Activities of LL-37, a cathelin-associated antimicrobial peptide of human neutrophils

Abstract

Human neutrophils contain two structurally distinct types of antimicrobial peptides, beta-sheet defensins (HNP-1 to HNP-4) and the alpha-helical peptide LL-37. We used radial diffusion assays and an improved National Committee for Clinical Laboratory Standards-type broth microdilution assay to compare the antimicrobial properties of LL-37, HNP-1, and protegrin (PG-1). Although generally less potent than PG-1, LL-37 showed considerable activity (MIC, <10 microgram/ml) against Pseudomonas aeruginosa, Salmonella typhimurium, Escherichia coli, Listeria monocytogenes, Staphylococcus epidermidis, Staphylococcus aureus, and vancomycin-resistant enterococci, even in media that contained 100 mM NaCl. Certain organisms (methicillin-resistant S. aureus, Proteus mirabilis, and Candida albicans) were resistant to LL-37 in media that contained 100 mM NaCl but were susceptible in low-salt media. Burkholderia cepacia was resistant to LL-37, PG-1, and HNP-1 in low- or high-salt media. LL-37 caused outer and inner membrane permeabilization of E. coli ML-35p. Chromogenic Limulus assays revealed that LL-37 bound to E. coli O111:B4 lipopolysaccharide (LPS) with a high affinity and that this binding showed positive cooperativity (Hill coefficient = 2.02). Circular dichroism spectrometry disclosed that LL-37 underwent conformational change in the presence of lipid A, transitioning from a random coil to an alpha-helical structure. The broad-spectrum antimicrobial properties of LL-37, its presence in neutrophils, and its inducibility in keratinocytes all suggest that this peptide and its precursor (hCAP-18) may protect skin and other tissues from bacterial intrusions and LPS-induced toxicity. The potent activity of LL-37 against P. aeruginosa, including mucoid and antibiotic-resistant strains, suggests that it or related molecules might have utility as topical bronchopulmonary microbicides in cystic fibrosis.

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.

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.

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 (▵).

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.

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 [(F_I)/(1.0 − F_I)] 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.

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.

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.