Rationale for the Design of Shortened Derivatives of the NK-lysin-derived Antimicrobial Peptide NK-2 with Improved Activity against Gram-negative Pathogens

Abstract

The peptide NK-2 is an effective antimicrobial agent with low hemolytic and cytotoxic activities and is thus a promising candidate for clinical applications. It comprises the ␣-helical, cationic core region of porcine NK-lysin a homolog of human granulysin and of amoebapores of pathogenic amoeba. Here we visualized the impact of NK-2 on Escherichia coli by electron microscopy and used NK-2 as a template for sequence variations to improve the peptide stability and activity and to gain insight into the structure/ function relationships. We synthesized 18 new peptides and tested their activities on seven Gram-negative and one Gram-positive bacterial strains, human erythrocytes, and HeLa cells. Although all peptides appeared unordered in buffer, those active against bacteria adopted an ␣-helical conformation in membrane-mimetic environments like trifluoroethanol and negatively charged phosphatidylglycerol (PG) liposomes that mimick the cytoplasmic membrane of bacteria. This conformation was not observed in the presence of liposomes consisting of zwitterionic phosphatidylcholine (PC) typical for the human cell plasma membrane. The interaction was paralleled by intercalation of these peptides into PG liposomes as determined by FRET spectroscopy. A comparative analysis between biological activity and the calculated peptide parameters revealed that the decisive factor for a broad spectrum activity is not the peptide overall hydrophobicity or amphipathicity, but the possession of a minimal positive net charge plus a highly amphipathic anchor point of only seven amino acid residues (two helical turns).

Interaction of an Antimicrobial Peptide with Membranes: Experiments and Simulations with NKCS

“…According to previous studies, 16 our CD measurements, and the MC simulations, NKCS was mostly unstructured in water but adopted an R-helical conformation in the membrane-mimetic environment. Moreover, the simulations of the peptide in POPE membranes showed a helix disruption at residues Thr-13 and Leu-14.…”

“…15 The replacement of cysteine residue within the NK-2 sequence with a serine (C7S), resulted in a peptide with an improved antibacterial activity referred to as NKCS in the current study. 16 Both peptides are randomly coiled in water and adopt a helical structure upon interaction with the lipid bilayer. 11,16 Several approaches are used to investigate the interaction between antibacterial peptides and lipid membranes.…”

“…16 Both peptides are randomly coiled in water and adopt a helical structure upon interaction with the lipid bilayer. 11,16 Several approaches are used to investigate the interaction between antibacterial peptides and lipid membranes. These include biophysical studies using techniques such as differential scanning calorimetry (DSC), 17 Fourier transform infrared (FTIR) spectroscopy, 18 circular dichroism (CD) spectroscopy, 19 scattering techniques (X-ray and neutron scattering), 17,20 NMR, 21 and surface plasmon resonance (SPR), 22 as well as computational studies including molecular dynamics simulations, 23,24 continuum solvent models, 25,26 and Monte Carlo (MC) simulations.…”

Structural Features Governing the Activity of Lactoferricin-Derived Peptides That Act in Synergy with Antibiotics against Pseudomonas aeruginosa In Vitro and In Vivo

“…While considerable effort to study how to rationally improve the antibacterial activity and selectivity of AMPs has been made (2,8,14,15,16,26,28,29,30,31,45,48,50), to our knowledge, this is the first report investigating the structural basis on May 10, 2018 by guest http://aac.asm.org/ of peptide-mediated OM permeabilization as a function of the peptide's ability to enhance antibiotic activity. In this regard, our results confirm (53,56,64) that antibacterial activity and antibiotic-enhancing ability are not necessarily associated, since peptides with poor antimicrobial activity levels showed significant novobiocin-enhancing potential (e.g., compounds P1-14 and P1-48).…”

The Antimicrobial Peptide NK-2, the Core Region of Mammalian NK-Lysin, Kills Intraerythrocytic Plasmodium falciparum

“…Albeit their existence on earth dates back millions of years, these weapons are still effective, pointing to the fact that their mode of action, i.e., the permeabilization of the microbial membrane (44), makes the emergence of complete microbial resistance highly unlikely. Currently more than 700 antimicrobial peptides have been identified from natural sources (http: //aps.unmc.edu/AP/main.php), and a plethora of synthetic analogs with improved activity and selectivity have been constructed (1,4,7,9,13,18,31,32,39,45). Moreover, cellpenetrating peptides have become interesting, as they are capable of delivering a broad range of compounds into a target cell (16,23).…”