Amphipathic Α-helical Structure Research Articles

Transcriptome analysis of Corvus splendens reveals a repertoire of antimicrobial peptides

Multidrug resistance has become a global health problem associated with high morbidity and mortality. Antimicrobial peptides have been acknowledged as potential leads for prospective anti-infectives. Owing to their scavenging lifestyle, Corvus splendens is thought to have developed robust immunity to pathogens found in their diet, implying that they have evolved mechanisms to resist infection. In the current study, the transcriptome of C. splendens was sequenced, and de novo assembled to identify the presence of antimicrobial peptide genes. 72.09 million high-quality clean reads were obtained which were then de novo assembled into 3,43,503 transcripts and 74,958 unigenes. About 37,559 unigenes were successfully annotated using SwissProt, Pfam, GO, and KEGG databases. A search against APD3, CAMPR3 and LAMP databases identified 63 AMP candidates belonging to more than 20 diverse families and functional classes. mRNA of AvBD-2, AvBD-13 and CATH-2 were found to be differentially expressed between the three tested crows as well as among the tissues. We also characterized Corvus Cathelicidin 2 (CATH-2) to gain knowledge of its antimicrobial mechanisms. The CD spectroscopy of synthesized mature Corvus CATH-2 peptide displayed an amphipathic α-helical structure. Though the synthetic CATH-2 caused hemolysis of human RBC, it also exhibited antimicrobial activity against E. coli, S. aureus, and B. cereus. Docking simulation results revealed that this peptide could bind to the LPS binding site of MD-2, which may prevent LPS from entering the MD-2 binding pocket, and trigger TLR4 signaling pathway. The Corvus CATH-2 characterized in this study could aid in the development of novel therapeutics.

An inducible amphipathic α-helix mediates subcellular targeting and membrane binding of RPE65.

RPE65 retinol isomerase is an indispensable player in the visual cycle between the vertebrate retina and RPE. Although membrane association is critical for RPE65 function, its mechanism is not clear. Residues 107-125 are believed to interact with membranes but are unresolved in all RPE65 crystal structures, whereas palmitoylation at C112 also plays a role. We report the mechanism of membrane recognition and binding by RPE65. Binding of aa107-125 synthetic peptide with membrane-mimicking micellar surfaces induces transition from unstructured loop to amphipathic α-helical (AH) structure but this transition is automatic in the C112-palmitoylated peptide. We demonstrate that the AH significantly affects palmitoylation level, membrane association, and isomerization activity of RPE65. Furthermore, aa107-125 functions as a membrane sensor and the AH as a membrane-targeting motif. Molecular dynamic simulations clearly show AH-membrane insertion, supporting our experimental findings. Collectively, these studies allow us to propose a working model for RPE65-membrane binding, and to provide a novel role for cysteine palmitoylation.

Simulation Study of the Effect of Antimicrobial Peptide Associations on the Mechanism of Action with Bacterial and Eukaryotic Membranes.

Antimicrobial peptides (AMPs) can be directed to specific membranes based on differences in lipid composition. In this study, we performed atomistic and coarse-grained simulations of different numbers of the designed AMP adepantin-1 with a eukaryotic membrane, cytoplasmic Gram-positive and Gram-negative membranes, and an outer Gram-negative membrane. At the core of adepantin-1’s behavior is its amphipathic α-helical structure, which was implemented in its design. The amphipathic structure promotes rapid self-association of peptide in water or upon binding to bacterial membranes. Aggregates initially make contact with the membrane via positively charged residues, but with insertion, the hydrophobic residues are exposed to the membrane’s hydrophobic core. This adaptation alters the aggregate’s stability, causing the peptides to diffuse in the polar region of the membrane, mostly remaining as a single peptide or pairing up to form an antiparallel dimer. Thus, the aggregate’s proposed role is to aid in positioning the peptide into a favorable conformation for insertion. Simulations revealed the molecular basics of adepantin-1 binding to various membranes, and highlighted peptide aggregation as an important factor. These findings contribute to the development of novel anti-infective agents to combat the rapidly growing problem of bacterial resistance to antibiotics.

The Central PXXP Motif Is Crucial for PMAP-23 Translocation across the Lipid Bilayer.

PMAP-23, a cathelicidin-derived host defense peptide, does not cause severe membrane permeabilization, but exerts strong and broad-spectrum bactericidal activity. We have previously shown that it forms an amphipathic α-helical structure with a central hinge induced by the PXXP motif, which is implicated in the interaction of PMAP-23 with negatively charged bacterial membranes. Here, we studied the potential roles of the PXXP motif in PMAP-23 translocation across the lipid bilayer by replacing Pro residues with either α-helix former Ala (PMAP-PA) or α-helix breaker Gly (PMAP-PG). Although both PMAP-PA and PMAP-PG led to effective membrane depolarization and permeabilization, they showed less antimicrobial activity than wild-type PMAP-23. Interestingly, we observed that PMAP-23 crossed lipid bilayers much more efficiently than its Pro-substituted derivatives. The fact that the Gly-induced hinge was unable to replace the PXXP motif in PMAP-23 translocation suggests that the PXXP motif has unique structural properties other than the central hinge. Surface plasmon resonance sensorgrams showed that the running buffer almost entirely dissociated PMAP-23 from the membrane surface, while its Pro-substituted derivatives remained significantly bound to the membrane. In addition, kinetic analysis of the sensorgrams revealed that the central PXXP motif allows PMAP-23 to rapidly translocate at the interface between the hydrophilic and hydrophobic phases. Taken together, we propose that the structural and kinetic understanding of the PXXP motif in peptide translocation could greatly aid the development of novel antimicrobial peptides with intracellular targets by promoting peptide entry into bacterial cells.

Novel antimicrobial cecropins derived from O. curvicornis and D. satanas dung beetles

Antibiotic resistance is an increasing global problem and therapeutic alternatives to traditional antibiotics are needed. Antimicrobial and host defense peptides represent an attractive source for new therapeutic strategies, given their wide range of activities including antimicrobial, antitumoral and immunomodulatory. Insects produce several families of these peptides, including cecropins. Herein, we characterized the sequence, structure, and biological activity of three cecropins called satanin 1, 2, and curvicin, found in the transcriptome of two dung beetle species Dichotomius satanas and Onthophagus curvicornis. Sequence and circular dichroism analyses show that they have typical features of the cecropin family: short length (38–39 amino acids), positive charge, and amphipathic α-helical structure. They are active mainly against Gram-negative bacteria (3.12–12.5 μg/mL), with low toxicity on eukaryotic cells resulting in high therapeutic indexes (TI > 30). Peptides also showed effects on TNFα production in LPS-stimulated PBMCs. The biological activity of Satanin 1, 2 and Curvicin makes them interesting leads for antimicrobial strategies.

Antimicrobial Peptide Modifications against Clinically Isolated Antibiotic-Resistant Salmonella.

Antimicrobial peptides are promising molecules to address the global antibiotic resistance problem, however, optimization to achieve favorable potency and safety is required. Here, a peptide-template modification approach was employed to design physicochemical variants based on net charge, hydrophobicity, enantiomer, and terminal group. All variants of the scorpion venom peptide BmKn-2 with amphipathic α-helical cationic structure exhibited an increased antibacterial potency when evaluated against multidrug-resistant Salmonella isolates at a MIC range of 4–8 µM. They revealed antibiofilm activity in a dose-dependent manner. Sheep red blood cells were used to evaluate hemolytic and cell selectivity properties. Peptide Kn2-5R-NH2, dKn2-5R-NH2, and 2F-Kn2-5R-NH2 (variants with +6 charges carrying amidated C-terminus) showed stronger antibacterial activity than Kn2-5R (a variant with +5 charges bearing free-carboxyl group at C-terminus). Peptide dKn2-5R-NH2 (d-enantiomer) exhibited slightly weaker antibacterial activity with much less hemolytic activity (higher hemolytic concentration 50) than Kn2-5R-NH2 (l-enantiomer). Furthermore, peptide Kn2-5R with the least hydrophobicity had the lowest hemolytic activity and showed the highest specificity to Salmonella (the highest selectivity index). This study also explained the relationship of peptide physicochemical properties and bioactivities that would fulfill and accelerate progress in peptide antibiotic research and development.

Comparative Antimicrobial Activity of Hp404 Peptide and Its Analogs against Acinetobacter baumannii.

An amphipathic α-helical peptide, Hp1404, was isolated from the venomous gland of the scorpion Heterometrus petersii. Hp1404 exhibits antimicrobial activity against methicillin-resistant Staphylococcus aureus but is cytotoxic. In this study, we designed antimicrobial peptides by substituting amino acids at the 14 C-terminal residues of Hp1404 to reduce toxicity and improve antibacterial activity. The analog peptides, which had an amphipathic α-helical structure, were active against gram-positive and gram-negative bacteria, particularly multidrug-resistant Acinetobacter baumannii, and showed lower cytotoxicity than Hp1404. N-phenyl-1-naphthylamine uptake and DisC3-5 assays demonstrated that the peptides kill bacteria by effectively permeating the outer and cytoplasmic membranes. Additionally, the analog peptides inhibited biofilm formation largely than Hp1404 at low concentrations. These results suggest that the analog peptides of Hp1404 can be used as therapeutic agents against A. baumannii infection.

Characterization of 2 linear peptides without disulfide bridges from the venom of the spider Lycosa poonaensis (Lycosidae).

Spider venom is a complex mixture of bioactive components, in which peptides play an important role by showing neurotoxicity or cytotoxicity. Disulfide-rich peptides are major components in the venom, but linear peptides without disulfide bridges are also present and often show antimicrobial activity. In this study, we analyzed the venom of the spider Lycosa poonaensis (Lycosidae) to find novel antimicrobial peptides using mass spectrometry. The result revealed that 120 out of 401 detected components were nondisulfide-bridged peptides. From them, the sequence of 2 peptides (lyp2370 and lyp1987) were determined by MS/MS analysis. The biological activity test revealed that lyp2370 has only weak antibacterial activity. On the other hand, lyp1987, which is identical to M-lycotoxin-Ls3b from the Lycosa singoriensi venom, showed significant antibacterial activity. The weak activity of lyp2370 was found to be due to the presence of a Glu residue on the hydrophilic face of its amphipathic α-helical structure.

Lipid headgroups alter huntingtin aggregation on membranes

Huntington's Disease is a fatal neurodegenerative disorder caused by expansion of a glutamine repeat region (polyQ) beyond a critical threshold within exon1 of the huntingtin protein (htt). As a consequence of polyQ expansion, htt associates into a variety of aggregate species that are thought to underlie cellular toxicity. Within cells, htt associates with numerous membranous organelles and surfaces that exert influence on the aggregation process. In particular, the first 17 amino acids at the N-terminus of htt (Nt17) serve as a lipid-binding domain that is intrinsically disordered in bulk solution but adopts an amphipathic α-helical structure upon binding membranes. Beyond this, Nt17 is implicated in initiating htt fibrillization. As the interaction between Nt17 and lipid membranes is likely influenced by lipid properties, the impact of lipid headgroups on htt-exon1 aggregation, membrane activity, and the ability to form protein:lipid complexes was determined. Htt-exon1 with a disease-length polyQ domain (46Q) was exposed to lipid vesicles comprised of lipids with either zwitterionic (POPC and POPE) or anionic (POPG and POPS) headgroups. With zwitterionic head groups, large lipid to peptide ratios were required to have a statistically significant impact on htt aggregation. Anionic lipids enhanced htt fibrillization, even at low lipid:protein ratios, and this was accompanied by changes in aggregate morphology. Despite the larger impact of anionic lipids, htt-exon1(46Q) was more membrane active with zwitterionic lipid systems. The ability of Nt17 to form complexes with lipids was also mediated by lipid headgroups as zwitterionic ionic lipids more readily associated with multimeric forms of Nt17 in comparison with anionic lipids. Collectively, these results highlight the complexity of htt/membrane interactions and the resulting impact on the aggregation process.

Interaction of Alpha-Synuclein and Its Mutants with Rigid Lipid Vesicle Mimics of Varying Surface Curvature.

Abnormal aggregation of alpha-synuclein (α-syn), an intrinsically disordered neuronal protein, is strongly implicated in the development of Parkinson's disease. Efforts to better understand α-syn's native function and its pathogenic role in neurodegeneration have revealed that the protein interacts with anionic lipid vesicles via adoption of an amphipathic α-helical structure; however, the ability of α-syn to remodel lipid membranes has made it difficult to decipher the role of vesicle surface curvature in protein binding behavior. In this study, sodium dodecyl sulfate (SDS)-coated gold nanoparticles (AuNPs), which mimic bilayer vesicle architecture, were synthesized in order to conduct a systematic investigation into the binding interaction of α-syn and two of its mutants (A30P and E46K) with rigid lipid vesicle mimics of defined surface curvature. By incorporating a rigid AuNP core (∼10-100 nm), the ability of α-syn to remodel the vesicle mimics was removed and their surface curvature could be fixed. Proteomics studies showed that, upon binding of free α-syn to the surface of SDS-AuNPs, the N-terminus of α-syn became less solvent accessible, whereas its C-terminus became more accessible. Interestingly, α-syn's non-amyloid-β component (NAC) region also exhibited increased solvent accessibility, suggesting that α-syn bound to rigid vesicle-like structures could possess heightened aggregation propensity and therefore pathogenicity. Additionally, both the A30P and E46K mutations were found to adopt distinct binding modes on the mimics' surface. In contrast with previous reports, similar binding affinities were observed for WT, A30P, and E46K α-syn toward SDS-AuNPs of all sizes, indicating the potential importance of vesicle deformability in determining α-syn binding behavior.

Non-perfectly Amphipathic α-Helical Structure Containing the XXYXX Sequence Improves the Biological Activity of Bovine αs2-Casein Antimicrobial Peptides

Non-amphiphilic WIQPKTKVIPYVRYL (WI-6) derived from bovine αs2-casein f (193-207) was modified by a defined mutation method to obtain five engineered peptides with mirror symmetry structures. The five engineered peptide sequences were WF-1 (WFQVKTRVRTKVQFW), FW-2 (FWRRYKKVKKYRRWF), FW-3 (FWQVIKKVKKIVQWF), FK-4 (FKQFYRRVRRYFQKF), and FR-5 (FRQWYRRVRRYWQRF). However, FW-2, FW-3, FK-4, and FR-5 had obvious XXYXX sequences. Among these, FW-3 was demonstrated to have the highest antibacterial activity, which indicates that the non-perfectly amphipathic α-helical structure containing the XXYXX sequence has a better bactericidal effect. Therefore, peptide FW-3 could be widely used as a substitute for antibiotics in food, medicine, and other fields. These findings provide a potential method for designing novel antimicrobial peptides.

Huntingtin Aggregation and Lipid Binding are Influenced by Physicochemical Properties of Membranes

Huntington's Disease is a fatal neurodegenerative disorder caused by an expanded glutamine repeat region (polyQ) within the huntingtin protein (htt), resulting in the misfolding and aggregation of htt into toxic aggregate species. The first 17 amino acids at the N-terminus of htt (Nt17) are intrinsically disordered in bulk solution, but adopt an amphipathic α-helical structure in the presence of binding partners, such as lipid membranes. In addition to comprising a lipid binding domain, this amphipathic α-helix has also been directly implicated in the initiation of htt aggregation via self-association with other Nt17 α-helices. Due to this, htt/lipid interaction likely has a large impact on the rate and extent of htt aggregate formation, with potential implications for HD pathogenesis. The interaction between Nt17 and lipid membranes is likely influenced by lipid properties, such as fluidity and lateral pressure. As a result, we undertook a series of experiments to determine the role that membrane physicochemical properties have on htt aggregation and lipid binding. Htt aggregation in the presence and absence of DMPC, POPC, and DOPC lipid vesicles was investigated using thioflavin T aggregation assays and atomic force microscopy (AFM). Huntingtin lipid binding was investigated using electrospray ionization mass spectrometry (ESI-MS), PDA lipid binding assays, and molecular dynamics simulations. Overall, our results suggest that membrane physicochemical properties associated with lipid tail groups alter htt lipid binding and aggregate formation.

Epinecidin-1, an Antimicrobial Peptide Derived From Grouper (Epinephelus coioides): Pharmacological Activities and Applications.

Epinecidin-1 is an antimicrobial peptide derived from the orange-spotted grouper (Epinephelus coioides). The mature epinecidin-1 peptide is predicted to have an amphipathic α-helical structure and a non-helical hydrophilic domain at the C-terminal RRRH. The majority of work studying the potential pharmacological activities of epinecidin-1, utilize synthesized epinecidin-1 (Epi-1), which is made up of 21 amino acids, from the amino acid sequence of 22–42 residues of Epi-1—GFIFHIIKGLFHAGKMIHGLV. The synthetized Epi-1 peptide has been demonstrated to possess diverse pharmacological activities, including antimicrobial, immunomodulatory, anticancer, and wound healing properties. It has also been utilized in different clinical and agricultural fields, including topical applications in wound healing therapy as well as the enhancement of fish immunity in aquaculture. Hence, the present work aims to consolidate the current knowledge and findings on the characteristics and pharmacological properties of epinecidin-1 and its potential applications.

Biological Function and Application of Picornaviral 2B Protein: A New Target for Antiviral Drug Development.

Picornaviruses are associated with acute and chronic diseases. The clinical manifestations of infections are often mild, but infections may also lead to respiratory symptoms, gastroenteritis, myocarditis, meningitis, hepatitis, and poliomyelitis, with serious impacts on human health and economic losses in animal husbandry. Thus far, research on picornaviruses has mainly focused on structural proteins such as VP1, whereas the non-structural protein 2B, which plays vital roles in the life cycle of the viruses and exhibits a viroporin or viroporin-like activity, has been overlooked. Viroporins are viral proteins containing at least one amphipathic α-helical structure, which oligomerizes to form transmembrane hydrophilic pores. In this review, we mainly summarize recent research data on the viroporin or viroporin-like activity of 2B proteins, which affects the biological function of the membrane, regulates cell death, and affects the host immune response. Considering these mechanisms, the potential application of the 2B protein as a candidate target for antiviral drug development is discussed, along with research challenges and prospects toward realizing a novel treatment strategy for picornavirus infections.

A Photo-Activatable Peptide Mimicking Functions of Apolipoprotein A-I.

Apolipoprotein A-I (apoA-I) plays a critical role in high-density lipoprotein (HDL) biogenesis, function and structural dynamics. Peptides that mimic apoA-I have a short amphipathic α-helical structure that can functionally recapitulate many of the same biologic properties of full-length apoA-I in HDL. Hence, they might be expected to have clinical applications in the reduction of atherosclerosis. However, nonspecific cellular efflux of cholesterol induced by apoA-I mimetic peptides might cause side effects that are, as yet, unidentified. In this study, we developed a photo-activatable peptide, 2F*, which is an 18 amino acid peptide mimicking apoA-I bearing an internal photocleavable caging group that is designed to assume an α-helical structure in response to a light stimulus and trigger efflux of cholesterol from cells. Without light irradiation, 2F* peptide showed a low tendency for the formation of α-helices, and therefore did not associate with lipids and failed to induce efflux of cholesterol. In addition, 2F* did not cause hemolysis under our experimental condition. Mass spectrometry indicated that, after light exposure, the caging group detached from 2F* and it assumed the α-helical structure in the presence of lipids, and enhanced cholesterol efflux from cells. Photo-activatable peptides such as 2F* that control cholesterol efflux following light stimulus may be useful for future atherosclerosis-reducing therapies.

High-Resolution Structural Studies Elucidate Antiatherogenic and Anti-Inflammatory Properties of Peptides Designed to Mimic Amphipathic α-Helical Domains of Apolipoprotein A-I.

Peptides designed to mimic the antiatherogenic and anti-inflammatory properties of apolipoprotein A-I show that although lipid association is required, not all lipid-associating peptides exhibit these properties. Our studies of a series of peptides showed that peptides with aromatic residues at the center of the nonpolar face were able to interact with inflammatory lipids and inhibited inflammation, which resulted in the amelioration of several lipid-mediated disorders such as lesion development, tumor formation, and Alzheimer’s plaque formation. The pKa values determined using 13C nuclear magnetic resonance (NMR) spectroscopy of K residues located at the polar-nonpolar interface provided the first clue to the relative orientations of the peptide helices with respect to each other and around the edge of the lipid discoidal complexes. High-resolution 1H-NMR studies of peptide-lipid discoidal complex confirmed the amphipathic α-helical structure of the peptide, location of aromatic residues of the peptide closer to the polar-nonpolar interface, and head-to-tail arrangement of the peptide helices around the edge of the disc. Amphipathic α-helical structure and the location of aromatic residues (F, W, Y) closer to the polar-nonpolar interface in a lipid environment allow the peptide to strongly bind oxidized lipids resulting in its anti-inflammatory properties.

Development of a novel short 12-meric papiliocin-derived peptide that is effective against Gram-negative sepsis

The development of novel peptide antibiotics with potent activity against multidrug-resistant Gram-negative bacteria and anti-septic activity is urgently needed. In this study, we designed short, 12-meric antimicrobial peptides by substituting amino acids from the N-terminal 12 residues of the papiliocin (Pap12-1) peptide to alter cationicity and amphipathicity and improve antibacterial activity and bacterial membrane interactions. Pap12-6, with an amphipathic α-helical structure and Trp12 at the C-terminus, showed broad-spectrum antibacterial activity, especially against multidrug-resistant Gram-negative bacteria. Dye leakage, membrane depolarization, and electron microscopy data proved that Pap12-6 kills bacteria by permeabilizing the bacterial membrane. Additionally, Pap12-6 significantly reduced the secretion of NO, TNF-α, and IL-6 and secreted alkaline phosphatase reporter gene activity confirmed that Pap12-6 shows anti-inflammatory activity via a TLR4-mediated NF-κB signaling pathway. In a mouse sepsis model, Pap12-6 significantly improved survival, reduced bacterial growth in organs, and reduced LPS and inflammatory cytokine levels in the serum and organs. Pap12-6 showed minimal cytotoxicity towards mammalian cells and controlled liver and kidney damage, proving its high bacterial selectivity. Our results suggest that Pap12-6 is a promising peptide antibiotic for the therapeutic treatment of Gram-negative sepsis via dual bactericidal and immunomodulatory effects on the host.

Pse-T2, an Antimicrobial Peptide with High-Level, Broad-Spectrum Antimicrobial Potency and Skin Biocompatibility against Multidrug-Resistant Pseudomonas aeruginosa Infection.

Pseudin-2, isolated from the frog Pseudis paradoxa, exhibits potent antibacterial activity but also cytotoxicity. In an effort to develop clinically applicable antimicrobial peptides (AMPs), we designed pseudin-2 analogs with Lys substitutions, resulting in elevated amphipathic α-helical structure and cationicity. In addition, truncated analogs of pseudin-2 and Lys-substituted peptides were synthesized to produce linear 18-residue amphipathic α-helices, which were further investigated for their mechanism and functions. These truncated analogs exhibited higher antimicrobial activity and lower cytotoxicity than pseudin-2. In particular, Pse-T2 showed marked pore formation, permeabilization of the outer/inner bacterial membranes, and DNA binding. Fluorescence spectroscopy and scanning electron microscopy showed that Pse-T2 kills bacterial cells by disrupting membrane integrity. In vivo, wounds infected with multidrug-resistant (MDR) Pseudomonas aeruginosa healed significantly faster when treated with Pse-T2 than did untreated wounds or wounds treated with ciprofloxacin. Moreover, Pse-T2 facilitated infected-wound closure by reducing inflammation through suppression of interleukin-1β (IL-1β), IL-6, and tumor necrosis factor alpha (TNF-α). These data suggest that the small antimicrobial peptide Pse-T2 could be useful for future development of therapeutic agents effective against MDR bacterial strains.

Functional Characterization of a Novel Lipopolysaccharide-Binding Antimicrobial and Anti-Inflammatory Peptide in Vitro and in Vivo.

Antimicrobial peptides (AMPs) are key components of host immune defense of vertebrates against microbial invasions. Here, we report a new AMP (esculentin-1GN) characterized from the skin of the frog Hylarana guentheri. Esculentin-1GN (GLFSKKGGKGGKSWIKGVFKGIKGIGKEVGGDVIRTGIEIAACKIKGEC) with high amphipathic α-helical structure in membrane-mimetic environments has the microbial-killing activity by destruction of the cell membrane. Moreover, esculentin-1GN inhibits LPS-induced expression of proinflammatory nitric oxide, interleukin-1β, interleukin-6, and tumor necrosis factor while it enhances expression of interleukin-10. Furthermore, esculentin-1GN can bind to d-(+)-galacturonic acid and LPS. Meanwhile, esculentin-1GN suppresses the activation of inflammatory response pathway induced by LPS. In addition, esculentin-1GN significantly reduces acute inflammation in carrageenan-induced mice paw. Taken together, the novel LPS-binding esculentin-1GN with antimicrobial and anti-inflammatory activities will be an excellent temple for designing new antibiotic formulations.

Leptodactylus latrans Amphibian Skin Secretions as a Novel Source for the Isolation of Antibacterial Peptides.

Amphibians´ skin produces a diverse array of antimicrobial peptides that play a crucial role as the first line of defense against microbial invasion. Despite the immense richness of wild amphibians in Argentina, current knowledge about the presence of peptides with antimicrobial properties is limited to a only few species. Here we used LC-MS-MS to identify antimicrobial peptides with masses ranging from 1000 to 4000 Da from samples of skin secretions of Leptodactylus latrans (Anura: Leptodactylidae). Three novel amino acid sequences were selected for chemical synthesis and further studies. The three synthetic peptides, named P1-Ll-1577, P2-Ll-1298, and P3-Ll-2085, inhibited the growth of two ATCC strains, namely Escherichia coli and Staphylococcus aureus. P3-Ll-2085 was the most active peptide. In the presence of trifluoroethanol (TFE) and anionic liposomes, it adopted an amphipathic α-helical structure. P2-Ll-1298 showed slightly lower activity than P3-Ll-2085. Comparison of the MIC values of these two peptides revealed that the addition of seven amino acid residues (GLLDFLK) on the N-terminal of P2-Ll-1298 significantly improved activity against both strains. P1-Ll-1577, which remarkably is an anionic peptide, showed interesting antimicrobial activity against E. coli and S. aureus strain, showing marked membrane selectivity and non-hemolysis. Due to this, P1-L1-1577 emerges as a potential candidate for the development of new antibacterial drugs.