Structural and functional analysis of horse cathelicidin peptides

Abstract

Cathelicidin-derived antimicrobial peptides are a component of the peptide-based host defense of neutrophils and epithelia, with a widespread distribution in mammals. We recently reported the cDNA sequences of three putative horse myeloid cathelicidins, named eCATH-1, -2, and -3. A Western analysis was performed to investigate their presence in neutrophils and processing to mature peptides. eCATH-2 and eCATH-3, but not eCATH-1, were found to be present in uncleaved forms in horse neutrophils. The corresponding mature peptides were detected in inflammatory sites, suggesting that processing of the propeptides takes place upon neutrophil activation. A functional characterization was then performed with synthetic eCATH peptides. Circular dichroism measurements indicated an amphipathic alpha-helical conformation of these peptides in an anisotropic environment, and in vitro assays revealed a potent activity and a broad spectrum of antimicrobial activity for eCATH-1 and a somewhat more restricted spectrum of activity for eCATH-2. Conversely, a strong dependence on salt concentration was observed when the activity of eCATH-3 was tested. This peptide efficiently killed bacteria and some fungal species, i.e., Cryptococcus neoformans and Rhodotorula rubra, in low-ionic-strength media, but the activity was inhibited in the presence of physiological salt medium. This behavior could be modified by modulating the amphipathicity of the molecule. In fact, the synthetic analogue LLK-eCATH-3, with a slightly modified sequence that increases the hydrophobic moment of the peptide, displayed a potent activity in physiological salt medium against the strains resistant to eCATH-3 under these conditions.

FIG. 1

Amino acid sequences of the equine cathelicidin peptides eCATH-1, -2, and -3, as deduced from cDNA (30). The analogue LLK-eCATH-3 was synthesized to improve the amphipathicity of the parent eCATH-3 peptide (see Fig. 3). Dashes denote identical residues in the LLK-eCATH-3 and eCATH-3 sequences.

FIG. 2

(A) Western analysis of bronchial secretions using antibodies to eCATH-2 (lanes a to c) and eCATH-3 (lanes d to f). Lane a, synthetic eCATH-2 peptide; lane d, synthetic eCATH-3 peptide; lanes b and e, tracheobronchial secretions from a horse affected by COPD; lanes c and f, tracheobronchial secretions from a horse affected by acute bronchiolitis. (B) Western analysis of total granule populations from horse neutrophils (lanes a to c) and whole horse neutrophils (lanes d to f) using antibodies to eCATH-2. Lane a, TCA-precipitated granules; lane b, neutrophil granules after incubation with TX-100 for 10 min; lane c, neutrophil granules after incubation with TX-100 for 10 min in the presence of the elastase inhibitor AAPV-CMK; lane d, synthetic eCATH-2; lane e, TCA-precipitated neutrophils; lane f, neutrophils after incubation with TX-100 for 120 min.

FIG. 3

Helical wheel representation of the eCATH peptides. Charged residues are in boldface, hydrophobic residues are boxed, and small, hydrophilic, neutral residues are in italics. Arrows in the eCATH-3 wheel indicate substitutions introduced to obtain the LLK-eCATH-3 analogue with improved amphipathicity.

FIG. 4

Helical content of the eCATH peptides in the absence or presence of TFE. The α-helical content was estimated from mean residue ellipticity at 222 nm. ●, eCATH-1; ▵, eCATH-2; ○, eCATH-3; □, LLK-eCATH-3.

FIG. 5

Kinetics of the inner membrane permeabilization of E. coli ML-35 by the eCATH peptides. Permeabilization was determined by recording spectrophotometrically the hydrolysis of o-nitrophenyl-β-d-galactopyranoside, a normally impermeant substrate of the cytoplasmic β-galactosidase. Experiments were carried out in 10 mM sodium phosphate buffer (pH 7.4) with 100 mM NaCl. Traces a, e, and i, untreated bacteria; bacteria were treated with 0.6, 3, and 15 μg of eCATH 1 per ml (traces b, c, and d, respectively); 0.7, 3.5, and 7 μg of eCATH-2 per ml (traces f, g, and h, respectively); 90 μg of eCATH-3 per ml (trace j); and 2, 4.5, and 9 μg of LLK-eCATH-3 per ml (traces k, l, and m, respectively). The time of addition of peptides is indicated by arrows. The results are representative of two to three independent determinations.

FIG. 6

Antibacterial activity of eCATH-3 in low-salt medium. E. coli ATCC 25922 (open bars), S. enterica serovar Typhimurium ATCC 14028 (dark bars), and S. aureus ATCC 25923 (dotted bars). Bacterial cells (2 × 10⁵ to 5 × 10⁵ CFU/ml) were incubated with the indicated amounts of eCATH-3 in 10 mM sodium phosphate buffer (pH 7.4) for 1 h at 37°C. The cells were then serially diluted in sterile saline, plated in Mueller-Hinton agar, and incubated for 16 to 18 h to allow colony counts to be performed. The results are the means of three independent determinations. Error bars indicate standard deviations.

FIG. 7

Kinetics of inactivation of C. neoformans by eCATH peptides. Fungi were incubated at 30°C for the indicated times in the absence or presence of 31 μg of eCATH-1 per ml and 46 μg of eCATH-3 or LLK-eCATH-3 per ml (10 μM peptides), serially diluted in buffered saline, and plated in solid Sabouraud medium to allow colony counts to be performed. The experiment on the left side was performed in Sabouraud medium in the absence (○) or presence of eCATH-3 (▵) or eCATH-1 (□). The experiment shown on the right side was performed in RPMI 1640 medium in the absence (○) or presence of LLK-eCATH-3 (▵) or eCATH-1 (□). Each point is the mean of three independent experiments. Error bars indicate standard deviations.