Studies on Lactoferricin-derived Escherichia coli Membrane-active Peptides Reveal Differences in the Mechanism of N-Acylated Versus Nonacylated Peptides

Abstract

To improve the low antimicrobial activity of LF11, an 11-mer peptide derived from human lactoferricin, mutant sequences were designed based on the defined structure of LF11 in the lipidic environment. Thus, deletion of noncharged polar residues and strengthening of the hydrophobic N-terminal part upon adding a bulky hydrophobic amino acid or N-acylation resulted in enhanced antimicrobial activity against Escherichia coli, which correlated with the peptides' degree of perturbation of bacterial membrane mimics. Nonacylated and N-acylated peptides exhibited different effects at a molecular level. Nonacylated peptides induced segregation of peptide-enriched and peptide-poor lipid domains in negatively charged bilayers, although N-acylated peptides formed small heterogeneous domains resulting in a higher degree of packing defects. Additionally, only N-acylated peptides perturbed the lateral packing of neutral lipids and exhibited increased permeability of E. coli lipid vesicles. The latter did not correlate with the extent of improvement of the antimicrobial activity, which could be explained by the fact that elevated binding of N-acylated peptides to lipopolysaccharides of the outer membrane of Gram-negative bacteria seems to counteract the elevated membrane permeabilization, reflected in the respective minimal inhibitory concentration for E. coli. The antimicrobial activity of the peptides correlated with an increase of membrane curvature stress and hence bilayer instability. Transmission electron microscopy revealed that only the N-acylated peptides induced tubular protrusions from the outer membrane, whereas all peptides caused detachment of the outer and inner membrane of E. coli bacteria. Viability tests demonstrated that these bacteria were dead before onset of visible cell lysis. Lactoferrin (LF)5 is an iron-binding glycoprotein found in milk (1) and in exocrine secretions of mammals as well as in granules and neutrophils during inflammatory responses (2, 3). The pepsin cleavage fragment lactoferricin, comprising amino acid residues 1-45 of the N terminus of human LF and 17-41 of bovine LF, is one example of naturally occurring antimicrobial peptides (4). Lactoferricin displays activity against a diverse range of microorganisms such as Gram-negative bacteria, Gram-positive bacteria, yeast, and filamentous fungi, including a number of antibiotic-resistant pathogens (4). It is assumed that lactoferricin permeabilizes cell membranes and exerts anti-endotoxic activity by binding to lipopolysaccharides (4). In earlier studies, we demonstrated that the stretch of 11 amino acid residues (LF11, FQWQRNIRKVR-NH 2 ) corresponding to LF residues 21-31 exhibits only weak antibacterial activity against a number of Gram-positive and Gram-negative bacterial strains (5, 6). A series of about 150 LF11 mutants was designed with the aim to strengthen membrane-peptide interactions and hence to generate highly antimicrobial and endotoxin-neutralizing peptides, while having no or little antigenic or toxic effect on humans. Our stra...

Phospholipid-driven differences determine the action of the synthetic antimicrobial peptide OP-145 on Gram-positive bacterial and mammalian membrane model systems

“…Leakage of the aqueous content of the 8-aminonaphthalene-1,3,6-trisulfonic acid/p-xylene-bis-pyridinium bromide (ANTS/DPX)-loaded large unilamellar vesicles (LUVs) composed of POPG, POPG/LTA, POPC or POPC/cholesterol upon incubation with peptide was determined as described [36]. In the case of POPG/PGN vesicles, 0.1% PGN has been added to the 50 μM POPG large unilamellar vesicles prior to the incubation with peptide.…”

“…LTA concentration has been calculated with a molecular weight of 6200 g/mol estimated for 24 repeating units of the polyglycerolphosphate chain [35]. Large unilamellar vesicles were obtained by several cycles of extrusion of the hydrated liposomes through a polycarbonate filter (Millipore-Isopore™) of 0.1 μm pore size following a protocol described previously [36].…”

Small cationic antimicrobial peptides delocalize peripheral membrane proteins

“…Changes in pattern are indicative of perturbation of the membrane bilayer and packaging of the phospholipids' fatty acyl chains and, therefore, of peptide integration. Deconvolution of the thermograms indicates that MP196 integrates into the phospholipid bilayer, generating "peptide-affected domains" (14). These are characterized by a broadening of the existing transition and the appearance of a small transition at lower temperatures, reflecting rearrangement of the lipid mixture, and, consequently, alterations in packaging of the lipids at the emerging borderlines.…”

“…They are characterized by positively charged and by hydrophobic amino acids (9,10). Previous mechanistic studies in vitro have examined the interactions of the peptides with membranes or membrane extracts (11)(12)(13)(14)(15), but their effects on bacterial physiology have been largely underexplored.…”

A Moraxella catarrhalis Two-Component Signal Transduction System Necessary for Growth in Liquid Media Affects Production of Two Lysozyme Inhibitors

“…Lack of expression of lysozyme inhibitors can reduce the resistance of Gram-negative bacteria to killing by lysozyme, especially in the presence of lactoferrin (47,72,73), which increases the permeability of the outer membrane (74,75). Incubation of the wild-type M. catarrhalis O35 strain and the O35E lipA lipB double mutant in the presence of both lactoferrin (2.0 mg/ml) and human lysozyme (300 g/ml) for 6 h resulted in a decrease in the viability of both strains, with the mutant being killed more extensively than the wild-type parent strain ( Fig.…”