Isolation of an ovine pulmonary surfactant-associated anionic peptide bactericidal for Pasteurella haemolytica.

Abstract

Ovine pulmonary surfactant is bactericidal for Pasteurella haemolytica when surfactant and bacteria mixtures are incubated with normal ovine serum. To isolate this component, surfactant (1 mg/ml) was centrifuged at 100,000 X gav, and the supernatant was fractionated by HPLC. Fractions were eluted with acetonitrile (10-100%)/0.1% trifluoracetic acid and tested for bactericidal activity. Amino acid and sequence analysis of three bactericidal fractions showed that fraction 2 contained H-GDDDDDD-OH, fraction 3 contained H-DDDDDDD-OH, and fraction 6 contained H-GADDDDD-OH. Peptides in 0.14 M NaCl/10 ,iM ZnC12 (zinc saline solution) induced killing of P. haemolytica and other bacteria comparable to defensins and 13-defensins [minimal bactericidal concentration (MBC)50 range, 0.01-0.06 mM] but not in 0.14 M NaCi/10 mM sodium phosphate buffer, pH 7.2/0.5 mM CaCl2/0.15 mM MgCl2 (MBC50 range, 2.8-11.5 mM). Bactericidal activity resided in the core aspartate hexapeptide homopolymeric region, and MBC50 values of aspartate dipeptide-to-heptapeptide homopolymers were inversely proportional to the number of aspartate residues in the peptide. P. haemolytica incubated with H-DDDDDD-OH in zinc saline solution was killed within 30 min. Ultrastructurally, cells contained flocculated intracellular constituents. In contrast to cationic defensins and 18-defensins, surfactantassociated anionic peptides are smaller in size, opposite in charge, and are bactericidal in zinc saline solution. They are members of another class of peptide antibiotics containing aspartate, which when present in pulmonary secretions may help clear bacteria as a part of the innate pulmonary defense system.

Molecular Characterization of Two Novel Antibacterial Peptides Inducible upon Bacterial Challenge in an Annelid, the Leech Theromyzon tessulatum

“…This is the first report of an anionic antibacterial peptide in invertebrates. In vertebrates, antimicrobial peptides with anionic properties were evidenced in the human and in sheep lung (4). Anionic properties are anionic because of the homopolymeric regions of aspartate and require zinc as a cofactor for bactericidal activity.…”

“…This amphipathic structure allows them to interact with bacterial membrane. Brogden et al (4) have determined in ovine that a novel class of peptides with anionic properties also has antimicrobial properties (4). In marked contrast to cationic antimicrobial peptides, no anionic antibacterial molecules have been described hitherto in invertebrates.…”

Suppression of Mannheimia (Pasteurella) haemolytica serovar 1 infection in lambs by intrapulmonary administration of ovine antimicrobial anionic peptide

“…Anionic peptides were originally detected in sheep and three small peptides, H-GADDDDD-OH, H-GDDDDDD-OH and H-DDDDDDD-OH, were isolated from ovine surfactant extracts [4]. Monoclonal antibodies prepared to AP were used to detect AP in the bronchoalveolar lavage fluid and respiratory epithelial cells of sheep [5], cattle, and humans [6].…”

“…Monoclonal antibodies prepared to AP were used to detect AP in the bronchoalveolar lavage fluid and respiratory epithelial cells of sheep [5], cattle, and humans [6]. APs occur in mM concentrations [5], require zinc as a cofactor for antimicrobial activity [4] and peptides (natural and synthetic) are rapidly antimicrobial against both Grampositive and Gram-negative organisms (range 0.15 -1.06 mM).…”

Comparison of bronchoalveolar lavage fluid obtained from Mannheimia haemolytica-inoculated calves with and without prior treatment with the selectin inhibitor TBC1269

“…This peptide is smaller than most AMP, and its anionic properties differ from most AMP, which are cationic. 31 It is composed of consecutive aspartates and requires zinc as a cofactor. 31 Antimicrobial anionic peptide initially was isolated from ovine surfactant.…”