Dimerization of the ß-Hairpin Membrane-Active Cationic Antimicrobial Peptide Capitellacin from Marine Polychaeta: An NMR Structural and Thermodynamic Study.

Capitellacin is the ß-hairpin membrane-active cationic antimicrobial peptide from the marine polychaeta Capitella teleta. Capitellacin exhibits antibacterial activity, including against drug-resistant strains. To gain insight into the mechanism of capitellacin action, we investigated the structure of the peptide in the membrane-mimicking environment of dodecylphosphocholine (DPC) micelles using high-resolution NMR spectroscopy. In DPC solution, two structural forms of capitellacin were observed: a monomeric ß-hairpin was in equilibrium with a dimer formed by the antiparallel association of the N-terminal ß-strands and stabilized by intermonomer hydrogen bonds and Van der Waals interactions. The thermodynamics of the enthalpy-driven dimerization process was studied by varying the temperature and molar ratios of the peptide to detergent. Cooling the peptide/detergent system promoted capitellacin dimerization. Paramagnetic relaxation enhancement induced by lipid-soluble 12-doxylstearate showed that monomeric and dimeric capitellacin interacted with the surface of the micelle and did not penetrate into the micelle interior, which is consistent with the "carpet" mode of membrane activity. An analysis of the known structures of ß-hairpin AMP dimers showed that their dimerization in a membrane-like environment occurs through the association of polar or weakly hydrophobic surfaces. A comparative analysis of the physicochemical properties of ß-hairpin AMPs revealed that dimer stability and hemolytic activity are positively correlated with surface hydrophobicity. An additional positive correlation was observed between hemolytic activity and AMP charge. The data obtained allowed for the provision of a more accurate description of the mechanism of the oligomerization of ß-structural peptides in biological membranes.

Novel BRICHOS-Related Antimicrobial Peptides from the Marine Worm Heteromastus filiformis: Transcriptome Mining, Synthesis, Biological Activities, and Therapeutic Potential.

Marine polychaetes represent an extremely rich and underexplored source of novel families of antimicrobial peptides (AMPs). The rapid development of next generation sequencing technologies and modern bioinformatics approaches allows us to apply them for characterization of AMP-derived genes and the identification of encoded immune-related peptides with the aid of genome and transcriptome mining. Here, we describe a universal bioinformatic approach based on the conserved BRICHOS domain as a search query for the identification of novel structurally unique AMP families in annelids. In this paper, we report the discovery of 13 novel BRICHOS-related peptides, ranging from 18 to 91 amino acid residues in length, in the cosmopolitan marine worm Heteromastus filiformis with the assistance of transcriptome mining. Two characteristic peptides with a low homology in relation to known AMPs-the α-helical amphiphilic linear peptide, consisting of 28 amino acid residues and designated as HfBRI-28, and the 25-mer ß-hairpin peptide, specified as HfBRI-25 and having a unique structure stabilized by two disulfide bonds-were obtained and analyzed as potential antimicrobials. Interestingly, both peptides showed the ability to kill bacteria via membrane damage, but mechanisms of their action and spectra of their activity differed significantly. Being non-cytotoxic towards mammalian cells and stable to proteolysis in the blood serum, HfBRI-25 was selected for further in vivo studies in a lethal murine model of the Escherichia coli infection, where the peptide contributed to the 100% survival rate in animals. A high activity against uropathogenic strains of E. coli (UPEC) as well as a strong ability to kill bacteria within biofilms allow us to consider the novel peptide HfBRI-25 as a promising candidate for the clinical therapy of urinary tract infections (UTI) associated with UPEC.

Epithelial-Immune Cell Crosstalk Determines the Activation of Immune Cells In Vitro by the Human Cathelicidin LL-37 at Low Physiological Concentrations.

The only human cathelicidin, LL-37, is a host defense antimicrobial peptide with antimicrobial activities against protozoans, fungi, Gram(+) and Gram(-) bacteria, and enveloped viruses. It has been shown in experiments in vitro that LL-37 is able to induce the production of various inflammatory and anti-inflammatory cytokines and chemokines by different human cell types. However, it remains an open question whether such cytokine induction is physiologically relevant, as LL-37 exhibited its immunomodulatory properties at concentrations that are much higher (>20 µg/mL) than those observed in non-inflamed tissues (1-5 µg/mL). In the current study, we assessed the permeability of LL-37 across the Caco-2 polarized monolayer and showed that this peptide could pass through the Caco-2 monolayer with low efficiency, which predetermined its low absorption in the gut. We showed that LL-37 at low physiological concentrations (<5 µg/mL) was not able to directly activate monocytes. However, in the presence of polarized epithelial monolayers, LL-37 is able to activate monocytes through the MAPK/ERK signaling pathway and induce the production of cytokines, as assessed by a multiplex assay at the protein level. We have demonstrated that LL-37 is able to fulfill its immunomodulatory action in vivo in non-inflamed tissues at low physiological concentrations. In the present work, we revealed a key role of epithelial-immune cell crosstalk in the implementation of immunomodulatory functions of the human cathelicidin LL-37, which might shed light on its physiological action in vivo.

Design of Protegrin-1 Analogs with Improved Antibacterial Selectivity.

Protegrin-1 (PG-1) is a cationic ß-hairpin pore-forming antimicrobial peptide having a membranolytic mechanism of action. It possesses in vitro a potent antimicrobial activity against a panel of clinically relevant MDR ESKAPE pathogens. However, its extremely high hemolytic activity and cytotoxicity toward mammalian cells prevent the further development of the protegrin-based antibiotic for systemic administration. In this study, we rationally modulated the PG-1 charge and hydrophobicity by substituting selected residues in the central ß-sheet region of PG-1 to design its analogs, which retain a high antimicrobial activity but have a reduced toxicity toward mammalian cells. In this work, eight PG-1 analogs with single amino acid substitutions and five analogs with double substitutions were obtained. These analogs were produced as thioredoxin fusions in Escherichia coli. It was shown that a significant reduction in hemolytic activity without any loss of antimicrobial activity could be achieved by a single amino acid substitution, V16R in the C-terminal ß-strand, which is responsible for the PG-1 oligomerization. As the result, a selective analog with a ≥30-fold improved therapeutic index was obtained. FTIR spectroscopy analysis of analog, [V16R], revealed that the peptide is unable to form oligomeric structures in a membrane-mimicking environment, in contrast to wild-type PG-1. Analog [V16R] showed a reasonable efficacy in septicemia infection mice model as a systemic antibiotic and could be considered as a promising lead for further drug design.

Genomic Insights into Bacterial Resistance to Proline-Rich Antimicrobial Peptide Bac7.

Proline-rich antimicrobial peptides (PrAMPs) having a potent antimicrobial activity and a modest toxicity toward mammalian cells attract much attention as new templates for the development of antibiotic drugs. However, a comprehensive understanding of mechanisms of bacterial resistance development to PrAMPs is necessary before their clinical application. In this study, development of the resistance to the proline-rich bovine cathelicidin Bac71-22 derivative was characterized in the multidrug-resistant Escherichia coli clinical isolate causing the urinary tract infection. Three Bac71-22-resistant strains with ≥16-fold increase in minimal inhibitory concentrations (MICs) were selected by serially passaging after four-week experimental evolution. It was shown that in salt-containing medium, the resistance was mediated by inactivation of the SbmA transporter. The absence of salt in the selection media affected both dynamics and main molecular targets under selective pressure: a point mutation leading to the amino acid substitution N159H in the WaaP kinase responsible for heptose I phosphorylation in the LPS structure was also found. This mutation led to a phenotype with a decreased susceptibility to both the Bac71-22 and polymyxin B. Screening of antimicrobial activities with the use of a wide panel of known AMPs, including the human cathelicidin LL-37 and conventional antibiotics, against selected strains indicated no significant cross-resistance effects.

In Vitro Modulation of Complement Activation by Therapeutically Prospective Analogues of the Marine Polychaeta Arenicin Peptides.

The widespread resistance to antibiotics in pathogenic bacteria makes the development of a new generation of antimicrobials an urgent task. The development of new antibiotics must be accompanied by a comprehensive study of all of their biological activities in order to avoid adverse side-effects from their application. Some promising antibiotic prototypes derived from the structures of arenicins, antimicrobial peptides from the lugworm Arenicola marina, have been developed. Previously, we described the ability of natural arenicins -1 and -2 to modulate the human complement system activation in vitro. In this regard, it seems important to evaluate the effect of therapeutically promising arenicin analogues on complement activation. Here, we describe the complement-modulating activity of three such analogues, Ar-1[V8R], ALP1, and AA139. We found that the mode of action of Ar-1[V8R] and ALP1 on the complement was similar to that of natural arenicins, which can both activate and inhibit the complement, depending on the concentration. However, Ar-1[V8R] behaved predominantly as an inhibitor, showing only a moderate increase in C3a production in the alternative pathway model and no enhancement at all of the classical pathway of complement activation. In contrast, the action of ALP1 was characterized by a marked increase in the complement activation through the classical pathway in the concentration range of 2.5-20 µg/mL. At the same time, at higher concentrations (80-160 µg/mL), this peptide exhibited a complement inhibitory effect characteristic of the other arenicins. Peptide AA139, like other arenicins, exhibited an inhibitory effect on complement at a concentration of 160 µg/mL, but was much less pronounced. Overall, our results suggest that the effect on the complement system should be taken into account in the development of antibiotics based on arenicins.

Novel ß-Hairpin Peptide from Marine Polychaeta with a High Efficacy against Gram-Negative Pathogens.

In recent years, new antibiotics targeting multidrug resistant Gram-negative bacteria have become urgently needed. Therefore, antimicrobial peptides are considered to be a novel perspective class of antibacterial agents. In this study, a panel of novel BRICHOS-related ß-hairpin antimicrobial peptides were identified in transcriptomes of marine polychaeta species. Two of them-abarenicin from Abarenicola pacifica and UuBRI-21 from Urechis unicinctus-possess strong antibacterial potential in vitro against a wide panel of Gram-negative bacteria including drug-resistant strains. Mechanism of action assays demonstrate that peptides disrupt bacterial and mammalian membrane integrity. Considering the stronger antibacterial potential and a low ability of abarenicin to be bound by components of serum, this peptide was selected for further modification. We conducted an alanine and arginine scanning of abarenicin by replacing individual amino acids and modulating hydrophobicity so as to improve its antibacterial potency and membrane selectivity. This design approach allowed us to obtain the Ap9 analog displaying a high efficacy in vivo in the mice septicemia and neutropenic mice peritonitis models. We demonstrated that abarenicin analogs did not significantly induce bacterial resistance after a four-week selection experiment and acted on different steps of the biofilm formation: (a) killing bacteria at their planktonic stage and preventing biofilm formation and (b) degrading pre-formed biofilm and killing embedded bacteria. The potent antibacterial and antibiofilm activity of the abarenicin analog Ap9 with its high efficacy in vivo against Gram-negative infection in mice models makes this peptide an attractive candidate for further preclinical investigation.

A Novel Proline-Rich Cathelicidin from the Alpaca Vicugna pacos with Potency to Combat Antibiotic-Resistant Bacteria: Mechanism of Action and the Functional Role of the C-Terminal Region.

Over recent years, a growing number of bacterial species have become resistant to clinically relevant antibiotics. Proline-rich antimicrobial peptides (PrAMPs) having a potent antimicrobial activity and a negligible toxicity toward mammalian cells attract attention as new templates for the development of antibiotic drugs. Here, we mined genomes of all living Camelidae species and found a novel family of Bac7-like proline-rich cathelicidins which inhibited bacterial protein synthesis. The N-terminal region of a novel peptide from the alpaca Vicugna pacos named VicBac is responsible for inhibition of bacterial protein synthesis with an IC50 value of 0.5 µM in the E. coli cell-free system whereas the C-terminal region allows the peptide to penetrate bacterial membranes effectively. We also found that the full-length VicBac did not induce bacterial resistance after a two-week selection experiment, unlike the N-terminal truncated analog, which depended on the SbmA transport system. Both pro- and anti-inflammatory action of VicBac and its N-terminal truncated variant on various human cell types was found by multiplex immunoassay. The presence of the C-terminal tail in the natural VicBac does not provide for specific immune-modulatory effects in vitro but enhances the observed impact compared with the truncated analog. The pronounced antibacterial activity of VicBac, along with its moderate adverse effects on mammalian cells, make this molecule a promising scaffold for the development of peptide antibiotics.

Mechanism of Action and Therapeutic Potential of the ß-Hairpin Antimicrobial Peptide Capitellacin from the Marine Polychaeta Capitella teleta.

Among the most potent and proteolytically resistant antimicrobial peptides (AMPs) of animal origin are molecules forming a ß-hairpin structure stabilized by disulfide bonds. In this study, we investigated the mechanism of action and therapeutic potential of the ß-hairpin AMP from the marine polychaeta Capitella teleta, named capitellacin. The peptide exhibits a low cytotoxicity toward mammalian cells and a pronounced activity against a wide range of bacterial pathogens including multi-resistant bacteria, but the mechanism of its antibacterial action is still obscure. In view of this, we obtained analogs of capitellacin and tachyplesin-inspired chimeric variants to identify amino acid residues important for biological activities. A low hydrophobicity of the ß-turn region in capitellacin determines its modest membranotropic activity and slow membrane permeabilization. Electrochemical measurements in planar lipid bilayers mimicking the E. coli membrane were consistent with the detergent-like mechanism of action rather than with binding to a specific molecular target in the cell. The peptide did not induce bacterial resistance after a 21-day selection experiment, which also pointed at a membranotropic mechanism of action. We also found that capitellacin can both prevent E. coli biofilm formation and destroy preformed mature biofilms. The marked antibacterial and antibiofilm activity of capitellacin along with its moderate adverse effects on mammalian cells make this peptide a promising scaffold for the development of drugs for the treatment of chronic E. coli infections, in particular those caused by the formation of biofilms.

Dodecapeptide Cathelicidins of Cetartiodactyla: Structure, Mechanism of Antimicrobial Action, and Synergistic Interaction With Other Cathelicidins.

In this study, dodecapeptide cathelicidins were shown to be widespread antimicrobial peptides among the Cetruminantia clade. In particular, we investigated the dodecapeptide from the domestic goat Capra hircus, designated as ChDode and its unique ortholog from the sperm whale Physeter catodon (PcDode). ChDode contains two cysteine residues, while PcDode consists of two dodecapeptide building blocks and contains four cysteine residues. The recombinant analogs of the peptides were obtained by heterologous expression in Escherichia coli cells. The structures of the peptides were studied by circular dichroism (CD), FTIR, and NMR spectroscopy. It was demonstrated that PcDode adopts a ß-hairpin structure in water and resembles ß-hairpin antimicrobial peptides, while ChDode forms a ß-structural antiparallel covalent dimer, stabilized by two intermonomer disulfide bonds. Both peptides reveal a significant right-handed twist about 200 degrees per 8 residues. In DPC micelles ChDode forms flat ß-structural tetramers by antiparallel non-covalent association of the dimers. The tetramers incorporate into the micelles in transmembrane orientation. Incorporation into the micelles and dimerization significantly diminished the amplitude of backbone motions of ChDode at the picosecond-nanosecond timescale. When interacting with negatively charged membranes containing phosphatidylethanolamine (PE) and phosphatidylglycerol (PG), the ChDode peptide adopted similar oligomeric structure and was capable to form ion-conducting pores without membrane lysis. Despite modest antibacterial activity of ChDode, a considerable synergistic effect of this peptide in combination with another goat cathelicidin - the α-helical peptide ChMAP-28 was observed. This effect is based on an increase in permeability of bacterial membranes. In turn, this mechanism can lead to an increase in the efficiency of the combined action of the synergistic pair ChMAP-28 with the Pro-rich peptide mini-ChBac7.5Nα targeting the bacterial ribosome.

Antimicrobial Peptide Arenicin-1 Derivative Ar-1-(C/A) as Complement System Modulator.

Antimicrobial peptides (AMPs) are not only cytotoxic towards host pathogens or cancer cells but also are able to act as immunomodulators. It was shown that some human and non-human AMPs can interact with complement proteins and thereby modulate complement activity. Thus, AMPs could be considered as the base for complement-targeted therapeutics development. Arenicins from the sea polychaete Arenicola marina, the classical example of peptides with a ß-hairpin structure stabilized by a disulfide bond, were shown earlier to be among the most prospective regulators. Here, we investigate the link between arenicins' structure and their antimicrobial, hemolytic and complement-modulating activities using the derivative Ar-1-(C/A) without a disulfide bond. Despite the absence of this bond, the peptide retains all important functional activities and also appears less hemolytic in comparison with the natural forms. These findings could help to investigate new complement drugs for regulation using arenicin derivatives.

Structure Elucidation and Functional Studies of a Novel ß-hairpin Antimicrobial Peptide from the Marine Polychaeta Capitella teleta.

Endogenous antimicrobial peptides (AMPs) are evolutionary ancient molecular factors of innate immunity that play a key role in host defense. Among the most active and stable under physiological conditions AMPs are the peptides of animal origin that adopt a ß-hairpin conformation stabilized by disulfide bridges. In this study, a novel BRICHOS-domain related AMP from the marine polychaeta Capitella teleta, named capitellacin, was produced as the recombinant analogue and investigated. The mature capitellacin exhibits high homology with the known ß-hairpin AMP family-tachyplesins and polyphemusins from the horseshoe crabs. The ß-hairpin structure of the recombinant capitellacin was proved by CD and NMR spectroscopy. In aqueous solution the peptide exists as monomeric right-handed twisted ß-hairpin and its structure does not reveal significant amphipathicity. Moreover, the peptide retains this conformation in membrane environment and incorporates into lipid bilayer. Capitellacin exhibits a strong antimicrobial activity in vitro against a wide panel of bacteria including extensively drug-resistant strains. In contrast to other known ß-hairpin AMPs, this peptide acts apparently via non-lytic mechanism at concentrations inhibiting bacterial growth. The molecular mechanism of the peptide antimicrobial action does not seem to be related to the inhibition of bacterial translation therefore other molecular targets may be assumed. The reduced cytotoxicity against human cells and high antibacterial cell selectivity as compared to tachyplesin-1 make it an attractive candidate compound for an anti-infective drug design.

Caprine Bactenecins as Promising Tools for Developing New Antimicrobial and Antitumor Drugs.

Proline-rich antimicrobial peptides (PR-AMPs) having a potent antimicrobial activity predominantly toward Gram-negative bacteria and negligible toxicity toward host cells, are attracting attention as new templates for developing antibiotic drugs. We have previously isolated and characterized several bactenecins that are promising in this respect, from the leukocytes of the domestic goat Capra hircus: ChBac5, miniChBac7.5N-α, and -ß, as well as ChBac3.4. Unlike the others, ChBac3.4 shows a somewhat unusual pattern of activities for a mammalian PR-AMP: it is more active against bacterial membranes as well as tumor and, to the lesser extent, normal cells. Here we describe a SAR study of ChBac3.4 (RFRLPFRRPPIRIHPPPFYPPFRRFL-NH2) which elucidates its peculiarities and evaluates its potential as a lead for antimicrobial or anticancer drugs based on this peptide. A set of designed structural analogues of ChBac3.4 was explored for antibacterial activity toward drug-resistant clinical isolates and antitumor properties. The N-terminal region was found to be important for the antimicrobial action, but not responsible for the toxicity toward mammalian cells. A shortened variant with the best selectivity index toward bacteria demonstrated a pronounced synergy in combination with antibiotics against Gram-negative strains, albeit with a somewhat reduced ability to inhibit biofilm formation compared to native peptide. C-terminal amidation was examined for some analogues, which did not affect antimicrobial activity, but somewhat altered the cytotoxicity toward host cells. Interestingly, non-amidated peptides showed a slight delay in their impact on bacterial membrane integrity. Peptides with enhanced hydrophobicity showed increased toxicity, but in most cases their selectivity toward tumor cells also improved. While most analogues lacked hemolytic properties, a ChBac3.4 variant with two additional tryptophan residues demonstrated an appreciable activity toward human erythrocytes. The variant demonstrating the best tumor/nontumor cell selectivity was found to more actively initiate apoptosis in target cells, though its action was slower than that of the native ChBac3.4. Its antitumor effectiveness was successfully verified in vivo in a murine Ehrlich ascites carcinoma model. The obtained results demonstrate the potential of structural modification to manage caprine bactenecins' selectivity and activity spectrum and confirm that they are promising prototypes for antimicrobial and anticancer drugs design.

Redesigning Arenicin-1, an Antimicrobial Peptide from the Marine Polychaeta Arenicola marina, by Strand Rearrangement or Branching, Substitution of Specific Residues, and Backbone Linearization or Cyclization.

Arenicin-1, a ß-sheet antimicrobial peptide isolated from the marine polychaeta Arenicola marina coelomocytes, has a potent, broad-spectrum microbicidal activity and also shows significant toxicity towards mammalian cells. Several variants were rationally designed to elucidate the role of structural features such as cyclization, a certain symmetry of the residue arrangement, or the presence of specific residues in the sequence, in its membranolytic activity and the consequent effect on microbicidal efficacy and toxicity. The effect of variations on the structure was probed using molecular dynamics simulations, which indicated a significant stability of the ß-hairpin scaffold and showed that modifying residue symmetry and ß-strand arrangement affected both the twist and the kink present in the native structure. In vitro assays against a panel of Gram-negative and Gram-positive bacteria, including drug-resistant clinical isolates, showed that inversion of the residue arrangement improved the activity against Gram-negative strains but decreased it towards Gram-positive ones. Variants with increased symmetry were somewhat less active, whereas both backbone-cyclized and linear versions of the peptides, as well as variants with R→K and W→F replacement, showed antimicrobial activity comparable with that of the native peptide. All these variants permeabilized both the outer and the inner membranes of Escherichia coli, suggesting that a membranolytic mechanism of action was maintained. Our results indicate that the arenicin scaffold can support a considerable degree of variation while maintaining useful biological properties and can thus serve as a template for the elaboration of novel anti-infective agents.

Combined Antibacterial Effects of Goat Cathelicidins With Different Mechanisms of Action.

Being essential components of innate immune system, animal antimicrobial peptides (AMPs) also known as host-defense peptides came into sharp focus as possible alternatives to conventional antibiotics due to their high efficacy against a broad range of MDR pathogens and low rate of resistance development. Mammalian species can produce a set of co-localized AMPs with different structures and mechanisms of actions. Here we examined the combined antibacterial effects of cathelicidins, structurally diverse family of host-defense peptides found in vertebrate species. As a model we have used structurally distinct cathelicidins expressed in the leukocytes of goat Capra hircus. The recombinant analogs of natural peptides were obtained by heterologous expression in bacterial system and biological activities as well as the major mechanisms of antibacterial action of the peptides were investigated. As the result, the marked synergistic effect against wide panel of bacterial strains including extensively drug-resistant ones was observed for the pair of membranolytic α-helical amphipathic peptide ChMAP-28 and Pro-rich peptide mini-ChBac7.5Nα targeting a bacterial ribosome. ChMAP-28 was shown to damage the outer bacterial membrane at sub-inhibitory concentrations that could facilitate Pro-rich peptide translocation into the cell. Finally, resistance changes under a long-term continuous selective pressure of each individual peptide and the synergistic combination of both peptides were tested against Escherichia coli strains. The combination was shown to keep a high activity after the 26-days selection experiment in contrast to mini-ChBac7.5Nα used alone and the reference antibiotic polymyxin B. We identified the point mutation leading to amino acid substitution V102E in the membrane transport protein SbmA of the mini-ChBac7.5Nα-resistant strain obtained by selection. The experiments revealed that the presence of sub-inhibitory concentrations of ChMAP-28 restored the activity of mini-ChBac7.5Nα against this strain and clinical isolate with a weak sensitivity to mini-ChBac7.5Nα. The obtained results suggest a potential medical application of synergistic combinations of natural cathelicidins, which allows using a lower therapeutic dose and minimizes the risk of resistance development.

Cytotoxic Potential of the Novel Horseshoe Crab Peptide Polyphemusin III.

Biological activity of the new antimicrobial peptide polyphemusin III from the horseshoe crab Limulus polyphemus was examined against bacterial strains and human cancer, transformed, and normal cell cultures. Polyphemusin III has the amino acid sequence RRGCFRVCYRGFCFQRCR and is homologous to other ß-hairpin peptides from the horseshoe crab. Antimicrobial activity of the peptide was evaluated and MIC (minimal inhibitory concentration) values were determined. IC50 (half-maximal inhibitory concentration) values measured toward human cells revealed that polyphemusin III showed a potent cytotoxic activity at concentrations of <10 µM. Polyphemusin III caused fast permeabilization of the cytoplasmic membrane of human leukemia cells HL-60, which was measured with trypan blue exclusion assay and lactate dehydrogenase-release assay. Flow cytometry experiments for annexin V-FITC/ propidium iodide double staining revealed that the caspase inhibitor, Z-VAD-FMK, did not abrogate disruption of the plasma membrane by polyphemusin III. Our data suggest that polyphemusin III disrupts the plasma membrane integrity and induces cell death that is apparently not related to apoptosis. In comparison to known polyphemusins and tachyplesins, polyphemusin III demonstrates a similar or lower antimicrobial effect, but significantly higher cytotoxicity against human cancer and transformed cells in vitro.

Novel Antimicrobial Peptides from the Arctic Polychaeta Nicomache minor Provide New Molecular Insight into Biological Role of the BRICHOS Domain.

Endogenous antimicrobial peptides (AMPs) are among the earliest molecular factors in the evolution of animal innate immunity. In this study, novel AMPs named nicomicins were identified in the small marine polychaeta Nicomache minor in the Maldanidae family. Full-length mRNA sequences encoded 239-residue prepropeptides consisting of a putative signal sequence region, the BRICHOS domain within an acidic proregion, and 33-residue mature cationic peptides. Nicomicin-1 was expressed in the bacterial system, and its spatial structure was analyzed by circular dichroism and nuclear magnetic resonance spectroscopy. Nicomicins are unique among polychaeta AMPs scaffolds, combining an amphipathic N-terminal α-helix and C-terminal extended part with a six-residue loop stabilized by a disulfide bridge. This structural arrangement resembles the Rana-box motif observed in the α-helical host-defense peptides isolated from frog skin. Nicomicin-1 exhibited strong in vitro antimicrobial activity against Gram-positive bacteria at submicromolar concentrations. The main mechanism of nicomicin-1 action is based on membrane damage but not on the inhibition of bacterial translation. The peptide possessed cytotoxicity against cancer and normal adherent cells as well as toward human erythrocytes.

Comparative in vitro study on cytotoxicity of recombinant ß-hairpin peptides.

Natural antimicrobial peptides (AMPs) are important components of the innate immune system with a wide spectrum of biological activity. In this study, we investigated the cytotoxic effect of three recombinant ß-hairpin cationic AMPs: arenicin-1 from the polychaeta Arenicola marina, tachyplesin I from the horseshoe crab Tachypleus tridentatus, and gomesin from the spider Acanthoscurria gomesiana. All the three ß-hairpin AMPs were overexpressed in Escherichia coli. Different cell lines were incubated with various concentrations of the investigated AMPs in order to evaluate their cytotoxic activity. Double staining with subsequent flow cytometric analysis was used to determine the predominant way of cell death mediated by each AMP. Hemolytic activity of the peptides was tested against fresh human red blood cells. Our results indicated that all the three AMPs exhibited significant cytotoxic effect against cancer cells that varied depending on the cell line type and, in most cases, on the presence of serum components. Flow cytometric analysis implicitly indicated that tachyplesin I mostly promoted late apoptosis/necrosis, while arenicin-1 and gomesin induced early apoptosis under the same conditions. Tachyplesin I proved to be the most promising therapeutic candidate as it displayed the highest specific cytotoxicity against cancer cell lines, independent of the serum presence.

Anticancer Activity of the Goat Antimicrobial Peptide ChMAP-28.

Cytotoxic effect of the antimicrobial peptide ChMAP-28 from leucocytes of the goat Capra hircus was examined against five cancer and two normal human cell lines. ChMAP-28 has the amino acid sequence GRFKRFRKKLKRLWHKVGPFVGPILHY and is homologous to other α-helical mammalian antimicrobial peptides. ChMAP-28 shows considerably higher cytotoxicity against cultured tumor cells than toward normal cells at concentrations of <10 µM. Our findings suggest that ChMAP-28 can initiate necrotic death of cancer cells. Its cytotoxic effect is accomplished due to disruption of the plasma membrane integrity and is not abrogated by the addition of the caspase inhibitor Z-VAD-FMK. ChMAP-28 causes permeabilization of cytoplasmic membrane of human leukemia cells HL-60 already after 15 min of incubation. Here, we show that ChMAP-28 has one of the highest antitumor activity in vitro among all known antimicrobial peptides. We speculate that the observed specificity of ChMAP-28 cytotoxic effect against tumor cells is due to its relatively low hydrophobicity and high cationicity. In the meantime, this peptide has low hemolytic activity, which generates a potential for its use as a therapeutic agent.

Marine antimicrobial peptide arenicin adopts a monomeric twisted ß-hairpin structure and forms low conductivity pores in zwitterionic lipid bilayers.

Arenicins are 21-residue ß-hairpin antimicrobial peptides (AMPs) isolated from the marine lugworm Arenicola marina [Ovchinnikova et al., FEBS Lett. 2004;577:209-214]. The peptides have a high positive charge (+6) and display a broad spectrum of antimicrobial activities against bacteria and fungi. Arenicins adopt the monomeric highly twisted ß-hairpin in water or planar ß-structural dimers in anionic liposomes and detergent micelles. Until now, the interaction of cationic ß-structural AMPs with zwitterionic phospholipid bilayers mimicking eukaryotic membranes is not well understood. To study the structural basis of arenicins activity against eukaryotic cells, we investigated arenicin-2 in the solvents of low polarity (ethanol, 4% dioxane) and in zwitterionic soybean PC and PC/PE liposomes by CD and FTIR spectroscopy. It was shown that arenicin-2 adopted the twisted ß-hairpin structure in all the environments studied. Measurements of the Trp fluorescence and H→D exchange in soybean PC liposomes and boundary potential in the planar DPhPC bilayers confirmed the partitioning of the arenicin-2 monomers into interfacial region of the zwitterionic membranes. The low-conductivity (0.12 nS) arenicin-2 pores were detected in the DPhPC bilayers. The lifetime of the open state (up to 260 ms) was significantly longer than lifetime of low-conductivity (0.23 nS) pores previously described in partially anionic membranes (44 ms). The formation of narrow arenicin-2 pores without disruption of the membrane was discussed in the light of the disordered toroidal pore model previously proposed for ß-structural AMPs [Jean - Francois et al. Biophys. J. 2008;95:5748 - 5756]. A novel non-lytic mechanism of the arenicin-2 action was proposed.