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Background: The consistently increasing reports of bacterial resistance and the reemergence of bacterial epidemics have inspired the health and scientific community to discover new molecules with antibacterial potential continuously. Frog-skin secretions constitute bioactive compounds essential for finding new biopharmaceuticals. The exact antibacterial characterization of dermaseptin related peptides derived from Agalychnis annae, is limited. The resemblance in their conserved and functionally linked genomes indicates an unprecedented opportunity to obtain novel bioactive compounds. Objective: In this study, we derived a novel peptide sequence and determined its antibacterial potentials. Method: Consensus sequence strategy was used to design the novel and active antibacterial peptide named 'AGAAN' from skin secretions of Agalychnis annae. The In-vitro activities of the novel peptide against some bacterial strains were investigated. Time kill studies, DNA retardation, cytotoxicity, beta-galactosidase, and molecular computational studies were conducted. Results: AGAAN inhibited P. aeruginosa, E. faecalis, and S. typhimurium at 20 µM concentration. E. coli and S. aureus were inhibited at 25 µM, and lastly, B. subtilis at 50 µM. Kinetics of inactivation against exponential and stationary growing bacteria was found to be rapid within 1-5 hours of peptide exposure, depending on time and concentration. The peptide displayed weak hemolytic activity between 0.01%–7.31% at the antibacterial concentrations. AGAAN efficiently induced bacterial membrane damage with subsequent cell lysis. The peptide's DNA binding shows that it also targets intracellular DNA by retarding its movement. Our in-silico molecular docking analysis displayed a strong affinity to the bacterial cytoplasmic membrane. Conclusion: AGAAN exhibits potential antibacterial properties that could be used to combat bacterial resistance.

The current emergence of the COVID-19 virus has originated a global disaster due to the unavailability of any type of vaccine or drug which can be effective and determined to combat it. Naturally, various probabilities (such as herbal remedies with known medical importance) have been investigated by scientists. Research of systematic scientific (starting from silico research) of herbal medicine in specific and any type of drug in common is now possible that is building blocks (proteins) of COVID-19 which are already being identified. Mpro or 3CLpro is the main protease of the COVID-19 virus which have an important CoV enzyme and an attractive drug target because it plays a very important role in the viral replication and transcription. By using molecular docking scientist’s aims to explore bioactive chemicals which are found in medicinal plants such as potential COVID-19 Mpro inhibitors. Molecular docking was performed by Autodock 4.2, and the Lamarckian Genetic Algorithm, for analyzing the possibility of a dock. Although, further research is needed here to investigate their uses of potential drug. And the search for new drugs, especially through the use of antiviral peptides, is a very possible and protein-protein docking were performed using silico methods to identify, test, and evaluate cell interactions and interactions of dermaseptin peptide molecules produced by Phyllomedusa frogs against SARS-CoV-2 spike protein macromolecule, with its effect on the surface attachment of the ACE-2 (Angiotensin Converting Enzyme-2) receptor. Therefore, it is hoped that the dermaseptin-S9 peptide molecule may continue to be studied in the formulation of antibodies to anti-virus peptides that will be able to control COVID-19 infectious diseases.

Dermaseptins are peptides found in the skin secretions of Phyllomedusinae frogs. These peptides exert a lytic action on various microorganisms and have no considerable hemolytic effect except dermaseptin S4 (DS4) which exhibits a powerful cytotoxic effect. Therefore, we synthesized several analogs of DS4 in an attempt to find molecules with a weak hemolytic effect and significant bioactivities. In this study, we performed the synthesis of truncated peptides by introducing C-terminal and N-terminal amino acid deletions of the native sequence. All peptide analogs, in comparison with parental peptide, were tested firstly on human red blood cells to work out their cytotoxicity, secondly on the multidrug-resistant bacteria by trying to find MICs, and finally on colon cancer tumor cell line SW620 using the MTT test so as to investigate the anti-proliferative effect. Our results showed that, on the one hand, the N terminus of the native peptide was necessary for the antibacterial activity and the anti-proliferative effect of the peptide. On the other hand, the hemolytic activity was more notable in the sequences broken down on the C-terminal side.

Background: Rhabdomyosarcoma (RMS) is the most prevalent soft tissue sarcoma in children, representing approximately 50% of pediatric sarcomas and can develop in any part of the body though more frequently at the extremities. Aim: Evaluating the in vitro anti-proliferative activity of Dermaseptin B2 on Rhabdomyosarcoma RD (CCL-136TM) cells and its effect on the expression of MYC, FGFR1, NOTCH1, and CXCR7 genes involve in processes including proliferation, angiogenesis and metastasis. Methods: RD cells were grown in Dulbecco’s Modified Eagle’s Medium supplemented with 10% Fetal Bovine Serum. Exponentially growing cells were treated with Dermaseptin B2 and Antiproliferative activity was assayed using the resazurin and migration assays at three time-points. In order to determine the gene expression profiles of MYC, NOTCH1, FGFR1 and CXCR7, total RNA was extracted from the cells and q-RT-PCR was performed with β-Actin as reference gene. Results: Dermaseptin B2 inhibited the proliferation of RD cells in a time and concentration dependent manner as with IC50 values of 7.679 μM, 7.235 μM, 5.993 μM. The 2-dimentional wound healing assay showed inhibition of migration and motility of the RD cells at time-points of 6, 24, 48 and 72-hours with the greatest inhibition observed at 72-hours. Dermaseptin B2 downregulated the target MYC (fc; 1.5013, 1.5185, 2.4144), CXCR7 (fc; 2.8818, 4.4430, 3.9924), FGFR1 (fc; 2.3515, 2.0809, 2.2543), NOTCH1 (fc; 2.4667, 4.6274, 4.3352) genes for the three-time points respectively. NOTCH1 and CXCR7 showed higher fold changes with respect to β-Actin than MYC and FGFR1. Conclusion: The results of this study indicate that Dermaseptin B2 is a target molecule for signaling pathways including PI3K/AKT, RTK and NOTCH pathways that could affect the transcription of these genes and overall inhibition of cancer progression. Further studies are needed to give a better understanding of the detailed mechanisms of action as well as the effects of the Dermaseptin B2 peptide in vivo.

A pandemic coronavirus disease of 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has now been declared a global pandemic by the World Health Organization. The search for new drugs, especially by utilizing antiviral peptides is a very potential area. Through this study, protein-peptide docking and protein-protein docking simulations were conducted using in silico methods to identify, evaluate, and explore the molecular affinity and interaction of dermaseptin peptide molecules produced by frogs of the genus Phyllomedusa against the SARS-CoV-2 spike protein macromolecule, and its effect on attachment to the surface of the ACE-2 (Angiotensin Converting Enzyme-2) receptor. Protein-peptide docking simulation results show that dermaseptin-S9 peptide molecule has the best affinity to the active site of SARS-CoV-2 spike protein macromolecule binding site, with a binding free energy value of −792.93 kJ/mol. Then the results of protein-protein docking simulations proved that dermaseptin-S9 peptide molecule was able to prevent the attachment of SARS-CoV-2 spike protein to the surface of the ACE-2 receptor, with a total energy value of 517.85 kJ/mol. Therefore, it is hoped that dermaseptin-S9 peptide molecule can be further studied in the development of novel antiviral peptide candidates for the control of COVID-19 infectious disease.

The objectives of this study were to examine the physicochemical and structural properties of peptide derivatives of dermaseptin S4, investigate their detrimental effects on red blood and sperm cells and ascertain their antibacterial potency to control bacterial contaminants in fresh bovine semen. The dermaseptin S4 peptide derivatives used in this study were K4S4, S4(5-28), S4(5-28)a, K20S4(5-28), K4S4(1-16)a, K4S4(1-15)a and K4S4(1-15). Peptides K4S4, S4(5-28)a, K20S4(5-28), K4S4(1-15)a and K4S4(1-16)a, with a higher positive charge, were the most potent against the bacterial strains tested, with the lowest minimum inhibitory concentration (MIC), whereas S4(5-28) and K4S4(1-15), with a lower positive charge, showed the highest MIC (p < .01). Haemolysis percentage depended on peptide concentration (p < .01). The K4S4 was the most powerful haemolytic peptide, showing the highest haemolysis percentage at all peptide concentrations (p < .01). In contrast, S4(5-28), S4(5-28)a, K20S4(5-28) and K4S4(1-15) were not able to produce 50% cell lysis up to 100 µM (p < .01). All peptides reduced sperm motility in a dose-dependent manner when used in concentrations from 16 to 64 μM (p < .01). The highest reduction was seen due to K4S4 activity, and the lowest reductions of sperm motility were observed due to K4S4(1-16)a and K4S4(1-15)a activity (p < .01). Hence, we can conclude that K4S4(1-16)a and K4S4(1-15)a at a concentration of approximately 15 µM are the most promising peptides as antibacterial agents in fresh bovine semen, because at this concentration, they showed the most potent antibacterial activity against evaluated strains without significant effects on haemolysis or a reduction in sperm motility.

Modification of the poplar defense pathway through pathogen-induced expression of an amphibian host defense peptide modulates plant innate immunity and confers robust and reliable resistance against a major poplar pathogen, Septoria musiva. Host defense peptides (HDPs), also known as cationic antimicrobial peptides, represent a diverse group of small membrane-active molecules that are part of the innate defense system of their hosts against pathogen invasion. Here we describe a strategy for development of poplar plants with enhanced HDP production and resistance to the commercially significant fungal pathogen Septoria musiva. The naturally occurring linear amphipathic α-helical HDP dermaseptin B1, which has 31 residues and originated from the skin secretion of arboreal frogs, was N-terminally modified (MsrA2) and evaluated in vitro for antifungal activity and phytotoxicity. The MsrA2 peptide inhibited germination of S. musiva conidia at physiologically relevant low micromolar concentrations that were non-toxic to poplar protoplasts. The nucleotide sequence of MsrA2, optimized for expression in plants, was introduced into the commercial hybrid poplar Populus nigra L. × P. maximowiczii A. Henry (NM6) via Agrobacterium-mediated transformation. Transgene expression was regulated by the pathogen-inducible poplar promoter win3.12T, a part of the poplar innate defense system. Most importantly, the induced accumulation of MsrA2 peptide in poplar leaves was sufficient to confer resistance against S. musiva. The antifungal resistance of plants with high MsrA2 expression and MsrA2 accumulation was strong and reproducible, and without deleterious effects on plant growth and development. These results provide an insight into development of new technologies for engineering durable disease resistance against major pathogens of poplar and other plants.

Expression of strong antimicrobial peptides in plants is of great interest to combat a wide range of plant pathogens. To bring the Dermaseptin B1 (DrsB1) peptide to the intimate contact of the plant pathogens cell wall surface, the DrsB1 encoding sequence was fused to the C-terminal part of the two copies of the chitin-binding domain (CBD) of the Avr4 effector protein and used for Agrobacterium rhizogenes-mediated transformation. The expression of the recombinant protein in the tobacco hairy roots (HRs) was confirmed by molecular analysis. Antimicrobial activity analysis of the recombinant protein purified from the transgenic HRs showed that the (CBD)2-DrsB1 recombinant protein had a significant (p

To reduce pesticide use while preserving crop productivity, alternative pest and disease control measures are needed. We thought of an alternative way of functionalizing leaves of soybean to fight its most severe disease, Asian soybean rust (Phakopsora pachyrhizi). To do so, we produced bifunctional peptides that adhere to the soybean leaf surface and prevent the germination of P. pachyrhizi spores. In detail, amphiphilic peptides liquid chromatography peak I (LCI), thanatin (THA), tachystatin A2 (TA2), and lactoferricin B (LFB) were all fused to enhanced green fluorescent protein (eGFP). Of these fusion peptides, eGFP–LCI and eGFP–THA bound strongly and in a rainfast manner to the surface of soybean, barley, and corn leaves. eGFP–THA binding to soybean also withstood high temperature, sunlight and biotic degradation for at least 17 days. The dipeptides seem to bind mainly to the surface wax layer of leaves because eGFP–THA and eGFP–LCI did not stick to the wax-depleted cer-j59 mutant of barley or to corn leaves with their surface wax removed. A fusion of the antimicrobial peptide dermaseptin 01 and THA (DS01–THA) inhibits the germination of P. pachyrhizi spores in vitro and reduces Asian soybean rust disease in a rainfast manner. Therefore, this study reveals that bifunctional peptides can be used to functionalize the crop surface for sustainable disease management.

Lyotropic bicontinuous cubic (QII) phases have attracted increasing attention as nanostructured fluidic matrices for some bio-polymers including enzymes and DNA. Owing to the 3D nature of the QII phases, the incorporated polymers show unique molecular behavior in the matrices. In many cases, monoolein (MO) is frequently employed as a lipid molecule because of its advanced ability to form QII phases under a wide range of conditions. However, there are still several aspects that require improvements in MO/water-based systems. In the present study, we have focused on the improvement of the temporal stability of MO/water-based QII matrices by the addition of a zwitterion. We have found that (I) the addition of 3-(1-methyl-3-imidazolio)propanesulfonate (ImZI) results in a decrease in the water activity of the MO/water-based matrices; (II) ImZI is a kosmotropic salt with structure-stabilizing effects on proteins; (III) a polypeptide, dermaseptin, forms a secondary structure in the MO/water/ImZI systems. These results suggest that the present lyotropic systems have great potential to be an ideal matrix for the lipidic cubic phase method that is known as a method to create single crystals of membrane proteins.

International audience; Dermaseptin B2 (DRS-B2) is an antimicrobial peptide secreted by Phyllomedusa bicolor, which is an Amazonian tree frog. Here, we show that the adsorption of DRS-B2 on alginate nanoparticles (Alg NPs) results in a formulation (Alg NPs + DRS-B2) with a remarkable antibacterial activity against Escherichia coli ATCC 8739 and E. coli 184 strains, which are sensitive and resistant, respectively, to colistin. The antibacterial activity, obtained with this new formulation, is higher than that obtained with DRS-B2 alone. Of note, the addition of lactic acid or menthol to this new formulation augments its antibacterial activity against the aforementioned Gram-negative bacilli. The safety of DRS-B2, and also that of the new formulation supplemented or not with a small molecule such as lactic acid or menthol has been proven on the human erythrocytes and the eukaryotic cell line types HT29 (human) and IPEC-1 (animal). Similarly, their stability was determined under the conditions mimicking the gastrointestinal tract with different conditions: pH, temperature, and the presence of digestive enzymes. Based on all the obtained data, we assume that these new formulations are promising and could be suggested, after in vivo approval and completing regulation aspects, as alternatives to antibiotics to fight infections caused by Gram-negative bacilli such as E. coli.

Antibiotic resistance represents a tremendous contemporary clinical challenge. Given this challenge, antimicrobial peptides (AMPs) are regarded as one of the most promising new options for next-generation lead antibiotics. Here, we describe the antibacterial activities of a cationic peptide named DRP-AC4, obtained from frog skin secretion using shotgun cloning. Two modified peptides were derived by substituting the sequence of amino acids to complete the hydrophobic face (DRP-AC4b) and increase net charge (DRP-AC4a), respectively. The activity and cytotoxicity of these two peptides were compared. DRP-AC4a displayed significantly increased potency against bacteria compared to the natural peptide. It should be noted, however, that both analogue peptides demonstrated higher lytic ability than the natural peptide against the membranes of mammalian erythrocytes. At the same time, all three peptides displayed lower hemolytic activity compared to their antibacterial activity. Here, we demonstrate that AMPs have more complex activity mechanisms and faster bactericidal rates than traditional antibiotics, which may be one of the reasons why bacteria do not develop resistance to them. These discoveries provide interesting insights into the discovery and development of novel drugs from natural sources.

Antimicrobial peptides (AMP) secreted by the granular glands of frog skin have been widely reported to exhibit strong bacteriostatic and bactericidal activities. Many of them have been documented with potent antiproliferative effects on multiple cancer cells, many studies also suggested that AMPs exert their functions via disrupting cell membranes. However, whether and how other cell death induction mechanism is involved in mammalian cancer cells has rarely been investigated. In this study, a novel AMP named Dermaseptin‐PS1 was isolated and identified from Phyllomedusa sauvagei, it showed strong antimicrobial activities against three types of microorganisms. In vitro antiproliferative studies on human glioblastoma U‐251 MG cells indicated that Dermaseptin‐PS1 disrupted cell membranes at the concentrations of 10−5 M and above, while the cell membrane integrity was not affected when concentrations were decreased to 10−6 M or lower. Further examinations revealed that, at the relatively low concentration (10−6 M), Dermaseptin‐PS1 induced apoptosis through mitochondrial‐related signal pathway in U‐251 MG cells. Thus, for the first time, we report a novel frog skin derived AMP with anticancer property by distinct mechanisms, which largely depends on its concentration. Together, our study provides new insights into the mechanism‐illustrated drug design and the optimisation of dose control for cancer treatment in clinic.

Las secreciones de la piel de Agalychnis spurrelli han probado tener una marcada actividad antimicrobiana sobre diferentes microrganismos patógenos por la presencia de biomoléculas en ellas. Se realizaron análisis moleculares del ARN mensajero de la piel de A. spurrelli para determinar el tipo de péptido antimicrobiano presente en las secreciones cutáneas de esta especie. Para amplificar las secuencias precursoras de los péptidos maduros, se utilizaron iniciadores específicos que contienen secuencias altamente conservadas. Como resultado se obtuvo una secuencia de ADN complementario de 357 pb, la cual fue comparada con sus ortólogos de otras especies de la misma subfamilia Phyllomedusinae, se lograron índices de identidad muy altos para precursores de dermaseptinas. Finalmente, para el análisis de la secuencia de ADN complementario, se tradujo la secuencia nucleotídica por codones, con lo que se obtuvo una secuencia aminoacídica. Dicha secuencia posee las características particulares de péptidos de la familia de las dermaseptinas: una secuencia altamente conservada, una propieza acídica con los aminoácidos Lisina y Arginina y el extremo C-terminal variable. En conclusión, la secreción total de Agalychnis spurrelli contiene un péptido de la familia de las dermaseptinas o un péptido relacionado con ellas.

Severe infections associated with antibiotic-resistant bacteria and biofilms have attracted increasing interest as these diseases are difficult to treat with current antibiotics. Typical cationic antimicrobial peptides dermaseptins are considered to be the most promising next-generation antibiotics because of their broad-spectrum antimicrobial activities and minor side effects. Two new dermaseptin peptides, DMS-PS1 and DMS-PS2, have been identified by "shotgun" molecular cloning of encoding cDNAs in the crude skin secretions of the waxy monkey tree frog, Phyllomedusa sauvagei. The mature peptide sequences predicted from the cloned cDNAs were separated from crude skin secretions and confirmed by mass spectrometry. Chemically synthetic replicates were assessed for various biological activities. Both dermaseptins were potently effective against a broad spectrum of microorganisms including antibiotic-resistant bacteria and displayed significant potency against gram-positive and gram-negative bacterial biofilms with low toxicity towards mammalian red blood cells. Remarkably, DMS-PS2 was effective against infections in murine skin caused by methicillin-resistant Staphylococcus aureus as a result of an induced wound. The actions of DMS-PS2 were with a membrane permeabilization mode. Overall, the data provided convincing evidence for the development of anti-infectious agents and/or biomaterials as a new therapeutic approach against bacterial infections. STATEMENT OF SIGNIFICANCE: Bacterial adhesion to biomaterials remains a major problem. Antimicrobial peptides (AMPs) are well-known components of the innate immune system that can be applied to overcome biofilm-associated infections. Cationic dermaseptin peptides showed significant broad-spectrum antimicrobial activities and activities against bacterial biofilms of persistent infections in association with weak toxicity for mammalian red blood cells. The membrane permeabilizing ability of DMS-PS2 was confirmed, and importantly, it demonstrated potent efficiency of the treatment of MRSA infected murine skin model. Furthermore, beyond our expectation, DMS-PS2 showed a self-aggregating parameter, indicating a promising potential for the use of immobilized AMPs in clinical applications., which makes it also a promising suggestion for infection-proof biomaterial development.

A novel dermaseptin peptide, dermaseptin-PT9 (DPT9), was isolated and identified from Phyllomedusa tarsius by the combination of molecular cloning and LC-MS analysis. Chemically synthesised DPT9 was broadly effective against the tested microorganisms through the disruption of cell membranes and showed weak haemolytic activity towards horse erythrocytes. It also exhibited anti-proliferative effect against various human cancer cells. Moreover, an analogue with enhanced cationicity, K8, 23-DPT9, in which Asp8 and Glu23 were substituted by lysine residues, had a markedly increased antimicrobial effect against all tested microorganisms and disrupted microbial cell membranes. This analogue also showed no haemolysis at its effective antimicrobial concentrations. In addition, K8, 23-DPT9 displayed an enhanced anti-proliferative effect against cancer cells, while displayed weak activity against the normal human cell line, HMEC-1.

In this research, we present a preliminary computational study of four Dermaseptin-related peptides from the skin exudate of the gliding tree frog Agalychnis spurrelli. Experimentally, the amino acid sequence of these peptides was elucidated through molecular cloning and tandem mass spectrometry and synthetic peptides were assayed against E. coli, S. aureus, and C. albicans to determine their antimicrobial properties. With the sequences on hand, a computational study of the structures was carried out, obtaining their physicochemical properties, secondary structure, and their similarity to other known peptides. A molecular docking study of these peptides was also performed against cell membrane and several enzymes are known to be vital for the organisms. Results showed that Dermaseptin-related peptides are α-helical cationic peptides with an isoelectric point above 9.70 and a positive charge of physiological pH. Introducing theses peptides in a database, it was determined that their identity compared with known peptides range from 36 to 82% meaning these four Dermaseptins are novel peptides. This preliminary study of molecular docking suggests the mechanism of action of this peptide is not given by the inhibition of essential enzymatic pathways, but by cell lysis. Graphical abstract.

Antimicrobial peptides (AMPs) act either through membrane lysis or by attacking intracellular targets. Intracellular targeting AMPs are a resource for antimicrobial agent development. Several AMPs have been identified as intracellular targeting peptides; however, the intracellular targets of many of these peptides remain unknown. In the present study, we used an Escherichia coli proteome microarray to systematically identify the protein targets of three intracellular targeting AMPs: bactenecin 7 (Bac7), a hybrid of pleurocidin and dermaseptin (P-Der), and proline-arginine-rich peptide (PR-39). In addition, we also included the data of lactoferricin B (LfcinB) from our previous study for a more comprehensive analysis. We analyzed the unique protein hits of each AMP in the Kyoto Encyclopedia of Genes and Genomes. The results indicated that Bac7 targets purine metabolism and histidine kinase, LfcinB attacks the transcription-related activities and several cellular carbohydrate biosynthetic processes, P-Der affects several catabolic processes of small molecules, and PR-39 preferentially recognizes proteins involved in RNA- and folate-metabolism-related cellular processes. Moreover, both Bac7 and LfcinB target purine metabolism, whereas LfcinB and PR-39 target lipopolysaccharide biosynthesis. This suggested that LfcinB and Bac7 as well as LfcinB and PR-39 have a synergistic effect on antimicrobial activity, which was validated through antimicrobial assays. Furthermore, common hits of all four AMPs indicated that all of them target arginine decarboxylase, which is a crucial enzyme for Escherichia coli survival in extremely acidic environments. Thus, these AMPs may display greater inhibition to bacterial growth in extremely acidic environments. We have also confirmed this finding in bacterial growth inhibition assays. In conclusion, this comprehensive identification and systematic analysis of intracellular targeting AMPs reveals crucial insights into the intracellular mechanisms of the action of AMPs.

Bioactive peptides are active against the malaria parasite growth through targeting various stages of life cycle, but their mechanism of action is not fully understood. PFI1625c, a metalloprotease present in Plasmodium falciparum, is a potential drug target, and it shows best binding to peptide P550 (LVIVAKRA). Out of 20 selected antimalarial peptides, 4 peptides namely Dermaseptin S3, Cyclosporin A, Angiotensin II, and PcFK2 show high affinity with PFI1625c. Dermaseptin S3 is making polar contacts and hydrophobic interactions with the residues present within the catalytic site of PFI1625c. The affinity of bioactive peptides toward PFI1625c and anti-malarial activity of peptide shows a strong correlation (R = 0.921), indicating PFI1625c as a potential target behind their antimalarial activity. Thus, current study confirms the involvement of PFI1625c behind the antimalarial activity of tested bioactive peptides, and the result can be helpful to design better bio-compatible antimalarial drugs.

Several amyloid-forming and antimicrobial peptides (AMYs and AMPs) have the ability to bind to and damage cell membranes. In addition, some AMYs possess antimicrobial activity and some AMPs form amyloid-like fibrils, relating the two peptide types and their properties. However, a comparison of their sequence characteristics reveals important differences. The high β-strand and aggregation propensities typical of AMYs are largely absent in α-helix-forming AMPs, which are instead marked by a strong amphipathic moment not generally found in AMYs. Although a few peptides, for example, islet amyloid polypeptide and dermaseptin S9, combine some determinants of both groups, the structural distinctions suggest that antimicrobial activity and amyloid formation are separate features not generally associated.

The rising number of infections caused by biofilm formation and the difficulties associated with their treatment by conventional antimicrobial therapies have led to an intensive search for novel antibiofilm agents. Dermaseptins are antimicrobial peptides with a number of attractive properties that might offer alternative therapies against resistant microorganisms. In this study, we synthesized a set of dermaseptin-derived peptides and evaluated their activities against Gram-positive and Gram-negative bacterial biofilm formation. All dermaseptin-derived peptides demonstrated concentration-dependent antibiofilm activities at microgram concentrations, and their activities were dependent on the nature of the peptides, with the highest levels of activity being exhibited by highly charged molecules. Fluorescent binding and confocal microscopy demonstrated that dermaseptin K 4 S 4 , a substituted derivative of the native molecule S 4 , significantly decreased the viability of planktonic and surface-attached bacteria and stopped biofilm formation under dynamic flow conditions. Cytotoxicity assays with HeLa cells showed that some of the tested peptides were less cytotoxic than current antibiotics. Overall, these findings indicate that dermaseptin derivatives might constitute new lead structures for the development of potent antibiofilm agents.

The present study aimed to entrap and characterize the morphology and antitumor effects of a dermaseptin (DStomo01) peptide in chitosan nanoparticles, in vitro. DStomo01 nanoparticles showed moderate polydispersivity, excellent colloidal stability, and slow release. It was noted that free DStomo01 induced DNA fragmentation and mitochondrial hyperpolarization in HeLa cells. However, when entrapped in chitosan nanoparticles, DStomo01 was slightly more active against HeLa cells than the free peptide. In conclusion, the present sustained release system was efficient in entrapping the peptide and reducing tumor cell viability, which are promising steps for future studies involving specific targeting of nanoparticles and in-vivo treatments.

Antimicrobial peptides (AMPs) have recently attracted great attention due to their rapid action, broad spectrum of activity, and low propensity of resistance development. The successful application of AMPs in the treatment of intracellular infections, however, remains a challenge because of their low penetration efficiency into the pathogen's intracellular niche. Herein, we report that sub-micrometer-sized crystals of the protein Cry3Aa formed within Bacillus thuringiensis are readily and specifically taken up by macrophages. We demonstrate that these protein crystals efficiently encapsulate a known antileishmanial peptide, dermaseptin S1 (DS1), and thereby promote improved cellular uptake of DS1 and its lysosomal accumulation in macrophages. Notably, this targeted delivery of DS1 results in enhanced in vitro and in vivo antileishmanial activity, as well as reduced toxicity to the host macrophages. These findings suggest that the Cry3Aa crystal can be an effective delivery platform for AMPs to treat intramacrophage infections.

Biadhesive peptides (peptesives) are an attractive tool for assembling two chemically different materials-for example, stainless steel and polycaprolactone (PCL). Stainless steel is used in medical stents and PCL is used as a biodegradable polymer for fabrication of tissue growth scaffolds and drug delivering micro-containers. Biadhesive peptides are composed of two domains (e.g., dermaseptin S1 and LCI) with different material-binding properties that are separated through a stiff peptide-spacer. The peptesive dermaseptin S1-domain Z-LCI immobilizes antibiotic-loaded PCL micro-containers on stainless steel surfaces. Immobilization is visualized by microscopy and field emission scanning electron microscopy analysis and released antibiotic from the micro-containers is confirmed through growth inhibition of Escherichia coli cells.

Antimicrobial peptides (AMPs) have emerged as a promising class of antimicrobial agents that could potentially address the global antibiotic resistance. Generating mirror-like peptides by minimizing dermaseptin family sequences is an effective strategy for designing AMPs. However, the previous research still had some limitations such as lower effectiveness and a narrow spectrum of antibacterial activity. To further expand and hone this strategy, we designed a series of AMPs consisting of the WXMXW-NH2 motif (X represents V, I, F, and W, M represents KAAAKAAAK). The peptides formed &alpha, helices and displayed broad-spectrum antimicrobial activities against eleven types of clinical bacteria including both Gram-negative and Gram-positive bacteria. The optimized peptide WW exhibited high physical rupture by inducing membrane shrinkage, disruption, and lysis. Moreover, WW effectively neutralized endotoxins and inhibited the inflammatory response while having the highest therapeutic index. In conclusion, these results indicated that the peptide WW has potential as a broad-spectrum antimicrobial agent or preservative for overcoming the risk of multidrug resistance in localized or external therapeutic applications.

Antimicrobial peptides produced by multicellular organisms as part of their innate system of defense against microorganisms are currently considered potential alternatives to conventional antibiotics in case of infection by multiresistant bacteria. However, while the mode of action of antimicrobial peptides is relatively well described, resistance mechanisms potentially induced or selected by these peptides are still poorly understood. In this work, we studied the mechanisms of action and resistance potentially induced by ApoEdpL-W, a new antimicrobial peptide derived from human apolipoprotein E. Investigation of the genetic response of Escherichia coli upon exposure to sublethal concentrations of ApoEdpL-W revealed that this antimicrobial peptide triggers activation of RcsCDB, CpxAR, and σ E envelope stress pathways. This genetic response is not restricted to ApoEdpL-W, since several other antimicrobial peptides, including polymyxin B, melittin, LL-37, and modified S 4 dermaseptin, also activate several E. coli envelope stress pathways. Finally, we demonstrate that induction of the CpxAR two-component system directly contributes to E. coli tolerance toward ApoEdpL-W, polymyxin B, and melittin. These results therefore show that E. coli senses and responds to different antimicrobial peptides by activation of the CpxAR pathway. While this study further extends the understanding of the array of peptide-induced stress signaling systems, it also provides insight into the contribution of Cpx envelope stress pathway to E. coli tolerance to antimicrobial peptides.

Chlamydia trachomatis is an obligate intracellular bacterium responsible for a number of health problems, including sexually transmitted infection in humans. Efforts were made for the search of alternative therapies. Accordingly, the present study was undertaken to perform a systemic in vitro investigation on the anti-chlamydial potential of cationic peptides from frog’s skin, namely dermaseptin S4 (S4) and its derivatives. Several strains of Chlamydia trachomatis serovar E were used to detect the antimicrobial activity of the new compounds. The infections tests and the toxic effects of the new compounds were determined using McCoy cells monolayers. Our data show that S4 exhibited a potent anti-chlamydial activity and found that these peptides blocked infection of McCoy cells and reduced the numbers of inclusion-forming units (81 %) after 48 h at low concentration (5 μg/ml). Besides, the finding revealed that increasing the number of positive charges of the peptide resulted in a reduced cytotoxicity without affecting the antimicrobial effect. Among all peptides, the derivative K4K20S4 was the more potent to inhibit C. trachomatis growth with 96 % reduction in the number of chlamydial inclusions compared with an untreated control infection and, therefore, can be considered as potential agents for therapy of Chlamydia infectious diseases.

Sexually transmitted infections (STIs) and unplanned pregnancies have serious effects on the reproductive health of women. The present study was undertaken with a view to develop an effective means of prevention. The microbicidal and contraceptive potential of cationic peptides from frog's skin, namely, dermaseptin S4 and its derivatives were investigated in vitro.: STUDY DESIGN Different bacterial and fungal strains were resorted to for determining the antimicrobial activity of the new compounds. The spermicidal activities of the latter were assessed using normal human semen samples, and their toxic effects were identified in a HeLa culture.All S4 derivatives elicited concentration-dependent spermicidal and antimicrobial activities at microgram concentrations. The highest levels recorded for both types of activity were displayed by K4K20S4, and the lowest levels were exhibited by D4D20S4 and S4(5-28). Cytotoxicity assays revealed that some of these compounds were significantly safer than nonoxynol-9 (N-9).The ability of these peptides to instantaneously kill human sperm and STI pathogens at low concentrations indicates that their application as active ingredients in vaginal contraceptive preparations could induce considerably better effects than N-9.

Schistosomiasis is one of the world's major public health problems, and praziquantel is the only available drug to treat this notable neglected disease. Drug combinations have been considered an important strategy for treatment of infectious diseases, which might enhance therapeutic efficacy and delaying resistance. In this study, we have examined the in vitro activities of the amide piplartine and the antimicrobial peptide dermaseptin 01 administered singly or in combination against Schistosoma mansoni of different ages including 3-hour-old and 7-day-old schistosomula and 49-day-old adult schistosomes as well as on egg output by adult worms. We calculated the median lethal concentrations (LC(50)) of 7.87 and 17.99 μM on 49-day-old adults, 11.02 and 71.58 μM on 7-day-old schistosomula, and 70.87 and 98.42 μM on 3-hour-old schistosomula for piplartine and dermaseptin, respectively. Most Piplartine/dermaseptin combinations showed synergistic effect, with combination index (CI) values less than 0.9 when S. mansoni adults or schistosomula were simultaneously incubated with both drugs in vitro; synergy between these two compounds was also indicated using isobolograms. Additionally, we observed alterations on the tegumental surface of schistosomula and adult schistosomes by means of laser scanning confocal microscopy. Furthermore, egg laying of surviving worms was considerably more reduced when exposed to the piplartine/dermaseptin combinations than each drug alone, and this inhibition was irreversible. This is the first report on the synergistic effect between piplartine and dermaseptin against S. mansoni and opens the route to further studies (e.g. in vivo) to characterize this combination in greater detail.

To enable selection and characterization of highly potent pore-forming peptides, we developed a set of novel assays to probe 1) the potency of peptide pores at very low peptide concentration; 2) the presence or absence of pores in membranes after equilibration; 3) the interbilayer exchangeability of pore-forming peptides; and 4) the degree to which pore-forming peptides disrupt the bilayer organization at equilibrium. Here, we use these assays to characterize, in parallel, six membrane-permeabilizing peptides belonging to multiple classes. We tested the antimicrobial peptides LL37 and dermaseptin S1, the well-known natural lytic peptides melittin and alamethicin, and the very potent lentivirus lytic peptides LLP1 and LLP2 from the cytoplasmic domain of HIV GP41. The assays verified that that the antimicrobial peptides are not potent pore formers, and form only transient permeabilization pathways in bilayers which are not detectable at equilibrium. The other peptides are far more potent and form pores that are still detectable in vesicles after many hours. Among the peptides studies, alamethicin is unique in that it is very potent, readily exchanges between vesicles, and disturbs the local bilayer structure even at very low concentration. The equally potent LLP peptides do not exchange readily and do not perturb the bilayer at equilibrium. Comparison of these classes of pore forming peptides in parallel using the set of assays we developed demonstrates our ability to detect differences in their mechanism of action. Importantly, these assays will be very useful in high-throughput screening where highly potent pore-forming peptides can be selected based on their mechanism of action.

Solanum tuberosum plants were transformed with three genetic constructions expressing the Nicotiana tabacum AP24 osmotine, Phyllomedusa sauvagii dermaseptin and Gallus gallus lysozyme, and with a double-transgene construction expressing the AP24 and lysozyme sequences. Re-transformation of dermaseptin-transformed plants with the AP24/lysozyme construction allowed selection of plants simultaneously expressing the three transgenes. Potato lines expressing individual transgenes or double- and triple-transgene combinations were assayed for resistance to Erwinia carotovora using whole-plant and tuber infection assays. Resistance levels for both infection tests compared consistently for most potato lines and allowed selection of highly resistant phenotypes. Higher resistance levels were found in lines carrying the dermaseptin and lysozyme sequences, indicating that theses proteins are the major contributors to antibacterial activity. Similar results were obtained in tuber infection tests conducted with Streptomyces scabies. Plant lines showing the higher resistance to bacterial infections were challenged with Phytophthora infestans, Rhizoctonia solani and Fusarium solani. Considerable levels of resistance to each of these pathogens were evidenced employing semi-quantitative tests based in detached-leaf inoculation, fungal growth inhibition and in vitro plant inoculation. On the basis of these results, we propose that stacking of these transgenes is a promising approach to achieve resistance to both bacterial and fungal pathogens. Fil: Rivero, Mercedes. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; Argentina Fil: Furman, Nicolas. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; Argentina Fil: Mencacci, Nicolas. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; Argentina Fil: Picca, Pablo Ignacio. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Biodiversidad y Biología Experimental. Laboratorio del Grupo de Estudio de Plantas Vasculares; Argentina Fil: Toum, Laila. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; Argentina Fil: Lentz, Ezequiel Matias. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Bravo Almonacid, Fernando Felix. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Mentaberry, Alejandro Nestor. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; Argentina

This article addresses the interactions of the synthetic antimicrobial peptide dermaseptin 01 (GLWSTIKQKGKEAAIAAA- KAAGQAALGAL-NH2, DS 01) with phospholipid (PL) monolayers comprising (i) a lipid-rich extract of Leishmania amazonensis (LRE-La), (ii) zwitterionic PL (dipalmitoylphosphatidylcholine, DPPC), and (iii) negatively charged PL (dipalmitoylphosphatidylglycerol, DPPG). The degree of interaction of DS 01 with the different biomembrane models was quantified from equilibrium and dynamic liquid-air interface parameters. At low peptide concentrations, interactions between DS 01 and zwitterionic PL, as well as with the LRE-La monolayers were very weak, whereas with negatively charged PLs the interactions were stronger. For peptide concentrations above 1 µg/ml, a considerable expansion of negatively charged monolayers occurred. In the case of DPPC, it was possible to return to the original lipid area in the condensed phase, suggesting that the peptide was expelled from the monolayer. However, in the case of DPPG, the average area per lipid molecule in the presence of DS 01 was higher than pure PLs even at high surface pressures, suggesting that at least part of DS 01 remained incorporated in the monolayer. For the LRE-La monolayers, DS 01 also remained in the monolayer. This is the first report on the antiparasitic activity of AMPs using Langmuir monolayers of a natural lipid extract from L. amazonensis. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.

Different species of Leishmania are responsible for cutaneous, mucocutaneous or visceral leishmaniasis infections in millions of people around the world [14]. The adverse reactions caused by antileishmanial drugs, emergence of resistance and lack of a vaccine have motivated the search for new therapeutic options to control this disease. Different sources of antimicrobial molecules are under study as antileishmanial agents, including peptides with antimicrobial and/or immunomodulatory activity, which have been considered to be potentially active against diverse species of Leishmania[7,39]. This study evaluated the cytotoxicity on dendritic cells, hemolytic activity, leishmanicidal properties on Leishmania panamensis and Leishmania major promastigotes and effectiveness on parasite intracellular forms (dendritic cells infected with L. panamensis and L. major promastigotes), when each parasite in culture was exposed to different concentrations of a group of synthetic peptides with previously reported antimicrobial properties, which were synthesized based on their naturally occurring reported sequences. Dermaseptin, Pr-2 and Pr-3 showed inhibitory activity on the growth of L. panamensis promastigotes, while Andropin and Cecropin A (with a selectivity index of 4 and 40, respectively) showed specific activity against intracellular forms of this species. The activities of Andropin and Cecropin A were exclusively against the intracellular forms of the parasite, therefore indicating the relevance of these two peptides as potential antileishmanial agents. In the case of L. major promastigotes, Melittin and Dermaseptin showed inhibitory activity, the latter also showed a selectivity index of 8 against intracellular forms. These findings suggest Andropin, Cecropin A and Dermaseptin as potential therapeutic tools to treat New and Old World cutaneous leishmaniasis.

Immobilized metal ion affinity chromatography (IMAC) has been widely used for the enrichment of phosphopeptides, whereas no report exists describing the use of IMAC columns for the enrichment of sulfopeptides. In this study, we used IMAC-Ga microcolumns for the enrichment of sulfopeptides from a complex mixture of peptides, extracted from skin secretions of the Pachymedusa dacnicolor frog. The enriched fraction obtained by IMAC-Ga was analyzed by liquid chromatograpy/electrospray ionization mass spectrometry (LC/ESI-MS) in an Orbitrap XL and by matrix-assisted laser desorption/ionization time-of-flight time-of-flight (MALDI-TOF/TOF) in an ABI 4800 instrument. From this fraction, different sulfated and non-sulfated peptides belonging to the caerulin and bradykinin families were structurally characterized. Other interesting negatively charged groups, such as phosphate adducts of dermaseptins and pyridoxal phosphate attached to a protease inhibitor, were also characterized. Unexpectedly, some dermaseptin antimicrobial peptides were also enriched by IMAC-Ga and a Sauvatine-like peptide was also fully sequenced. Furthermore, neutral loss of sulfated peptides and their fragmentation patterns in the gas phase were also compared using collision-induced dissociation (CID) and high-energy collision dissociation (HCD). Our present study provides evidence that IMAC-Ga enrichment is a fast, useful and promising method for high-throughput analysis of sulfated-peptides, since high-resolution mass spectrometers can be used for this purpose. Copyright © 2011 John Wiley & Sons, Ltd.

Savoia D, Donalisio M, Civra A, Salvadori S, Guerrini R. In vitro activity of dermaseptin S1 derivatives against genital pathogens. APMIS 2010; 118: 674–80. The aim of this study was to evaluate the biological activity of nine dermaseptin-S1 (DRS-S1) derivatives (synthesized by solid-phase methods and purified) against different pathogens causing genital infections (Trichomonas vaginalis, Herpes simplex virus, Papillomavirus). The in vitro activity on T. vaginalis was determined by counting the protozoon in a hemocytometer after vital staining with trypan blue; antiviral activity of the compounds was tested on monolayers of Vero cells for Herpes simplex virus-1 (GFP) and on 293TT cells for human papillomavirus (HPV-16) pseudovirions (GFP). The cytotoxicity of the derivatives was assessed by evaluating both the hemolytic activity and the effect on Vero and 293TT cells. The DRS-S1 longer peptides demonstrated a superior activity on T. vaginalis but also a certain cytopathic effect. The compounds with 29 amino acids exhibited activity against the two viruses tested at concentrations not toxic to cells. The results obtained show that some of the synthetic peptides assessed have inhibitory activity against the pathogens tested, indicating a potential for the development of new molecules for use as topical microbicides to prevent the sexual transmission of microorganisms.

Screening for new bioactive peptides in South American anurans has been pioneered in frogs of the genus Phyllomedusa. All frogs of this genus have venomous skin secretions, i.e., a complex mixture of bioactive peptides against potential predators and pathogens that presumably evolved in a scenario of predator-prey interaction and defense against microbial invasion. For every new anuran species studied new peptides are found, with homologies to hormones, neurotransmitters, antimicrobials, and several other peptides with unknown biological activity. From Vittorio Erspamer findings, this genus has been reported as a "treasure store" of bioactive peptides, and several groups focus their research on these species. From 1966 to 2009, more than 200 peptide sequences from different Phyllomedusa species were deposited in UniProt and other databases. During the last decade, the emergence of high-throughput molecular technologies involving de novo peptide sequencing via tandem mass spectrometry, cDNA cloning, pharmacological screening, and surface plasmon resonance applied to peptide discovery, led to fast structural data acquisition and the generation of peptide molecular libraries. Research groups on bioactive peptides in Brazil using these new technologies, accounted for the exponential increase of new molecules described in the last decade, much higher than in any previous decades. Recently, these secretions were also reported as a rich source of multiple antimicrobial peptides effective against multidrug resistant strains of bacteria, fungi, protozoa, and virus, providing instructive lessons for the development of new and more efficient nanotechnological-based therapies for infectious diseases treatment. Therefore, novel drugs arising from the identification and analysis of bioactive peptides from South American anuran biodiversity have a promising future role on nanobiotechnology.

To identify novel anti-HIV-1 peptides based on the antimicrobial peptide database (APD; http://aps.unmc.edu/AP/main.php ), we have screened 30 candidates and found 11 peptides with 50% effective concentrations (EC 50 ) of 1, increases in the Arg contents of amphibian maximin H5 and dermaseptin S9 peptides and the database-derived GLK-19 peptide improved the TIs. These examples demonstrate that the APD is a rich resource and a useful tool for developing novel HIV-1-inhibitory peptides.

The differential in vitro antimicrobial activity of a 12-residue-long arginine-rich peptide derived from protamine was examined against bacterial and parasite microbes. A design of discrete peptide fragments based on the thermolysin-digestion map allowed us to propose three peptide fragments to be further assessed regarding their biological and secondary structural properties. Peptide structure allowed designing three arginine-rich fragments. All peptide fragments were assessed regarding their antimicrobial activity against Gram-positive and Gram-negative bacteria and a human malaria strain. Qualitative and quantitative assays carried out for determining all peptides' antibacterial activity at different concentration levels included radial diffusion and a time-controlled technique. Tests demonstrated that all assessed molecules inhibited invasion of Plasmodium falciparum parasites to human red blood cells. Cytolytic activity of the parent protamine peptide was completely abolished by strategically fragmenting its aminoacid sequence. Remarkably, the cationic C-fragment exhibited stronger biological activity than its parent peptide. Interestingly, the peptide fragment denoted as 2077 displays a typical ?-helix profile according to its CD spectrum. The results support proposing the protamine C-terminal fragment as a potential new antimicrobial peptide. © 2009 Elsevier Inc. All rights reserved.

Antimicrobial peptides participate in innate host defense by directly eliminating pathogens as a result of their ability to damage the microbial membrane and by providing danger signals that will recruit innate immune cells to the site of infection. Dermaseptin DA4 (DRS-DA4), a new antimicrobial peptide of the dermaseptin superfamily, was identified based on its chemotactic properties, contrasting with the currently used microbicidal properties assessment. The peptide was isolated and purified by size exclusion HPLC and RP-HPLC from the skin of the Mexican frog, Pachymedusa dacnicolor. MS and amino acid sequence analyses were consistent with the structure GMWSKIKNAGKAAKAAAKAAGKAALGAVSEAM. CD experiments showed that, unlike most antimicrobial peptides of the dermaseptin superfamily, DRS-DA4 is not structured in the presence of zwitterionic lipids. DRS-DA4 is a potent chemoattractant for human leukocytes and is devoid of hemolytic activity; in addition, bactericidal tests and membrane perturbation assays on model membranes and on Escherichia coli and Staphylococcus aureus strains have shown that the antibacterial effects of DRS-DA4 and permeabilization of the inner membrane are exclusively selective for Gram-negative bacteria. Interestingly, despite high sequence homology with dermaseptin S4, dermaseptin B2 was not able to induce directional migration of leukocytes, and displayed a broader bactericidal spectrum. A detailed structure-function analysis of closely related peptides with different capabilities, such as DRS-DA4 and dermaseptin B2, is critical for the design of new molecules with specific attributes to modulate immunity and/or act as microbicidal agents.

There is widespread acceptance that cationic antimicrobial peptides, apart from their membrane-permeabilizing/disrupting properties, also operate through interactions with intracellular targets, or disruption of key cellular processes. Examples of intracellular activity include inhibition of DNA and protein synthesis, inhibition of chaperone-assisted protein folding and enzymatic activity, and inhibition of cytoplasmic membrane septum formation and cell wall synthesis. The purpose of this minireview is to question some widely held views about intracellular-targeting antimicrobial peptides. In particular, I focus on the relative contributions of intracellular targeting and membrane disruption to the overall killing strategy of antimicrobial peptides, as well as on mechanisms whereby some peptides are able to translocate spontaneously across the plasma membrane. Currently, there are no more than three peptides that have been convincingly demonstrated to enter microbial cells without the involvement of stereospecific interactions with a receptor/docking molecule and, once in the cell, to interfere with cellular functions. From the limited data currently available, it seems unlikely that this property, which is isolated in particular peptide families, is also shared by the hundreds of naturally occurring antimicrobial peptides that differ in length, amino acid composition, sequence, hydrophobicity, amphipathicity, and membrane-bound conformation. Microbial cell entry and/or membrane damage associated with membrane phase/transient pore or long-lived transitions could be a feature common to intracellular-targeting antimicrobial peptides and mammalian cell-penetrating peptides that have an overrepresentation of one or two amino acids, i.e. Trp and Pro, His, or Arg. Differences in membrane lipid composition, as well as differential lipid recruitment by peptides, may provide a basis for microbial cell killing on one hand, and mammalian cell passage on the other.

Dermaseptin PD-3-7 (aDrs) from frog skin contains three aspartic acid residues resulting in a negative net charge at neutral pH, as opposed to numerous other dermaseptins which are cationic helical antimicrobial peptides. Still, this peptide can be fitted into an amphipathic alpha helix by an Edmundson wheel projection. However, folding to the proposed helix was induced to only a low extent by zwitterionic vesicles or even detergents. Furthermore, no evidence of antibacterial or cytotoxic activity from soluble aDrs could be obtained. The peptide has an inherent propensity to an extended conformation in aqueous solution and self-assembles into amyloid fibrils in a reversible pH-controlled fashion, which was studied in some detail; above pH 5, the amyloid fibrils disassemble in a cooperative manner. This is probably caused by deprotonation of both side chain and terminal carboxyl groups, which results in intermolecular electrostatic repulsion. At neutral pH, this process proceeds instantaneously to the soluble form. Within the transition interval (pH 5-6.5), however, 'backward' granular aggregates, 10-500 nm in size, are formed. Such metastable amorphous aggregates, which are quickly released from an amyloid depot by a shift in pH, can mediate a strong cytotoxic effect. This activity does not involve lysis or interference with the cellular redox status, but apparently acts via an as yet unidentified mechanism. In this study, we present a new member of an emerging class of self-assembling frog skin peptides with extraordinary self-aggregation properties, which may potentially be relevant for biological processes. Structured digital abstract: * MINT-7256467: Dermaseptin (uniprotkb:O93455) and Dermaseptin (uniprotkb:O93455) bind (MI:0407) by circular dichroism (MI:0016) * MINT-7255686: Dermaseptin (uniprotkb:O93455) and Dermaseptin (uniprotkb:O93455) bind (MI:0407) by biophysical (MI:0013) * MINT-7256439: Dermaseptin (uniprotkb:O93455) and Dermaseptin (uniprotkb:O93455) bind (MI:0407) by fluorescence microscopy (MI:0416) * MINT-7256449: Dermaseptin (uniprotkb:O93455) and Dermaseptin (uniprotkb:O93455) bind (MI:0407) by electron microscopy (MI:0040) * MINT-7256430: Dermaseptin (uniprotkb:O93455) and Dermaseptin (uniprotkb:O93455) bind (MI:0407) by fluorescence technologies (MI:0051).

Skin secretions of hylid frogs show amazing levels of interspecific and intraspecific diversity and are comprised of a cocktail of genetically-related, but markedly diverse antimicrobial peptides that are grouped into a superfamily, termed the dermaseptins, comprising several families: dermaseptins (sensu stricto), phylloseptins, plasticins, dermatoxins, phylloxins, hyposins, caerins, and aureins. Dermaseptin gene superfamily evolution is characterized by repeated gene duplications and focal hypermutations of the mature peptide coding sequence, followed by positive (diversifying) selection. We review here molecular mechanisms leading to these vast combinatorial peptide libraries, and structural and functional properties of antimicrobial peptides of the dermaseptin and plasticin families, as well as those of dermaseptin S9, an amyloidogenic peptide with antimicrobial and chemoattractant activities.

A novel family of antimicrobial peptides, named raniseptins, has been characterized from the skin secretion of the anuran Hypsiboas raniceps. Nine cDNA molecules have been successfully cloned, sequenced, and their respective polypeptides were characterized by mass spectrometry and Edman degradation. The encoded precursors share structural similarities with the dermaseptin prepropeptides from the Phyllomedusinae subfamily and the mature 28-29 residue long peptides undergo further proteolytic cleavage in the crude secretion yielding consistent fragments of 14-15 residues. The biological assays performed demonstrated that the Rsp-1 peptide has antimicrobial activity against different bacterial strains without significant lytic effect against human erythrocytes, whereas the peptide fragments generated by endoproteolysis show limited antibiotic potency. MALDI imaging mass spectrometry in situ studies have demonstrated that the mature raniseptin peptides are in fact secreted as intact molecules within a defined glandular domain of the dorsal skin, challenging the physiological role of the observed raniseptin fragments, identified only as part of the crude secretion. In this sense, stored and secreted antimicrobial peptides may confer distinct protective roles to the frog.

Three dermaseptins, DS 01, DD K, and DD L, were compared with respect to their structural features and interactions with liposomes. Circular dichroic spectra at alcohols of different chain lengths revealed that DS 01 has the higher helicogenic potential in hydrophobic media. Binding of DS 01, DD K, and DD L to liposomes induced significant blue shifts of the emission spectra of the single tryptophan located at position 3 of all sequences indicating association of the peptides with bilayers. Kinetics evaluation of atomic force microscopy images evidenced the strong fusogenic activity of DS 01 whereas DD K and DD L showed increased lytic activities.

By integrating systematic peptidome and transcriptome studies of the defensive skin secretion of the Central American red-eyed leaf frog, Agalychnis callidryas, we have identified novel members of three previously described antimicrobial peptide families, a 27-mer dermaseptinrelated peptide (designated DRP-AC4), a 33-mer adenoregulin-related peptide (designated ARP-AC1) and most unusually, a 27-mer caerin-related peptide (designated CRP-AC1). While dermaseptin and adenoregulin were originally isolated from phyllomedusine leaf frogs, the caerins, until now, had only been described in Australian frogs of the genus, Litoria. Both the dermaseptin and adenoregulin were C-terminally amidated and lacked the C-terminal tripeptide of the biosynthetic precursor sequence. In contrast, the caerin-related peptide, unlike the majority of Litoria analogs, was not C-terminally amidated. The present data emphasize the need for structural characterization of mature peptides to ensure that unexpected precursor cleavages and/or post-translational modifications do not produce mature peptides that differ in structure to those predicted from cloned biosynthetic precursor cDNA. Additionally, systematic study of the secretory peptidome can produce unexpected results such as the CRP described here that may have phylogenetic implications. It is thus of the utmost importance in the functional evaluation of novel peptides that the primary structure of the mature peptide is unequivocally established e something that is often facilitated by cloning biosynthetic precursor cDNAs but obviously not reliable using such data alone. 2008 Elsevier Masson SAS. All rights reserved.

Dermaseptin S9 (Drs S9), GLRSKIWLWVLLMIWQESNKFKKM, isolated from frog skin, does not resemble any of the cationic and amphipathic antimicrobial peptides identified to date, having a highly hydrophobic core sequence flanked at either side by cationic termini. Previous studies [Lequin O, Ladram A, Chabbert A, Bruston F, Convert O, Vanhoye D, Chassaing G, Nicolas P & Amiche M (2006) Biochemistry45, 468-480] demonstrated that this peptide adopted a non-amphipathic alpha-helical conformation in trifluoroethanol/water mixtures, but was highly aggregated in aqueous solutions and in the presence of sodium dodecyl sulfate micelles. Circular dichroism, FTIR and attenuated total reflectance FTIR spectroscopies, combined with a surface plasmon resonance study, show that Drs S9 forms stable and ordered beta-sheet aggregates in aqueous buffers or when bound to anionic or zwitterionic phospholipid vesicles. These structures slowly assembled into amyloid-like fibrils in aqueous environments via spherical intermediates, as revealed by electron microscopy and Congo red staining. Drs S9 induced the directional migration of neutrophils, T lymphocytes and monocytes. Interestingly, the antimicrobial and chemotactic activities of Drs S9 are modulated by its amyloid-like properties. Whereas spherical oligomers of Drs S9 exhibit antimicrobial activity, the soluble, weakly self-associated forms of Drs S9 act on human leukocytes to promote chemotaxis and/or immunological response activation in the same range of concentration as amyloidogenic peptides Abeta(1-42), the most fibrillogenic isoform of amyloid beta peptides, and the prion peptide PrP(106-126).

Studies conducted on amphibian skin secretions over the past 40 years have isolated and identified huge arrays of bioactive peptides, many of which have demonstrated potent anti-microbial activity. Such peptides are attracting increasing attention due to the growing problem of pathogenic microorganisms resistant to conventional antibiotics. The current study utilized a combined proteomic/genomic approach to facilitate the high throughput sequencing of five novel dermaseptins and four novel phylloseptins from the skin secretions of Phyllomedusa hypochondrialis azurea . Peptides were partially identified using Q-TOF MS/MS fragmentation and de novo sequencing, while a cDNA library was constructed from the lyophilized skin secretion. 3′-RACE reactions used primers designed for the highly conserved 5′-signal regions of previously deduced dermaseptin precursors. cDNA sequenced peptides were attributed to their respective fragmentation spectra to confirm the structure of the final processed peptides. Such an approach identified post-translational modifications in addition to deciphering isobaric amino acids. Several of the peptides were purified to homogeneity and displayed potent antimicrobial activity with minimum inhibitory concentrations starting at 0.4 μM when tested against and range of Gram-positive and Gram-negative bacteria including Escherichia coli , Staphylococcus aureus and Micrococcus luteus .