Cell selectivity and mechanism of action of antimicrobial model peptides containing peptoid residues

To develop a useful method for designing cell-selective antimicrobial peptides and to investigate the effect of incorporating peptoid residues into an alpha-helical model peptide on structure, function, and mode of action, we synthesized a series of model peptides incorporating Nala (Ala-peptoid) into different positions of an amphipathic alpha-helical model peptide (KLW). Incorporation of one or two Nala residues into the hydrophobic helix face of KLW was more effective at disrupting the alpha-helical structure and bacterial cell selectivity than incorporation into the hydrophilic helix face or hydrophobic/hydrophilic interface. Tryptophan fluorescence studies of peptide interaction with model membranes indicated that the cell selectivity of KLW-L9-a and KLW-L9,13-a is closely correlated with their selective interactions with negatively charged phospholipids. KLW-L9,13-a, which has two Nala residues in its hydrophobic helix face, showed a random structure in membrane-mimicking conditions. KLW-L9,13-a exhibited the highest selectivity toward bacterial cells, showing no hemolytic activity and no or less cytotoxicity compared with other peptides against four mammalian cell lines. Unlike other model peptides, KLW-L9,13-a caused no or little membrane depolarization in Staphylococcus aureus or lipid flip-flop in negatively charged vesicles. In addition, KLW-L9,13-a caused very little fluorescent dye leakage from negatively charged vesicles. Furthermore, confocal laser-scanning microscopy and DNA-binding assays showed that KLW-L9,13-a probably exerts its antibacterial action by penetrating the bacterial membrane and binding to cytoplasmic compounds (e.g., DNA), resulting in cell death. Collectively, our results demonstrate that the incorporation of two Nala residues into the central position of the hydrophobic helix face of noncell-selective alpha-helical peptides is a promising strategy for the rational design of intracellular, cell-selective antimicrobial peptides.     

Mechanism of antibacterial action of a synthetic peptide with an Ala-peptoid residue based on the scorpion-derived antimicrobial peptide IsCT

A novel bacterial cell-selective antimicrobial peptide, IsCT-P (ILKKIWKPIKKLF-NH₂), was designed based on the scorpion-derived α-helical antimicrobial peptide, IsCT. Here, we investigated the effect of substituting Pro⁸ of IsCT-P with the Ala-peptoid residue (N-methylglycine) on the peptide’s structure and mechanism of action. Circular dichroism analysis revealed that the modified peptide, IsCT-a, has a much lower α-helicity than IsCT-P in membrane mimicking conditions, suggesting the peptoid residue provides much more structural flexibility than the proline residue. IsCT-a was also much less effective than IsCT-P at causing leakage of fluorescent dye entrapped within negatively charged vesicles and at dissipating the membrane potential of Staphylococcus aureus. Collectively, our results suggest that the antibacterial action of IsCT-a is due to the inhibition of intracellular targets rather than the disruption and depolarization of bacterial cell membranes.Shin Saeng Lim and Sang-Pil Yoon contributed equally to this work.     

Improvement of bacterial cell selectivity of melittin by a single Trp mutation with a peptoid residue

To design melittin (ME) analogues that are not cytotoxic against mammalian cells but which possessing potent antimicrobial activity, we synthesized a ME analogue (ME-w) in which the Trp-19 residue of ME was replaced by a Trp-peptoid residue (Nhtrp). ME-w exhibited similar antimicrobial activity compared to ME against the tested six bacteria and C. albicans. However, it was much less cytotoxic against the hRBCs and HeLa and NIH-3T3 cells than ME. Tryptophan fluorescence and CD spectra revealed that the Trp-19 --> Nhtrp substitution in ME contributed to a much lower helical assembly in an aqueous environment and structural flexibility and exterior localization to zwitterionic membrane which modulates its selectivity toward bacterial cells.     

Investigating the effects of positive charge and hydrophobicity on the cell selectivity, mechanism of action and anti-inflammatory activity of a Trp-rich antimicrobial peptide indolicidin

To investigate the effects of positive charge and hydrophobicity on the cell selectivity, mechanism of action and anti-inflammatory activity of a Trp-rich antimicrobial peptide indolicidin (IN), a series of IN analogs with Trp→Lys substitution were synthesized. All IN analogs displayed an approximately 7- to 18-fold higher cell selectivity, compared with IN. IN, IN-1 and IN-2 depolarized (50−90%) the cytoplasmic membrane potential of Staphylococcus aureus close to minimal inhibitory concentration (5–10 μg mL⁻¹). However, other IN analogs (IN-3 and IN-4) displayed very low ability in membrane depolarization even at 40 μg mL⁻¹. Confocal laser-scanning microscopy revealed that IN-3 and IN-4 penetrated the Escherichia coli cell membrane, whereas IN, IN-1 and IN-2 did not enter the cell membrane. In the gel retardation assay, IN-3 and IN-4 bound more strongly to DNA compared with IN, IN-1 and IN-2. These findings suggest that the mechanism of antimicrobial action of IN-3 and IN-4 may be involved in the inhibition of intracellular functions via interference with DNA/RNA synthesis. Unlike IN, all IN analogs did not inhibit nitric oxide production or inducible nitric oxide synthase mRNA expression in lipopolysaccharide-stimulated mouse macrophage RAW264.7 cells, indicating that the hydrophobicity of IN is more important for anti-inflammatory activity in lipopolysaccharide-treated macrophage cells than the positive charge.     

Structure and mechanism of action of the antimicrobial peptide piscidin

Piscidin, an antibacterial peptide isolated from the mast cells of striped bass, has potent antimicrobial activity against a broad spectrum of pathogens in vitro. We investigated the mechanism of action of this 22-residue cationic peptide by carrying out structural studies and electrophysiological experiments in lipid bilayers. Circular dichroism experiments showed that piscidin was unstructured in water but had a high alpha-helix content in dodecylphosphocholine (DPC) micelles. 1H NMR data in water and TFE confirmed these results and demonstrated that the segment of residues 8-17 adopted an alpha-helical structure in a micellar environment. This molecule has a marked amphipathic character, due to well-defined hydrophobic and hydrophilic sectors. This structure is similar to those determined for other cationic peptides involved in permeabilization of the bacterial membrane. Multichannel experiments with piscidin incorporated into azolectin planar bilayers gave reproducible I-V curves at various peptide concentrations and unambiguously showed that this peptide permeabilized the membrane. This pore forming activity was confirmed by single-channel experiments, with well-defined ion channels obtained at different voltages. The characteristics of the ion channels (voltage dependence, only one or two states of conductance) clearly suggest that piscidin is more likely to permeabilize the membrane by toroidal pore formation rather than via the "barrel-stave" mechanism.     

Effect of Pro11Ala substitution on the structural and functional properties of antimicrobial peptide buforin 2

Molecular dynamics simulations were used to investigate the spatial structure of antimicrobial peptide buforin 2 in water and in toroidal transmembrane pores. It is shown that replacement of Pro11 with Ala straightens and stabilizes the helix and enables the peptide to form a stable transmembrane toroidal pore. The results obtained correlate with the changes observed when the modified buforin 2 interacts with cells.     

Mutations to alter Aspergillus awamori glucoamylase selectivity. IV. Combinations of Asn20-->Cys/Ala27-->Cys, Ser30-->Pro, Gly137-->Ala, 311-4 loop, Ser411-->Ala and Ser436-->Pro

Six previously constructed and nine newly constructed Aspergillus awamori glucoamylases with multiple mutations made by combining existing single mutations were tested for their ability to produce glucose from maltodextrins. Multiple mutations have cumulative effects on glucose yield, specific activity and thermostability. No general correlation between glucose yield and thermostability was observed, although mutations that presumably impede unfolding at high temperatures uniformly increase thermostability and generally increase glucose yield. Peak glucose yields decrease with increasing temperature. The best combination of high glucose yield, high specific activity and high thermostability occurs in Asn20-->Cys/Ala27-->Cys/Ser30-->Pro/Gly137-->Ala glucoamylase.     

Effects and mechanism of action of synthetic peptide octarphin

We have synthesized the peptide TPLVTLFK corresponding to the β-endorphin fragment 12-19 (the name given by the authors - octarphin), and its analogs (LPLVTLFK, TLLVTLFK, TPLVLLFK, TPLVTLLK, TPLVTLFL). The peptide octarphin was labeled with tritium (the specific activity of 28 Ci/mmol) and its binding to the murine peritoneal macrophages has been studied. [(3)H]Octarphin was found to bind to macrophages with high affinity (K(d) = 2.3 ± 0.2 nM) and specificity. The specific binding of [(3)H]octarphin is inhibited by unlabeled β-endorphin and selective agonist of non-opioid β-endorphin receptor synthetic peptide immunorphin (SLTCLVKGFY) (K(i) = 2.7 ± 0.2 and 2.4 ± 0.2 nM respectively) and not inhibited by unlabeled naloxone, α-endorphin, γ-endorphin and [Met(5)]enkephalin (K(i) > 10 μM). Inhibiting activity of unlabeled analogs of octarphin is more then 100 times lower the unlabeled octarphin. Octarphin stimulates activity of murine immunocompetent cells in vitro and in vivo: at the concentration of 1-10 nM enhances the adhesion and spreading of peritoneal macrophages as well as their capacity to digest bacteria of Salmonella typhimurium virulent strain 415 in vitro. Intraperitoneal administration of peptide at dose 20 μg/animal on day 7,3 and 1 prior to the isolation of cells increases activity of peritoneal macrophages as well as T- and B-spleen lymphocytes.     

Selectivity in the mechanism of action of antimicrobial mastoparan peptide Polybia-MP1

Many potent antimicrobial peptides also present hemolytic activity, an undesired collateral effect for the therapeutic application. Unlike other mastoparan peptides, Polybia-MP1 (IDWKKLLDAAKQIL), obtained from the venom of the social wasp Polybia paulista, is highly selective of bacterial cells. The study of its mechanism of action demonstrated that it permeates vesicles at a greater rate of leakage on the anionic over the zwitterionic, impaired by the presence of cholesterol or cardiolipin; its lytic activity is characterized by a threshold peptide to lipid molar ratio that depends on the phospholipid composition of the vesicles. At these particular threshold concentrations, the apparent average pore number is distinctive between anionic and zwitterionic vesicles, suggesting that pores are similarly formed depending on the ionic character of the bilayer. To prospect the molecular reasons for the strengthened selectivity in Polybia-MP1 and its absence in Mastoparan-X, MD simulations were carried out. Both peptides presented amphipathic alpha-helical structures, as previously observed in Circular Dichroism spectra, with important differences in the extension and stability of the helix; their backbone solvation analysis also indicate a different profile, suggesting that the selectivity of Polybia-MP1 is a consequence of the distribution of the charged and polar residues along the peptide helix, and on how the solvent molecules orient themselves according to these electrostatic interactions. We suggest that the lack of hemolytic activity of Polybia-MP1 is due to the presence and position of Asp residues that enable the equilibrium of electrostatic interactions and favor the preference for the more hydrophilic environment.     

Design and mechanism of action of a novel bacteria-selective antimicrobial peptide from the cell-penetrating peptide Pep-1

Here, we report the successful design of a novel bacteria-selective antimicrobial peptide, Pep-1-K (KKTWWKTWWTKWSQPKKKRKV). Pep-1-K was designed by replacing Glu-2, Glu-6, and Glu-11 in the cell-penetrating peptide Pep-1 with Lys. Pep-1-K showed strong antibacterial activity against reference strains (MIC = 1-2 microM) of Gram-positive and Gram-negative bacteria as well as against clinical isolates (MIC = 1-8 microM) of methicillin-resistant Staphylococcus aureus and multidrug-resistant Pseudomonas aeruginosa. In contrast, Pep-1-K did not cause hemolysis of human erythrocytes even at 200 microM. These results indicate that Pep-1-K may be a good candidate for antimicrobial drug development, especially as a topical agent against antibiotic-resistant microorganisms. Tryptophan fluorescence studies indicated that the lack of hemolytic activity of Pep-1-K correlated with its weak ability to penetrate zwitterionic phosphatidylcholine/cholesterol (10:1, w/w) vesicles, which mimic eukaryotic membranes. Furthermore, Pep-1-K caused little or no dye leakage from negatively charged phosphatidylethanolamine/phosphatidylglycerol (7:3, w/w) vesicles, which mimic bacterial membranes but had a potent ability to cause depolarization of the cytoplasmic membrane potential of intact S. aureus cells. These results suggested that Pep-1-K kills microorganisms by not the membrane-disrupting mode but the formation of small channels that permit transit of ions or protons but not molecules as large as calcein.     

Substitution of the leucine zipper sequence in melittin with peptoid residues affects self-association, cell selectivity, and mode of action

Melittin (ME), a non-cell-selective antimicrobial peptide, contains the leucine zipper motif, wherein every seventh amino acid is leucine or isolucine. Here, we attempted to generate novel cell-selective peptides by substituting amino acids in the leucine zipper sequence of ME with peptoid residues. We generated a series of ME analogues by replacing Leu-6, Lue-13 and Ile-20 with Nala, Nleu, Nphe, or Nlys, and we examined their secondary structure, self-association activity, cell selectivity and mode of action. Circular dichroism spectroscopy indicated that the substitutions disrupt the alpha-helical structure of ME in micelles of sodium dodecyl sulfate and on negatively charged and zwitterionic phospholipid vesicles. Substitution by Nleu, Nphe, or Nlys but not Nala disturbed the self-association in an aqueous environment, interaction with zwitterionic membranes, and toxicity to mammalian cells of ME but did not affect the interaction with negatively charged membranes or antibacterial activity. Notably, peptides with Nphe or Nlys substitution had the highest therapeutic indices, consistent with their lipid selectivity. In addition, all of peptoid residue-containing ME analogues had little or no ability to induce membrane disruption, membrane depolarization and lipid flip-flop. Taken together, our studies indicate that substitution of the leucine zipper motif in ME with peptoid residues increases its selectivity against bacterial cells by impairing self-association activity and changes its mode of antibacterial action from membrane-targeting mechanism to possible intracellular targeting mechanism. Furthermore, our ME analogues especially those with Nleu, Nphe, or Nlys substitutions, may be therapeutically useful antimicrobial peptides.     

Substitution of the leucine zipper sequence in melittin with peptoid residues affects self-association, cell selectivity, and mode of action

Melittin (ME), a non-cell-selective antimicrobial peptide, contains the leucine zipper motif, wherein every seventh amino acid is leucine or isolucine. Here, we attempted to generate novel cell-selective peptides by substituting amino acids in the leucine zipper sequence of ME with peptoid residues. We generated a series of ME analogues by replacing Leu-6, Lue-13 and Ile-20 with Nala, Nleu, Nphe, or Nlys, and we examined their secondary structure, self-association activity, cell selectivity and mode of action. Circular dichroism spectroscopy indicated that the substitutions disrupt the α-helical structure of ME in micelles of sodium dodecyl sulfate and on negatively charged and zwitterionic phospholipid vesicles. Substitution by Nleu, Nphe, or Nlys but not Nala disturbed the self-association in an aqueous environment, interaction with zwitterionic membranes, and toxicity to mammalian cells of ME but did not affect the interaction with negatively charged membranes or antibacterial activity. Notably, peptides with Nphe or Nlys substitution had the highest therapeutic indices, consistent with their lipid selectivity. In addition, all of peptoid residue-containing ME analogues had little or no ability to induce membrane disruption, membrane depolarization and lipid flip-flop. Taken together, our studies indicate that substitution of the leucine zipper motif in ME with peptoid residues increases its selectivity against bacterial cells by impairing self-association activity and changes its mode of antibacterial action from membrane-targeting mechanism to possible intracellular targeting mechanism. Furthermore, our ME analogues especially those with Nleu, Nphe, or Nlys substitutions, may be therapeutically useful antimicrobial peptides.     

Lipid headgroup discrimination by antimicrobial peptide LL-37: insight into mechanism of action

Interaction of the human antimicrobial peptide LL-37 with lipid monolayers has been investigated by a range of complementary techniques including pressure-area isotherms, insertion assay, epifluorescence microscopy, and synchrotron x-ray scattering, to analyze its mechanism of action. Lipid monolayers were formed at the air-liquid interface to mimic the surface of the bacterial cell wall and the outer leaflet of erythrocyte cell membrane by using phosphatidylglycerol (DPPG), phosphatidylcholine (DPPC), and phosphatidylethanolamine (DPPE) lipids. LL-37 is found to readily insert into DPPG monolayers, disrupting their structure and thus indicating bactericidal action. In contrast, DPPC and DPPE monolayers remained virtually unaffected by LL-37, demonstrating its nonhemolytic activity and lipid discrimination. Specular x-ray reflectivity data yielded considerable differences in layer thickness and electron-density profile after addition of the peptide to DPPG monolayers, but little change was seen after peptide injection when probing monolayers composed of DPPC and DPPE. Grazing incidence x-ray diffraction demonstrated significant peptide insertion and lateral packing order disruption of the DPPG monolayer by LL-37 insertion. Epifluorescence microscopy data support these findings.     

The role of the central L- or D-Pro residue on structure and mode of action of a cell-selective alpha-helical IsCT-derived antimicrobial peptide

IsCT-P (ILKKIWKPIKKLF-NH2) is a novel alpha-helical antimicrobial peptide with bacterial cell selectivity designed from a scorpion-derived peptide IsCT. To investigate the role of L- or D-Pro kink on the structure and the mode of action of a short alpha-helical antimicrobial peptide with bacterial cell selectivity, we synthesized IsCT-p, in which D-Pro is substituted for L-Pro8 of IsCT-P. CD spectra revealed that IsCT-P adopted a typical alpha-helical structure in various membrane-mimicking conditions, whereas IsCT-p showed a random structure. This result indicated that D-Pro in the central position of a short alpha-helical peptide provides more remarkable structural flexibility than L-Pro. Despite its higher antibacterial activity, IsCT-p was much less effective at inducing dye leakage in the negatively charged liposome mimicking bacterial membrane and induced no or little membrane potential depolarization of Staphylococcus aureus. Confocal laser scanning microscopy showed that IsCT-p penetrated the bacterial cell membrane and accumulated in the cytoplasm, whereas IsCT-P remained outside or on the cell membrane. These results suggested that the major target of IsCT-P and IsCT-p is the bacterial membranes and intracellular components, respectively. Collectively, our results demonstrated that the central D-Pro kink in alpha-helical antimicrobial peptides plays an important role in penetrating bacterial membrane as well as bacterial cell selectivity.     

Structure and mode of action of the antimicrobial peptide arenicin

The solution structure and the mode of action of arenicin isoform 1, an antimicrobial peptide with a unique 18-residue loop structure, from the lugworm Arenicola marina were elucidated here. Arenicin folds into a two-stranded antiparallel beta-sheet. It exhibits high antibacterial activity at 37 and 4 degrees C against Gram-negative bacteria, including polymyxin B-resistant Proteus mirabilis. Bacterial killing occurs within minutes and is accompanied by membrane permeabilization, membrane detachment and release of cytoplasm. Interaction of arenicin with reconstituted membranes that mimic the lipopolysaccharide-containing outer membrane or the phospholipid-containing plasma membrane of Gram-negative bacteria exhibited no pronounced lipid specificity. Arenicin-induced current fluctuations in planar lipid bilayers correspond to the formation of short-lived heterogeneously structured lesions. Our results strongly suggest that membrane interaction plays a pivotal role in the antibacterial activity of arenicin.     

Permeabilizing action of an antimicrobial lactoferricin-derived peptide on bacterial and artificial membranes

We have previously cloned the human RNA polymerase II subunit 11, as a doxorubicin sensitive gene product. We suggested multiple tasks for this subunit, including structural and regulatory roles. With the aim to clarify the human RNA polymerase II subunit 11 function, we have identified its interacting protein partners using the yeast two-hybrid system. Here, we show that human RNA polymerase II subunit 11 specifically binds keratin 19, a component of the intermediate filament protein family, which is expressed in a tissue and differentiation-specific manner. In particular, keratin 19 is a part of the nuclear matrix intermediate filaments. We provide evidence that human RNA polymerase II subunit 11 interacts with keratin 19 via its N-terminal alpha motif, the same motif necessary for its interaction with the human RNA polymerase II core subunit 3. We found that keratin 19 contains two putative leucine zipper domains sharing peculiar homology with the alpha motif of human RNA polymerase II subunit 3. Finally, we demonstrate that keratin 19 can compete for binding human RNA polymerase II subunit 11/human RNA polymerase II subunit 3 in vitro, suggesting a possible regulatory role for this molecule in RNA polymerase II assembly/activity.     

Antimicrobial HPA3NT3 peptide analogs: Placement of aromatic rings and positive charges are key determinants for cell selectivity and mechanism of action

In an earlier study, we determined that HP(2‐20) (residues 2‐20 of parental HP derived from the N-terminus of the Helicobacter pylori ribosomal protein L1) and its analog, HPA3NT3, had potent antimicrobial effects. However, HPA3NT3 also showed undesirable cytotoxicity against HaCaT cells. In the present study, we designed peptide analogs including HPA3NT3-F1A (‐F1A), HPA3NT3-F8A (‐F8A), HPA3NT3-F1AF8A (‐F1AF8A), HPA3NT3-A1 (‐A1) and HPA3NT3-A2 (‐A2) in an effort to investigate the effects of amino acid substitutions in reducing their hydrophobicity or increasing their cationicity, and any resulting effects on their selectivity in their interactions with human cells and pathogens, as well as their mechanism of antimicrobial action. With the exception of HPA3NT3-A1, all of these peptides showed potent antimicrobial activity. Moreover, substitution of Ala for Phe at positions 1 and/or 8 of the HPA3NT3 peptides (‐F1A, -F8A and -F1AF8A) dramatically reduced their cytotoxicity. Thus the cytotoxicity of HPA3NT3 appears to be related to its Phe residues (positions 1 and 8), which strongly interact with sphingomyelin in the mammalian cell membrane. HPA3NT3 exerted its bactericidal effects through membrane permeabilization mediated by pore formation. In contrast, fluorescent dye leakage and nucleic acid gel retardation assays showed that ‐A2 acted by penetrating into the cytoplasm, where it bound to nucleic acids and inhibited protein synthesis. Notably, Staphylococcus aureus did not develop resistance to -A2 as it did with rifampin. These results suggest that the -A2 peptide could potentially serve as an effective antibiotic agent against multidrug-resistant bacterial strains.     

Prokaryotic selectivity and LPS-neutralizing activity of short antimicrobial peptides designed from the human antimicrobial peptide LL-37

To develop novel antimicrobial peptides (AMPs) with shorter lengths, improved prokaryotic selectivity and retained lipolysaccharide (LPS)-neutralizing activity compared to human cathelicidin AMP, LL-37, a series of amino acid-substituted analogs based on IG-19 (residues 13-31 of LL-37) were synthesized. Among the IG-19 analogs, the analog a4 showed the highest prokaryotic selectivity, but much lower LPS-neutralizing activity compared to parental LL-37. The analogs, a5, a6, a7 and a8 with higher hydrophobicity displayed LPS-neutralizing activity comparable to that of LL-37, but much lesser prokaryotic selectivity. These results indicate that the proper hydrophobicity of the peptides is crucial to exert the amalgamated property of LPS-neutralizing activity and prokaryotic selectivity. Furthermore, to increase LPS-neutralizing activity of the analog a4 without a remarkable decrease in prokaryotic selectivity, we synthesized Trp-substituted analogs (a4-W1 and a4-W2), in which Phe(5) or Phe(15) of a4 is replaced by Trp. Despite their same prokaryotic selectivity, a4-W2 displayed much higher LPS-neutralizing activity compared to a4-W1. When compared with parental LL-37, a4-W2 showed retained LPS-neutralizing activity and 2.8-fold enhanced prokaryotic selectivity. These results suggest that the effective site for Trp-substitution when designing novel AMPs with higher LPS-neutralizing activity, without a remarkable reduction in prokaryotic selectivity, is the amphipathic interface between the end of the hydrophilic side and the start of the hydrophobic side rather than the central position of the hydrophobic side in their α-helical wheel projection. Taken together, the analog a4-W2 can serve as a promising template for the development of therapeutic agents for the treatment of endotoxic shock and bacterial infection.