Minibactenecins ChBac7.Nα and ChBac7. Nβ - Antimicrobial Peptides from Leukocytes of the Goat Capra hircus

Abstract

Antimicrobial peptides (AMPs) of neutrophils play an important role in the animal and human host defenses. We have isolated two AMPs (average molecular masses of 2895.5 and 2739.3 Da), with potent antimicrobial activity from neutrophils of the domestic goat (Capra hircus). A structural analysis of the obtained peptides revealed that they encompass N-terminal fragments (1-21 and 1-22) of the proline-rich peptide bactenecin 7.5. The primary structure of caprine bactenecin 7.5 had been previously deduced from the nucleotide sequence, but the corresponding protein had not been isolated from leukocytes until now. The obtained caprine AMPs were designated as mini-batenecins (mini-ChBac7.5Nα and mini-ChBac7.5Nβ), analogously to the reported C-terminal fragment of the ovine bactenecin 7.5 named Bac7.5mini [Anderson, Yu, 2003]. Caprine mini-ChBac7.5Nα and mini-ChBac7.5Nβ exhibit significant antimicrobial activity against Gram-negative bacteria, including drug-resistant strains of Pseudomonas aeruginosa, Klebsiella spp., Acinetobacter baumannii at a range of concentrations of 0.5-4 μM, as well as against some species of Gram-positive bacteria (Listeria monocytogenes EGD, Micrococcus luteus). The peptides demonstrate lipopolysaccharide-binding activity. Similarly to most proline-rich AMPs, caprine peptides inactivate bacteria without appreciable damage of their membranes. Mini-ChBac7.5Nα and mini-ChBac7.5Nβ have no hemolytic effect on human red blood cells and are nontoxic to various cultured human cells. Therefore, they might be considered as promising templates for the development of novel antibiotic pharmaceuticals. Isolation of highly active fragments of the antimicrobial peptide from goat neutrophils supports the hypothesis that fragmentation of cathelicidin-related AMPs is an important process that results in the generation of potent effector molecules, which are in some cases more active than full-size AMPs. These truncated AMPs may play a crucial role in host defense reactions.

Keywords: antimicrobial peptides; cathelicidins; mini-bactenecins.

Fig. 1

Purification of antimicrobial peptides from extracts of goat leukocytes. A – Preparative continuous elution electrophoresis (CEE) of YM10 ultrafiltrate of goat leukocyte extract in a polyacrylamide gel (current strength 30 mA, flow rate 36 ml/h, fraction volume 3 ml). Peak 1 – fractions of peptides with a molecular mass of 2.8 – 6 kDa, containing mini-bactenecins; peak 2 – ChBac3.4; peak 3 – ChBac5. CEE fractions were tested for antimicrobial activity against Listeria monocytogenes EGD and E.coli ML35p in radial diffusion assays (right X axis – antimicrobial activity units). B – RP-HPLC of CEE fractions 19–24, using a linear gradient of acetonitrile (0–60%; 1%/min; 0.1% trifluoroacetic acid) on the Vydac C18-column (0.46 x 25 cm). C – RP-HPLC of fractions 24–26 obtained after RP-HPLC is shown on panel B (acetonotrile gradient: 0–20% during 20 min, 20–50% during 60 min, 50–60% during 10 min, 0.13 % heptafluorobutyric acid). Peaks of peptides with average molecular masses of 2895.5 Da and 2739.3 Da designated as mini-ChBac7.5Nα and mini-ChBac7.5Nβ are shown by arrows.

Fig. 2

Amino acid sequences of the antimicrobial peptides isolated from goat leukocytes, mini-ChBac7.5Nα and ChBac7.5Nα-β, compared to the previously reported sequences of bactenecins: bovine Bac7 (BtBac7; diverse amino acid residues are underlined) [4], ovine OaBac7.5 [7] and OaBac7.5mini [2], caprine ChBac5 [11] and ChBac3.4 [12]. The structure of the full-size caprine ChBac7.5 is shown. * – amidated C-terminus of the molecule.

Fig. 3

Kinetics of changes in Escherichia coli ML-35p membrane permeability with respect to chromogenic markers resulting from incubation of bacteria with mini-ChBac7.5Nα taken in various concentrations: 1 – 0.6 μM; 2 – 1.2 μM; 3 – 2.5 μM; 4 – 5 μM; 5 – 10 μM; 6 – 20 μM. x-axis – incubation time, min. y-axis – optical density of the solution containing chromogenic markers: nitrocefin hydrolysis product at a wavelength of 486 nm (left panel displaying the outer membrane permeabilization) and ONPG hydrolysis product, o-nitrophenol, at 420 nm (right panel displaying the inner membrane permeabilization). Another caprine bactenecin, ChBac3.4, was used as a reference (at a final concentration of 5 μM, which is 2 x MIC); a membranolytic peptide, porcine protegrin 1 (PG-1), was used as a positive control (2.5 μM).

Fig. 4

lipopolysaccharide by mini- ChBac7.5Nα and mini-ChBac7.5Nβ as compared with caprine bactenecins ChBac3.4, ChBac5 and its inactive fragment ChBac5 (20- 43) in a quantitative chromogenic Limulus Amebocyte Lysate Assay. Polymyxin B (PmxB) was used as a positive control. Mean values ± S.D., n=6 are shown. EC₅₀ (i.e., peptide concentrations that bound 50% of the LPS) are shown in the inset.