
      • Seelig J.
      ). A more potent derivative of PGLa in which glycines at positions 1 and 8 are replaced by lysines contains three heptamer repeats of sequence KXXXKXX, where X represents a nonpolar residue as shown in Table I. A 21-residue-amidated peptide containing three heptameric repeats of KIAGKIA possesses high antimicrobial and relatively low hemolytic activity (
      • Maloy W.L.
      • Kari U.P.
      ). When KIAGKIA adopts an α-helical conformation, the peptide is highly amphipathic with all six lysines clustered on the helical face (Fig.1). Using a consensus hydrophobicity scale, the hydrophobic moment, a quantitative measure of amphipathicity (
      • Eisenberg D.
      ) for this peptide, is much greater as an α-helix (0.40) as compared with a β-sheet (0.16).
      Table IAmino acid sequences of antimicrobial peptides
      15101520
      Magainin 2-NH2GIGKFLHSAKKPGKAFVGEIMNS-NH2
      PGLaGMASKAGAIAGKIAKVALKAL-NH2
      G1K,G8K-PGLaKLASKAGKIAGKIAKVALKAL-NH2
      KIAGKIAKIAGKIAKIAGKIAKIAGKIA-NH2
      W-KIAGKIAKIAGKIAKWAGKIAKIAGKIA-NH2
      KLAGLAKKLAGLAKKLAGLAKKLAGLAK-NH2
      W-KLAGLAKKLAGLAKKWAGLAKKLAGLAK-NH2
      KIGAKIKIGAKIKIGAKIKIGAKI-NH2
      W-KIGAKIKIGAKIKWGAKIKIGAKI-NH2
      Figure thumbnail gr1
      Figure 1Helical wheel and β-strand diagrams showing the distribution of amino acid side chains (+ = lysine; white= glycine; gray= alanine; and black = isoleucine or leucine). The hydrophobic moment (µH) calculated using the consensus hydrophobicity scale (
      • Wieprecht T.
      • Apostolov O.
      • Beyermann M.
      • Seelig J.
      ) is noted for each conformation. A, KIAGKIA, B, KLAGLAK, andC, KIGAKI.
      To determine whether a highly amphipathic α-helix is a prerequisite for potent antimicrobial activity, we synthesized a peptide KLAGLAK with a similar amino acid content but with a heptamer repeat that separates the six lysines into two groups of three on the helical face, resulting in a large decrease in hydrophobic moment to 0.25 (see TableI and Fig. 1). Like KIAGKIA, KLAGLAK cannot form a highly amphipathic β-sheet structure. Because all known naturally occurring linear antimicrobial peptides have the capability to form at least a reasonably amphipathic α-helical structure (
      • Epand R.M.
      • Vogel H.J.
      ,
      • Sitaram N.
      • Nagaraj R.
      ), we designed a new peptide that can form a highly amphipathic β-sheet rather than an α-helix. This 18-residue peptide contains the hexameric repeat KIGAKI (Table I). The values of hydrophobic moment as an α-helix and a β-sheet are 0 and 0.63, respectively, as shown in Fig. 1. The three model peptides, KIAGKIA, KLAGLAK, and KIGAKI, possess equal charge (+7) and nearly equal mean hydrophobicity values. We compared the antimicrobial and hemolytic activity of these peptides, used CD and FTIR spectroscopy to determine the conformation of the peptides in solution and when bound to lipid bilayers, and measured the ability of the peptides to induce leakage in and bind to LUV of varying lipid composition. Our results show that KIGAKI does indeed adopt a β-sheet conformation when bound to lipids and is comparable in antimicrobial activity to KIAGKIA and KLAGLAK. KIGAKI appears to possess greater selectivity for bacterialversus mammalian lipids as compared with the α-helical peptides tested.

      DISCUSSION

      PGLa and the three model peptides, KIAGKIA, KLAGLAK, and KIGAKI, possess no defined secondary structure in solution but adopt a conformation that appears to maximize amphipathic character upon interacting with lipid bilayers. Like PGLa, KIAGKIA and KLAGLAK can form an amphipathic α-helix at the bilayer surface. KIGAKI was designed to mimic KIAGKIA and KLAGLAK in terms of net charge and hydrophobicity but to form an amphipathic β-sheet instead of an α-helix. In 50% TFE, KIGAKI is mainly helical, but when bound to LUV, the drive to form an amphipathic structure dominates, and the resulting conformation is β-sheet as shown by CD and FTIR spectroscopy (Figs. 2 and 3). A comparison of antimicrobial activity (Table II) shows that KIAGKIA and KIGAKI are significantly more active than PGLa with KLAGLAK only slightly active. Notably, KIGAKI is the least hemolytic of the three model peptides and approximately the same as magainin 2-amide and PGLa.
      Several other linear amphipathic β-sheet peptides have been examined previously. An 18-residue Lys-Leu repeat was reported to have no appreciable antimicrobial or hemolytic activity (
      • Blondelle S.E.
      • Houghten R.A.
      ). Peptides containing 6–12 residues with repeats of either SVKV or Lys-Val were shown to adopt a β-sheet structure in the presence of lipid (
      • Ono S.
      • Lee S.
      • Mihara H.
      • Aoyagi H.
      • Kato T.
      • Yamasaki N.
      ). Although some of these peptides could induce leakage in lipid vesicles, none were antimicrobial below a concentration of 100 µg/ml. The peptide FKVKFKVKVK was able to inhibit the growth of E. coli, S. aureus, and P. aeruginosa at concentrations comparable to the peptides in this study, although the hemolytic activity of this peptide was not tested (
      • Oh J.E.
      • Hong S.Y.
      • Lee K.H.
      ). FKVKFKVKVK was shown by CD to adopt a β-sheet structure in the presence of either 50% TFE or 25 mm sodium dodecyl sulfate.
      Recently, a series of (KL)nK-NH2 peptides containing 9–15 residues (all dansylated at the NH2terminus) was studied by Castano et al. (
      • Castano S.
      • Desbat B.
      • Dufourcq J.
      ). The amide I' vibrational band in the infrared spectra (either dry, at the air/water interface, or inserted into a lipid monolayer) of these peptides was very similar to that of KIGAKI (Fig. 3), centered near 1620 cm−1. All of the peptides induced both leakage in lipid vesicles and hemolysis with activity increasing as a function of length. The antimicrobial activity of these peptides was not examined.
      Shai and co-workers (
      • Oren Z.
      • Hong J.
      • Shai Y.
      ,
      • Hong J.
      • Oren Z.
      • Shai Y.
      ,
      • Oren Z.
      • Hong J.
      • Shai Y.
      ) studied diastereomeric antimicrobial peptides based on 12-mers containing Lys and Leu or derivatives of pardaxin (
      • Oren Z.
      • Hong J.
      • Shai Y.
      ). These peptides were derived from all l-amino acid parent compounds that can form amphipathic α-helices. Based on changes in the amide I' infrared band, the conformation of the diastereomeric peptides was interpreted to be mainly β-sheet; however, the appearance of the amide I' infrared band is significantly different than that of the peptides in this study or those examined by Castano et al. (
      • Castano S.
      • Desbat B.
      • Dufourcq J.
      ). Instead of a relatively narrow band near 1620 cm−1, the diastereomeric peptides gave rise to broad bands centered between 1640 and 1650 cm−1. Clearly, the conformation of these peptides is significantly different than that of KIGAKI when bound to lipid bilayers.
      PGLa, KIAGKIA, KLAGLAK, and particularly KIGAKI were not very effective at inducing leakage in phosphatidylcholine LUV, but all were significantly more active with phosphatidylglycerol LUV. The binding of PGLa to membranes was shown recently to be dominated by electrostatic and not hydrophobic effects (
      • Wieprecht T.
      • Apostolov O.
      • Beyermann M.
      • Seelig J.
      ). Thus, the increased binding probably accounts for the greater leakage rates observed in POPGversus POPC LUV. However, at lower peptide levels, PGLa is more effective than the other peptides at inducing leakage in LUV containing POPG alone or POPC/POPG mixtures (Fig. 5). This contrasts with the antimicrobial activities (Table II) that show PGLa as the least potent peptide. However, in LUV composed of E. colipolar lipids, the activity of PGLa is markedly reduced, whereas that of KIGAKI is enhanced compared with the other peptides (Fig. 4).
      Because PE is the major uncharged polar lipid in E. coliplasma membranes, we examined the effect of replacing POPC by POPE. In LUV containing equimolar amounts of POPG and neutral lipid, only slight differences were observed. As the proportion of POPE in the LUV increased, however, the leakage rates resembled those in E. coli LUV more closely (Fig. 5). In a comparison of ternary mixtures of either POPE/POPG/DPG or POPC/POPG/DPG, the presence of phosphatidylcholine greatly enhances the activity of PGLa and the other α-helical peptides while reducing the activity of KIGAKI (Fig.6).
      One may legitimately question whether the relatively high level of peptide necessary to induce leakage in LUV composed of E. coli polar lipid extract and other lipid mixtures with a high proportion of PE is relevant to the inhibitory effect upon bacterial growth. We can estimate the number of peptide molecules/bacterium in the MIC assay. The assay mixture contains 105 bacteria in a volume of 0.2 ml. The lowest MIC value reported here is 8 µg/ml. With a molecular weight of ∼2000, the amount of peptide in the assay (1.6 µg) translates to >4 × 1014 molecules. Thus, there are ∼4 × 109 peptide molecules/bacterium even at the lowest MIC value. How does this relate to the number of lipid molecules in the plasma membrane? For a large bacterium of size of 2 × 4 µm (i.e. even larger than the bacteria tested here), the surface area is ∼3 × 107nm2. If the average surface area of a lipid molecule is estimated to be ∼0.7 nm2, then the number of lipid molecules on the outer surface of the plasma membrane is ∼4 × 107. Therefore, conservatively, there are ∼100 peptide molecules for each lipid molecule on the exterior of the bacterial plasma membrane. For smaller bacteria or for higher MIC values, the number of peptides/lipid is proportionally higher.
      This does not mean, however, that all of the peptides are bound to the plasma membrane. Many peptide molecules may be bound to lipopolysaccharide, peptidoglycan, teichoic acid, or other components of the cell envelope beyond the plasma membrane, whereas other peptides may remain free in solution. The estimate does point out, however, that a real potential exists for a very large number of peptides to interact with the plasma membrane surface at antimicrobial concentrations. Further experiments will be necessary to determine the binding affinity and location of the peptides on intact bacteria.
      What is the explanation for the observed differences between phosphatidylcholine and PE in the leakage experiments? One obvious possibility is that the peptides bind differently to LUV containing phosphatidylcholine or PE as the neutral lipid. We used tryptophan-containing analogs of the three model peptides to study their interactions with LUV. A blue shift in the maximum of the tryptophan emission band results from the decrease in polarity surrounding the indole side chain as the peptide binds to and penetrates the bilayer surface. Minimal and maximal blue shifts were observed in the presence of POPC and POPG LUV, respectively (Fig. 7). A comparison of LUV with either POPC/POPG or POPE/POPG reveals that W-KIAGKIA and W-KLAGLAK showed a larger blue shift with POPC/POPG. In contrast, W-KIGAKI showed a larger blue shift with POPE/POPG. An increased blue shift could result from more peptide molecules binding to LUV, a more hydrophobic environment of the tryptophan side chains in bound peptides (i.e. deeper insertion into the bilayers) or some combination of the two. Thus, only a qualitative assessment of peptide-lipid interaction can be inferred from the data. In the case of POPG, although all three peptides demonstrated a large shift (15–23 nm), suggesting that a significant fraction of the peptide molecules is bound to LUV, the precise fraction cannot be determined. Moreover, a small blue shift does not necessarily mean that little or no peptide is bound. If peptide binding to the surface of LUV is not associated with a change in polarity surrounding the indole ring, no change in the fluorescence emission spectrum would be expected. The large differences in blue shifts observed between the α-helical and β-sheet peptides suggest that the manner by which these peptides interact with LUV containing a mixture of neutral and acidic lipids is dissimilar. We plan to carry out surface plasmon resonance spectroscopy experiments to measure binding to LUV directly. Once binding is determined independently, a comparison of changes in the fluorescence emission spectrum will be more informative.
      The curvature-modulating property (
      • Epand R.M.
      ) of KLAGLAK differs from KIAGKIA and KIGAKI in that KLAGLAK promotes a more positive membrane curvature (Table III). This property appears to have consequences for the lipid dependence of the lytic activity of magainin 2, which was shown to induce positive curvature to a slightly greater extent than KLAGLAK (
      • Matsuzaki K.
      • Sugishita K.
      • Ishibe N.
      • Ueha M.
      • Nakata S.
      • Miyajima K.
      • Epand R.M.
      ). The curvature effects of these peptides can be rationalized in terms of their structure. In comparing the two α-helical model peptides, the six lysine residues are clustered together in KIAGKIA, whereas in KLAGLAK they are separated by three glycine residues in a helical wheel projection (Fig. 1). Lysines have a special role in the binding of peptides to bilayers because of the amphiphilic nature of their side chain (i.e. four hydrophobic methylene groups between the α-carbon atom and the side chain amino group) (
      • Mishra V.K.
      • Palgunachari M.N.
      • Segrest J.P.
      • Anantharamaiah G.M.
      ). In the case of KIAGKIA, the clustered lysine residues will allow the peptide to insert more deeply in the bilayer and thereby promote less positive curvature (
      • Tytler E.M.
      • Segrest J.P.
      • Epand R.M.
      • Nie S.Q.
      • Epand R.F.
      • Mishra V.K.
      • Venkatachalapathi Y.V.
      • Anantharamaiah G.M.
      ). In KLAGLAK, the two groups of lysine residues are at the interface between the hydrophobic and hydrophilic sides of the amphipathic helix as they are in class A peptides (
      • Segrest J.P.
      • De Loof H.
      • Dohlman J.G.
      • Brouillette C.G.
      • Anantharamaiah G.M.
      ), resulting in an increased positive curvature. This difference in insertion might not be reflected in the fluorescent properties of the tryptophan-substituted analogs, because the Trp residues should seek a position close to the interface regardless of the depth of insertion of the peptide as a whole (
      • White S.H.
      • Wimley W.C.
      • Ladokhin A.S.
      • Hristova K.
      ,
      • Yau W.M.
      • Wimley W.C.
      • Gawrisch K.
      • White S.H.
      ,
      • Wimley W.C.
      • White S.H.
      ). Also, in W-KIAGKIA, the substitution is closer to the hydrophilic face as compared with W-KLAGLAK, where it is near the center of the hydrophobic face (Fig. 1). Because the β-sheet peptide KIGAKI does not shift theT H of DiPoPE, its lytic activity may not be dependent on the curvature properties of the bilayer surface.
      The toroidal pore mechanism proposed by Matsuzaki et al.(
      • Matsuzaki K.
      • Murase O.
      • Fujii N.
      • Miyajima K.
      ) and Huang and co-workers (
      • Ludtke S.J.
      • He K.
      • Heller W.T.
      • Harroun T.A.
      • Yang L.
      • Huang H.W.
      ) for the antimicrobial action of magainin 2 is based upon the induction of sufficient positive curvature to create supramolecular pores. A similar “carpet” model, proposed earlier by Shai and co-workers (
      • Pouny Y.
      • Shai Y.
      ), also relies upon the changes in bilayer curvature to disrupt the membrane. Of the peptides examined in this work, only KLAGLAK resembles magainin 2 in its ability to generate positive curvature. Both KIAGKIA and KIGAKI have only a negligible effect on the T H of DiPoPE. If the interactions with DiPoPE can be generalized to other lipids, the membrane disruption caused by KIAGKIA and KIGAKI may well be different from magainin-like peptides.
      We have demonstrated that KIGAKI, designed to adopt a highly amphipathic β-sheet, possesses a combination of equivalent antimicrobial activity and superior selectivity compared with the α-helical peptides in this study. Since KIGAKI appears to bind preferentially to PE-containing bilayers and induces leakage in LUV-rich in PE to a greater extent than the α-helical peptides while maintaining low hemolytic activity, a more detailed examination of the mechanism of this peptide and an exploration of other peptides with amphipathic β-sheet potential is warranted.

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