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. a 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
). Binding of dansyl-polymyxin to unmodified P. aeruginosa
) was noted to be cooperative and of high affinity (Hill coefficient, 1.98; S0.5
[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 Kd
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 and ), leukocyte-derived antimicrobial proteins can also work synergistically, as recently demonstrated for rabbit neutrophil defensins BPI and “p15s” (30
). 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
) and in vivo (44
). 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. ). Nevertheless, their functions and secondary structures appear to be similar (Fig. ).
FIG. 7 Helical wheel. Residues 11 to 28 of the mature LL-37 peptide and the corresponding residues of rabbit CAP-18 (Fig. ) are shown. Note the sequestration of polar and apolar residues (all of the polar residues of LL-37 are clustered between (more ...)
Two-dimensional nuclear magnetic resonance (2D-NMR) and CD measurements have defined the solution structure (3
) of CAP-18106–137
, the 32-residue portion of CAP-18 that constitutes its LPS-binding domain (25
). Consistent with our findings for LL-37 (Fig. ), this CAP-18 peptide fragment (GLRKRLRKFRNKIKEKLKKI
GQKIQGLLPKLA) adopts an unordered, random coil conformation in aqueous solution and forms a long, straight, stable amphipathic α helix in 30% trifluoroethanol or in the presence of lipid A (3
Neural network predictions suggest that LL-37 has a long α-helical segment, similar to the residue specific structure of CAP-18 assumed by 2D-NMR in a membrane-like environment (3
). The high α-helical hydrophobic moments (μα
) of LL-37 residues 11 to 22 (μα
= 0.90) and CAP-18 residues 11 to 22 (μα
= 0.67) indicate similar sequence segment amphipathicity. The helical wheel axial projections shown in Fig. 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 CAP18106–125
(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-18106–137
also were active against several additional bacteria but lacked activity against C. albicans
). Recently, a covalent immunoglobulin G–CAP-18106–138
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
) 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.