INTRODUCTION

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The ability of neutrophils to ingest and kill bacteria and fungi is an important component of innate immunity. The microbicidal prowess of human neutrophils emanates from oxidative and nonoxidative mechanisms. The former results from activation of an enzyme complex that oxidizes NADPH to produce copious amounts of superoxide (5), whose dismutation yields hydrogen peroxide, which can form stronger oxidants by reacting with myeloperoxidase (15).

The nonoxidative mechanisms of human neutrophils are mediated by antimicrobial peptides and proteins stored within its various cytoplasmic granules. Cathepsin G, azurocidin (also called CAP37), BPI (also called CAP57), and defensins are restricted to the primary (azurophil) granules, which also contain myeloperoxidase, elastase, and proteinase 3 (10, 29, 35). Lactoferrin and hCAP-18 (the precursor of LL-37) are restricted to the neutrophil's secondary (specific) granules (40). Lysozyme, another antimicrobial molecule, occurs in both primary and secondary granules (10, 29). Whereas azurophil granule contents are delivered preferentially to intracellular phagolysosomes, the specific granule contents are largely secreted extracellularly.

The precursor of LL-37 is a 19.3-kDa prepropeptide (13) which, after losing its signal sequence, is called hCAP-18 (23). The cathelin domain of hCAP-18 (22, 36) places it within the cathelicidin family (51). Like other cathelicidins found in porcine, bovine, rabbit (51), and mouse (9), neutrophils, hCAP-18's cathelin domain is highly conserved and precedes the domain that encodes an antimicrobial peptide. Human hCAP-18 is expressed constitutively within neutrophils (40) and the testes (1) and is inducibly expressed by keratinocytes (8).

We compared the antimicrobial properties of three peptides: human LL-37, human defensin HNP-1, and porcine protegrin-1 (PG-1) (21). Protegrins are stored within the granules of porcine neutrophils as cathelin-containing precursors, whose proteolytic processing by elastase releases the microbicidal protegrin domain (34, 52). Similar elastase-mediated processing was also shown for bovine cathelicidins (39). The primary structures of the precursors of LL-37 and PG-1 are shown in Fig. 1. Also shown in Fig. 1 is CAP-18, an extensively studied rabbit cathelicidin that encodes an -helical antimicrobial peptide similar to LL-37. The structures of HNP-1 and other defensins have been described in recent reviews (10, 49).

View larger version (42K): [in this window] [in a new window]   FIG. 1.   Primary sequences of cathelicidins. The sequences of rabbit CAP-18, human hCAP-18 (the precursor of LL-37), and prepro-PG-1 are shown. Identical residues are connected by vertical lines, and similar residues are connected by dots.

	MATERIALS AND METHODS

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Peptides. LL-37 was synthesized at a 0.25-mmol scale with a Perkin-Elmer ABI 431 A synthesizer with prederivatized polyethylene glycol polystyrene serine resin (PerSeptive Biosystems, Framingham, Mass.), FastMoc chemistry, and single coupling for all residues. After its purification by reversed-phase high-pressure liquid chromatography, the peptide appeared to be homogeneous by capillary zone electrophoresis and had a mass of 4,493.16 by electrospray-mass spectrometry (expected mass, 4,493.3). HNP-1 was purified from human neutrophils as described previously (16), and protegrin PG-1 was purchased from SynPep (Dublin, Calif.). Polymyxin B sulfate (lot 25F-0078; 8,156 U/mg) was purchased from Sigma, and its content of polymyxin B base (0.1404 mg/mg of powder) was determined by quantitative analysis of the threonine, leucine, and phenylalanine contents of the powder. We also obtained a similar value (0.1525 of mg polymyxin B base/mg of powder) for the polymyxin B base content when a different polymyxin B preparation (lot 83H1112; 7,800 U/mg) was analyzed in this manner.

Antimicrobial testing. (i) Radial diffusion assays. The two-stage radial diffusion assay used in these studies has been fully described elsewhere (43). Briefly, the purified peptides were serially diluted in acidified water (0.01% acetic acid) that contained 0.1% human serum albumin (Sigma A-8763). The bacteria listed in Tables 1 and 2 were grown to the mid-logarithmic phase and washed. Approximately 2 × 10⁵ CFU/ml was incorporated into a thin (1.23-mm) agarose underlay gel that contained 1% (wt/vol) agarose (Sigma A-6013), 10 mM sodium phosphate buffer (pH 7.4), and 0.3 mg of Trypticase soy broth powder per ml with or without 100 mM NaCl. A regularly spaced, five-by-five array of wells was made in the underlay gel. The wells, 3.2 mm in diameter, had a 10-µl capacity. Six serially diluted samples of each peptide ranging in concentration from 0.79 to 250 µg/ml were prepared, and 5-µl aliquots were added to the wells. After 3 h, a 10-ml overlay gel composed of 6% Trypticase soy broth powder, 1% agarose, and 10 mM sodium phosphate buffer (pH 7.4) was poured onto the plates, and the plates were incubated overnight to allow the surviving organisms to form microcolonies.

(ii) Broth microdilution assays. Broth microdilution assays were performed according to the guidelines of the National Committee for Clinical Laboratory Standards (NCCLS), except that our 10× stock solutions of the peptides and control antibiotics were prepared in sterile 0.01% acetic acid with 0.2% bovine serum albumin instead of in Mueller-Hinton broth (MHB), because standard MHB formed a precipitate with PG-1 and LL-37 (see below). Bacterial inocula were prepared according to NCCLS guidelines (33a) and were adjusted appropriately by spectrophotometry at 620 nm to provide 2 × 10⁵ CFU ml¹ in the microplate wells. We tested two concentration series for each organism, such that the final antibiotic concentrations used for series 2 were 50% higher than those used for series 1.

(iii) Colony counting assays. Mid-logarithmic-phase E. coli ML-35p, grown as described above for the radial diffusion assays, was washed twice in 10 mM sodium phosphate buffer (pH 7.4) and was used at a final concentration of 2 × 10⁶ CFU/ml. These bacteria were exposed to a range of LL-37 concentrations in 100 µl of one of the following three media: (i) 10 mM sodium phosphate buffer (pH 7.4) containing 3 mg of Trypticase soy broth powder ml¹ with 100 mM sodium chloride, (ii) standard MHB, or (iii) refined MHB. After a 2-h incubation, 10-µl aliquots were removed, diluted 1:100 with the appropriate assay medium, and transferred to Trypticase soy agar plates with a SprialSpreader (SpiralTech, Rockville, Md.). Colonies were counted after incubation for 24 h at 37°C.

Membrane permeabilization. To examine the ability of antimicrobial peptides to permeabilize the inner and outer membranes of gram-negative bacteria, we simplified a previously described procedure that uses E. coli ML-35p (27, 28). As originally described, the assay was performed in a single cuvette that contained two substrates, PADAC (a cephalosporin) and o-nitrophenyl--D-galactose (ONPG; a -galactosidase substrate). The cuvette was monitored at multiple wavelengths, and the respective -lactamase and -galactosidase hydrolysis reactions were deconvoluted algebraically. In the present version, the assays were performed in 96-well microtiter plates (Corning, Corning, N.Y.) and were followed at a single wavelength (420 nm) that was sensitive to the hydrolysis both of ONPG and of PADAC. The reactions were monitored with a SpectraMax 250 Microplate Spectrophotometer (Molecular Devices, Sunnyvale, Calif.) with SOFTmaxPRO software supplied by the manufacturer. Both substrates were purchased from Calbiochem (La Jolla, Calif.).

LPS binding. A quantitative chromogenic Limulus amoebocyte assay was performed with the reagents contained in a QCL-1000 kit (BioWhittaker, Walkersville, Md.). Incubations were performed in flat-bottom, nonpyrogenic 96-well tissue culture plates (catalog no. 3596; Costar, Cambridge, Mass.). Stock solutions of LL-37 and polymyxin B (7,600 U/mg; Sigma) were prepared in endotoxin-free acidified water (0.01% acetic acid) at 40 µg/ml and were serially diluted in this solution. In step 1, 25 µl of the peptide solution and 25 µl of an E. coli O111:B4 lipopolysaccharide (LPS), containing 1 endotoxin U/ml, were mixed in a well, and the plate was incubated for 30 min at 37°C to permit peptide and LPS binding to occur. In step 2, 50 µl of the amoebocyte lysate reagent was added. In step 3, performed exactly 10 min later, 100 µl of chromogenic substrate (acetyl-Ile-Glu-Ala-Arg-p-nitroanilide) was introduced. Thereafter, the incubation was continued at 37°C for 20 min, while the liberation of p-nitroaniline was monitored every 60 s at 405 nm with a SpectraMax 250 Kinetic Microplate Spectrophotometer (Molecular Devices). The change in optical density (OD) between 10 and 16 min was calculated for the control sample (which contained peptide but no LPS), and this value was subtracted from the OD between 10 and 16 min for the experimental samples, which contained peptide plus LPS. The difference provided an index of LPS-mediated Limulus procoagulant activation during step 1 of the incubation. Since the assays were done kinetically, we could also monitor spontaneous procoagulant activation to ensure that LL-37 and melittin did not inhibit the procoagulant enzymes directly (neither did).

CD spectroscopy. Circular dichroism (CD) measurements were performed with an AVIV 62DS spectropolarimeter (AVIV Associates, Lakewood, N.J.) that was routinely calibrated with (+)-10-camphorsulfonic acid (1 mg/ml) in a 1-mm-path-length cell (19). The sample compartment was fitted with a customized cell holder to position the sample cuvette near the photomultiplier tube, thereby minimizing light-scattering artifacts induced by peptide-liposome dispersions (48). Peptide-solvent solutions were measured in 0.1- to 0.5-mm-light-path demountable cells that were scanned from 260 to 190 nm at a rate of 10 nm/min, with 0.2-nm intervals. The results were expressed as the mean residue ellipticity (MRE), []_MRE (degree centimeter² decimoles¹), and the percentage of -helical conformation was estimated by the following equation (4): % -helix = []_MRE222/{39,500 [1  (2.57/n)]} deg cm² dmol¹.

Preparation of liposomes. Palmitoyl-oleoyl-phosphatidylglycerol (POPG) was purchased from Avanti Polar Lipids (Alabaster, Ala.). The large unilamellar vesicles used in liposome lysis and CD measurements were formed by freezing-thawing followed by extrusion through an extrusion device (Lipex, Ottawa, Ontario, Canada), as described previously (17, 48). Briefly, liposomes were prepared by hydrating 2.5 mg of lipid with 1 ml of 10 mM phosphate buffer (pH 7.5), followed by seven freeze-thaw cycles and five extrusions through 100-nm-pore-size polycarbonate membranes. Diphosphoryl lipid A, prepared from E. coli F-583 (an Rd mutant), was purchased from Sigma, and dispersions were prepared as described by Chen et al. (3).

Secondary structural predictions. Neural network-based secondary structural predictions for the helical peptides were carried out on-line (http://www.embl-heidelberg.de) with the PHD Predict Protein program (37). Hydrophobic moments were calculated with the MOMENT computer program (6). An 11-residue amino acid sequence window, corresponding to the length of an average helical segment in a protein, was used to identify amphipathic helical motifs.

	RESULTS

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Antimicrobial activity. We tested LL-37, HNP-1, and PG-1 against a group of gram-positive bacteria, including Listeria monocytogenes, Staphylococcus epidermidis, S. aureus (four strains), methicillin-resistant S. aureus (MRSA; four strains), B. subtilis, and vancomycin-resistant strains of Enterococcus faecalis and Enterococcus faecium (Table 1). HNP-1 was broadly effective only when assayed in underlay gels that contained 10 mM phosphate buffer. When the underlay gels were supplemented with 100 mM NaCl, HNP-1 was active only against B. subtilis and vancomycin-resistant E. faecium. LL-37 was generally at least as potent as HNP-1 in low-salt underlays, and except against MRSA, it retained its activity in underlay gels containing 100 mM NaCl. PG-1 worked well against the entire panel under both the low- and high-salt conditions. Table 1 also indicates the sensitivities of these organisms to vancomycin, a conventional glycopeptide antibiotic that was used as a control. These data also show that only PG-1 killed Candida albicans under these experimental conditions.

Effect of divalent cations. To see if divalent cations affected the antibacterial effects of LL-37 and PG-1, we used E. coli ML-35p and L. monocytogenes EGD as targets. For E. coli, the addition 1 mM Ca²⁺ to the underlay gels increased the MIC of LL-37 substantially and the MIC of PG-1 slightly (Fig. 2). In contrast, 1 mM Ca²⁺ did not decrease either peptide's activity against L. monocytogenes. The addition of Mg²⁺ (1 mM) was not inhibitory for either peptide with either organism, and 1 mM Ca²⁺ plus 1 mM Mg²⁺ was no more inhibitory than Ca²⁺ alone for E. coli (data not shown).

View larger version (21K): [in this window] [in a new window]   FIG. 2.   Effect of calcium on antimicrobial activity. Solid symbols show the zone diameters from radial diffusion assays that were performed in underlay gels containing 10 mM phosphate buffer (pH 7.4), 100 mM NaCl, 0.3 mg of Trypticase soy broth powder per ml, 1% agarose, and either E. coli ML-35p (a and c) or L. monocytogenes (b and d). The open symbols indicate the results obtained when these underlay gels were supplemented with 1 mM calcium chloride. The intersections of the continuous or interrupted regression lines with the x axis define the respective MICs. The concentrations on the x axis refer to the concentration of peptide in the 5-µl volume that was initially added to the well. Note that the x axis (concentration) is drawn with a logarithmic scale.

Effect of sample volume. To minimize the amount of peptide consumed, all of the radial diffusion assays described in this report were performed with 5-µl samples that filled the sample wells to only half their height. Consequently, the introduced peptides were free to diffuse upward, as well as outward (radially), once they entered the underlay gels. Such upward diffusion would, in effect, allow up to a twofold additional dilution of the peptide concentration initially placed into the well. We therefore examined the effect of sample volume experimentally and found that the use of 10-µl sample volumes yielded MICs that were approximately 30 to 40% lower than those obtained with 5-µl samples (data not shown).

Broth microdilution assays. We also tested the activities of LL-37 and PG-1 against four organisms in broth microdilution assays that were set up according to NCCLS guidelines. In these assays, we used two types of MHB. One of these (standard MHB) was prepared in the customary manner, and the other (refined MHB) was subjected to anion-exchange chromatography in order to deplete it of (poly)anionic inhibitors, as discussed later. Table 3 shows the results obtained by these broth microdilution assays and compares them with the results obtained by radial diffusion assays. Note that the MIC determinations performed with standard MHB yielded values that were 3- to >20-fold higher, depending on the organism, than those obtained with refined MHB. When LL-37 was tested in refined MHB, two of the MICs were higher and two were lower than the values returned by radial diffusion assays. When LL-37 was tested in standard MHB, all of the values were at least threefold higher than those obtained in our radial diffusion assays.

View larger version (20K): [in this window] [in a new window]   FIG. 3.   Effects of various media on the activity of LL-37. Mid-logarithmic-phase E. coli ML-35p, approximately 2 × 106 CFU/ml, was incubated for 2 h with various concentrations of LL-37 in three different media: 10 mM sodium phosphate buffer plus 100 mM NaCl and dilute Trypticase soy broth (), standard (std) MHB (), and refined (ref) MHB ().

Membrane permeabilization. We previously described the use of E. coli ML-35p to examine the ability of antimicrobial peptides to permeabilize the membranes of a gram-negative organism by real-time spectrophotometry (27, 28). Because this bacterium constitutively expresses cytoplasmic -galactosidase but lacks the membrane permease that transports -galactosides across its inner (plasma) membrane, it cannot hydrolyze ONPG until its inner membrane undergoes permeabilization. E. coli ML-35p also expresses a periplasmic -lactamase, so that monitoring of the hydrolysis of a bulky and poorly penetrating cephalosporin (PADAC) also allows outer membrane permeabilization to be followed spectrophotometrically.

View larger version (34K): [in this window] [in a new window]   FIG. 4.   Membrane permeabilization. We tested the ability of LL-37 (5 µg/ml) to permeabilize the outer (a and c) and inner (b and d) membranes of E. coli ML-35p. Experiments were carried out either in 10 mM sodium phosphate buffer (a and b) or in 10 mM phosphate buffer plus 100 mM NaCl (panels c and d). Melittin (20 µg/ml) was used as a positive (lytic) standard, and acidified water was used as the negative control. OD420, optical density at 420 nm.

LPS binding. We used a sensitive and precise Limulus chromogenic assay to examine the ability of LL-37 to bind LPS from E. coli O111:B4 and to compare its binding to that of polymyxin B. As shown in Fig. 5a, approximately 0.36 µM LL-37 or 30 nM polymyxin B bound half of the E. coli LPS under the conditions used in our assay. Thus, on a molar basis, LL-37 was approximately 10% as potent as polymyxin B in binding LPS. Figure 5a also shows that the LPS-binding curves for polymyxin B and LL-37 differ in shape. Whereas the LL-37-binding isotherm is distinctly sigmoidal, the polymyxin B curve is not. Because sigmoidal curves suggest cooperativity, we also graphed the data as a Hill plot (Fig. 5b). By Hill plot analysis, the LL-37 binding data were linear (r = 0.992) and had a slope of 2.02, suggesting positive cooperativity between two ligand molecules and the LPS. In contrast, the polymyxin B line had a slope of 0.97, suggesting that polymyxin-LPS-binding events occurred independently rather than cooperatively.

View larger version (20K): [in this window] [in a new window]   FIG. 5.   LPS binding. (a) Binding of polymyxin B (PmxB) and LL-37 to LPS from E. coli O1111:B4, as determined by chromogenic Limulus assays. (b) The same data from panel a but as a Hill plot. The term log10 [(FI)/(1.0  FI)] on the ordinate is described in the Materials and Methods section. The Hill coefficient (n) shown in the figure corresponds to the slope of the continuous or interrupted regression lines.

Conformational studies. In CD spectroscopy measurements, -helical secondary structures are revealed by double dichroic minima at 208 and 222 nm (2, 50). Our CD studies revealed a largely random coil conformation with only 9.6 to 9.9%  helix in phosphate buffer with or without 100 mM NaCl unless structure-promoting exogenous elements were also present. These could be relatively simple, such as either 30% trifluoroethanol (48% -helical conformation) or 20 mM sodium dodecyl sulfate micelles (87.1% -helical conformation). They could also be more complex, such as POPG liposomes (94.6% -helical conformation), membranes whose anionicity simulates that present in bacterial membranes. As was reported for the antimicrobial peptide derived from rabbit CAP-18 (3), LL-37 also showed enhanced helical conformation (40.2%  helix) in the presence of diphosphoryl lipid A purified from an Rd E. coli mutant, strain F-583. Several examples of our CD findings can be seen in Fig. 6, including measurements taken in phosphate buffer (curve a), in the presence of diphosphoryl lipid A (curve b), and with POPG liposomes (curve c).

View larger version (20K): [in this window] [in a new window]   FIG. 6.   CD spectrometry. The spectra were measured in phosphate buffer (curve a), in the presence of diphosphoryl lipid A (curve b), and with POPG liposomes (curve c) and are described in the text.

	DISCUSSION

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Although our principal goals in these studies were to examine the properties of LL-37, we also performed NCCLS-type broth microdilution assays to substantiate the results of our radial diffusion assays. We found that the use of conventional MHB in a standard NCCLS broth microdilution assay vastly underestimates the activity of LL-37 and that this problem could be remedied by passing the MHB through an anion-exchange column before using it.

Since this point has important practical consequences for the testing of other polycationic antimicrobial molecules, this use of anion-exchange chromatography merits discussion and explanation. The major component of MHB is an acid hydrolysate of casein (17.5 g/liter), and its only other constituents are beef extract (3 g/liter) and starch (1.5 g/liter). Although bovine casein is a cheap and dependable nutrient source, it has an atypical amino acid composition. Glutamic acid and glutamine constitute 18.8% (39 of 208) of its residues, and it also contains 8 aspartic acid plus asparagine residues and 5 phosphorylated serines. Consequently, 25.0% (52 of 208) of the amino acids released by its total hydrolysis would be dicarboxylic or phosphorocarboxylic acids. Moreover, since the remarkable ESLSSSEE sequence (residues 14 to 20) of casein contains seven clustered, negatively charged glutamate and phosphoserine residues, incomplete hydrolysis could leave residual polyanionic peptides. Because most antimicrobial peptides are polycations, it should not be surprising that many are incompatible with conventional MHB, which would complex or even precipitate them. Subjecting MHB to simple anion-exchange chromatography to remove anionic inhibitors, as illustrated by these studies, enhanced the utility of MHB for broth microdilution studies for LL-37, a cationic antimicrobial molecule. Although ion-exchange cartridge columns were convenient for our small-scale experiments, one would probably use a batch process for larger-scale production of refined MHB.

Whereas multiple antimicrobial peptide precursors of the cathelicidin family have been described in cattle and pigs (51), hCAP-18 is believed to be the only human cathelicidin (1). Although it was discovered relatively recently, its concentration in the human neutrophil (0.63 mg of hCAP-18/10⁹ cells) makes hCAP-18 as abundant as lactoferrin, on a molar basis (41). Normal plasma contains 1.18 µg of hCAP-18/ml, which circulates in high-molecular-weight complexes (41). This concentration of circulating hCAP-18 might suffice to detoxify low concentrations of LPS that enter the concentration (Fig. 5a and b).

Our binding studies with LL-37 showed a Hill coefficient of 2.02, indicative of positive cooperativity between two molecules of the ligand (LL-37) and the receptor molecule (LPS). Polymyxin B showed a more typical hyperbolic binding curve, and the Hill coefficient of 1.08 suggested that its binding to LPS was simple and noncooperative. Although polymyxin B can bind a variety of anionic phospholipids, including phosphatidyl glycerol and cardiolipin (45), its interactions with the glucosamine phosphates and 2-keto-3-deoxyoctulosonic acid carboxylates found in bacterial LPS (33) have received considerably more scrutiny.

Previous studies of LPS binding have typically used radiolabelled or dansylated peptides or similarly modified LPS (33, 38). Binding of dansyl-polymyxin to unmodified P. aeruginosa LPS (33) was noted to be cooperative and of high affinity (Hill coefficient, 1.98; S_0.5 [an estimate of affinity] = 0.38 µM). The binding of polymyxin to dansylated LPS from Rc and Re mutants of S. typhimurium LT2 had a K_d of 0.3 to 0.5 µM and occurred without evident cooperativity (38). The use of a highly precise and sensitive version of the chromogenic Limulus assay allowed us to examine binding without structurally modifying either the peptides or the LPS. Further studies to define the precise portions of LPS that bind LL-37 could be of considerable interest.

Since the residues of LL-37 constitute approximately one-quarter of the hCAP-18 propeptide, conversion of circulating hCAP to LL-37 would liberate about 0.25 µg of LL-37 per ml. Although this concentration appears to be too low to exert microbicidal actions alone (Tables 1 and 2), leukocyte-derived antimicrobial proteins can also work synergistically, as recently demonstrated for rabbit neutrophil defensins BPI and "p15s" (30, 31). The possibility that LL-37 acts synergistically with other host-defense molecules also remains to be explored.

Rabbit CAP-18 (26), the homolog of human hCAP-18 found in rabbit granulocytes, has received extensive study because its C-terminal domain can bind and neutralize LPS and can prevent potentially deleterious consequences of LPS release in vitro (18, 25) and in vivo (44, 47). Whereas the signal sequence and cathelin domains of CAP-18 show marked primary sequence homology to hCAP-18 and other cathelicidins, primary structural homology between its C-terminal region and the corresponding domain of hCAP-18 is considerably less marked (Fig. 1). Nevertheless, their functions and secondary structures appear to be similar (Fig. 7).

View larger version (36K): [in this window] [in a new window]   FIG. 7.   Helical wheel. Residues 11 to 28 of the mature LL-37 peptide and the corresponding residues of rabbit CAP-18 (Fig. 1) are shown. Note the sequestration of polar and apolar residues (all of the polar residues of LL-37 are clustered between 11:30 and 5:30 when the figure is visualized as a clock face). CAP-18 shows a very similar pattern.

Two-dimensional nuclear magnetic resonance (2D-NMR) and CD measurements have defined the solution structure (3) of CAP-18_106-137, the 32-residue portion of CAP-18 that constitutes its LPS-binding domain (25). Consistent with our findings for LL-37 (Fig. 6), this CAP-18 peptide fragment (GLRKRLRKFRNKIKEKLKKI GQKIQGLLPKLA) adopts an unordered, random coil conformation in aqueous solution and forms a long, straight, stable amphipathic  helix in 30% trifluoroethanol or in the presence of lipid A (3).

Neural network predictions suggest that LL-37 has a long -helical segment, similar to the residue specific structure of CAP-18 assumed by 2D-NMR in a membrane-like environment (3). The high -helical hydrophobic moments (µ) of LL-37 residues 11 to 22 (µ = 0.90) and CAP-18 residues 11 to 22 (µ = 0.67) indicate similar sequence segment amphipathicity. The helical wheel axial projections shown in Fig. 7 indicate that the antimicrobial domains of CAP-18 and LL-37 demonstrate strikingly similar segregation of polar and strong nonpolar residues.

On the basis of structural predictions, a 20-residue peptide corresponding to CAP18_106-125 (GLRKRLRKFRNKIKEKKLKKI) was synthesized (46) and tested for its antimicrobial activity against E. coli, S. typhimurium, P. aeruginosa, Bacillus megaterium, and S. aureus. Although nonhemolytic for human erythrocytes, even at 50 mM, 1 mM peptide concentrations were reported to permeabilize the inner membrane of E. coli, and the peptides at concentrations of 0.4 to 4.0 µM killed all of the test organisms. The 32- and 37-residue peptides corresponding to CAP-18_106-137 and CAP-18_106-142 also were active against several additional bacteria but lacked activity against C. albicans (24). Recently, a covalent immunoglobulin G-CAP-18_106-138 conjugate was reported to bind to LPS, protect sensitized mice from LPS-induced mortality (7), and kill gram-negative bacteria (7).

The noteworthy resistance of B. cepacia to each of the antimicrobial peptides examined in our studies is consistent with observations showing the primacy of oxidative mechanisms in leukocyte-mediated host defenses against this opportunistic pathogen in both humans (42) and mice (32). Whereas the present studies with S. typhimurium 14028S (wild type) and 7953S (its phoP derivative) confirmed a previously reported correlation between phoP and resistance to human defensins (12), the wild-type and phoP strains showed similar susceptibilities to LL-37 and PG-1. Thus, the bacterial responses regulated by phoP (12, 14, 20) do not provide S. typhimurium with global immunity to leukocyte-derived antimicrobial peptides. The mechanisms responsible for the resistance of B. cepacia to LL-37 and PG-1 merit further investigation.

LL-37 and defensins are located in neutrophil granule populations that differ in content and in behavior. The placement of defensins in azurophil granules enables them to enter a locale, the phagosome, wherein the potentially inhibitory effects of NaCl could be modified by ion-transporting membrane pumps or overcome by high local concentrations of defensins. In contrast, LL-37 is stored as a propeptide (hCAP-18) and is placed in a secretory organelle, the secondary granule. As a result, extracellular bacteria (or free LPS) may encounter LL-37 primarily in its cathelin-containing precursor form (hCAP-18), whereas ingested bacteria may encounter LL-37, the microbicidal domain of hCAP-18, after proteolytic processing by enzymes of the neutrophil (or perhaps the microbial target). While much remains to be learned about both LL-37 and hCAP-18, the activity of LL-37 against P. aeruginosa, including mucoid and antibiotic-resistant strains, suggests that it could provide a suitable template for designing topical bronchopulmonary microbicides for use in conditions such as cystic fibrosis.