Amphibian Antimicrobial Peptides Research Articles

Investigation of the Mechanism of Action of AMPs from Amphibians to Identify Bacterial Protein Targets for Therapeutic Applications

Antimicrobial peptides (AMPs) from amphibians represent a promising source of novel antibacterial agents due to their potent and broad-spectrum antimicrobial activity, which positions them as valid alternatives to conventional antibiotics. This review provides a comprehensive analysis of the mechanisms through which amphibian-derived AMPs exert their effects against bacterial pathogens. We focus on the identification of bacterial protein targets implicated in the action of these peptides and on biological processes altered by the effect of AMPs. By examining recent advances in countering multidrug-resistant bacteria through multi-omics approaches, we elucidate how AMPs interact with bacterial membranes, enter bacterial cells, and target a specific protein. We discuss the implications of these interactions in developing targeted therapies and overcoming antibiotic resistance (ABR). This review aims to integrate the current knowledge on AMPs’ mechanisms, identify gaps in our understanding, and propose future directions for research to harness amphibian AMPs in clinical applications.

Assessment of the antimicrobial and immunomodulatory activity of QS-CATH, a promising therapeutic agent isolated from the Chinese spiny frogs (Quasipaa spinosa)

Cathelicidins are important antimicrobial peptides in various vertebrate species where they are crucial parts of the innate immune system. The current understanding of amphibian cathelicidins is limited, particularly with regard to their immunomodulatory effects. To address this knowledge gap, we produced the cDNA sequence of the cathelicidin gene from a skin transcriptome of the Chinese spiny frog Quasipaa spinosa. The amino acid sequence of the Quasipaa spinosa cathelicidin (QS-CATH) was predicted to consist of a signal peptide, a cathelin domain, and a mature peptide. Comparative analysis of the QS-CATH amino acid sequence with that of other amphibian cathelicidins revealed high variability in the functional mature peptide among amphibians, whereas the cathelin domain was conserved. The QS-CATH gene was expressed in several tissues, with the highest level of expression in the spleen. Upregulation of QS-CATH after Aeromonas hydrophila infection occurred in the kidney, gut, spleen, skin, and liver. Chemically synthesized QS-CATH exhibited pronounced antibacterial activity against Shigella flexneri, Staphylococcus warneri, Escherichia coli, Salmonella enterica, and Listeria monocytogenes. Furthermore, QS-CATH disrupted the cell membrane integrity of S. flexneri, as evidenced by a lactate dehydrogenase release assay, and it hydrolyzed the genomic DNA of S. flexneri. Additionally, QS-CATH elicited chemotaxis and modulated the expression of inflammatory cytokine genes in RAW264.7 mouse leukemic monocyte/macrophage cells. These findings confirm the antimicrobial effects of amphibian cathelicidin and its ability to influence immune cell function. This will expedite the potential utilization of amphibian antimicrobial peptides as therapeutic agents.

The importance of antimicrobial peptides (AMPs) in amphibian skin defense

Antimicrobial peptides (AMPs) are produced for defense in nearly all taxa from simple bacteria to complex mammalian species. Some amphibian families have developed this defensive strategy to a high level of sophistication by loading the AMPs into specialized granular glands within the dermis. Enervated by the sympathetic nervous system, the granular glands are poised to deliver an array of AMPs to cleanse the wound and facilitate healing. There have been a number of excellent review publications in recent years that describe amphibian AMPs with an emphasis on their possible uses for human medicine. Instead, my aim here is to review what is known about the nature of amphibian AMPs, the diversity of amphibian AMPs, regulation of their production, and to provide the accumulated evidence that they do, indeed, play an important role in the protection of amphibian skin, vital for survival. While much has been learned about amphibian AMPs, there are still important gaps in our understanding of peptide synthesis, storage, and functions.

Helical intermediate formation and its role in amyloids of an amphibian antimicrobial peptide.

Helical intermediates appear to be crucial in the amyloid formation of several amyloidogenic peptides, including Aβ, that are implicated in different neurodegenerative diseases. Intermediate species of amyloid formation have been reported to be more toxic than mature amyloid fibrils. Hence, the current work focuses on understanding the mechanistic roles of the helical intermediates in the early stages of amyloid self-assembly in amyloidogenic peptides. Molecular dynamics (MD) simulations and the adaptive biasing force (ABF) method were utilized to investigate structural changes that lead to amyloid formation in amphibian peptide uperin-3.5 (U3.5), an antimicrobial and amyloidogenic peptide. Microsecond time-scale MD simulations revealed that peptide aggregation, into β-sheet dominated aggregates, is centred on two important factors; evolution of α-helical intermediates and the critical role of local peptide concentration inside these aggregates. Electrostatic attraction between the oppositely charged aspartate (D) and arginine (R) residues located near the N-terminus induced hydrogen bonding resulting in the formation of precursor 310-helices close to the N-terminus. The 310-helices transitioned into α-helices, thereby imparting partial helical conformations to the peptides. In the initial stages of aggregation, U3.5 peptides with amphipathic, partial helices were driven closer by hydrophobic interactions to form small clusters of helical intermediates. These helices imparted stability to the helical intermediates, which promoted the growth of clusters by further addition of peptides. This led to an increase in the local peptide concentration, which enabled stronger peptide-peptide interactions and triggered a β-sheet transition in these aggregates. Thus, this study emphasized that the helical intermediates may be crucial to the evolution of β-sheet-rich amyloid structures.

The Inhibition of DNA Viruses by the Amphibian Antimicrobial Peptide Temporin G: A Virological Study Addressing HSV-1 and JPCyV.

Herpes simplex virus type-1 (HSV-1) and John Cunningham polyomavirus (JCPyV) are widely distributed DNA viruses causing mainly asymptomatic infection, but also mild to very severe diseases, especially when these viruses reach the brain. Some drugs have been developed to inhibit HSV-1 replication in host cells, but their prolonged use may induce resistance phenomena. In contrast, to date, there is no cure for JCPyV. The search for alternative drugs that can reduce viral infections without undermining the host cell is moving toward antimicrobial peptides (AMPs) of natural occurrence. These include amphibian AMPs belonging to the temporin family. Herein, we focus on temporin G (TG), showing that it strongly affects HSV-1 replication by acting either during the earliest stages of its life cycle or directly on the virion. Computational studies have revealed the ability of TG to interact with HSV-1 glycoprotein B. We also found that TG reduced JCPyV infection, probably affecting both the earliest phases of its life cycle and the viral particle, likely through an interaction with the viral capsid protein VP1. Overall, our results are promising for the development of short naturally occurring peptides as antiviral agents used to counteract diseases related to HSV-1 and JCPyV.

Kassporin-KS1: A Novel Pentadecapeptide from the Skin Secretion of Kassina senegalensis: Studies on the Structure-Activity Relationships of Site-Specific "Glycine-Lysine" Motif Insertions.

Due to the abuse of traditional antibiotics and the continuous mutation of microbial resistance genes, microbial infections have become serious problems for human health. Therefore, novel antibacterial agents are urgently required, and amphibian antimicrobial peptides (AMP) are among the most interesting potential antibacterial leads. In this research, a novel peptide, named kassporin-KS1 (generically QUB-1641), with moderate antibacterial activity against Gram-positive bacteria, was discovered in the skin secretion of the Senegal running frog, Kassina senegalensis. Using site-specific sequence enrichment with a motif “glycine-lysine” that frequently occurs in ranid frog temporin peptides, a series of QUB-1641 analogues were synthesized, and effects on selected bioactivities were studied. The greatest activity enhancement was obtained when the “glycine-lysine” motif was located at the eighth and ninth position as in QUB-1570.QUB-1570 had a broader antibacterial spectrum than QUB-1641, and was eight-fold more potent. Moreover, QUB-1570 inhibited S. aureus biofilm most effectively, and significantly enhanced the viability of insect larvae infected with S. aureus. When the “glycine-lysine” motif of QUB-1570 was substituted to reduce the helix ratio and positive charge, the antibacterial activities of these synthetic analogues decreased. These data revealed that the “glycine-lysine” motif at positions 8 and 9 had the greatest enhancing effect on the antibacterial properties of QUB-1570 through increasing positive charge and helix content. This research may provide strategies for the site’s selective amino acid modification of some natural peptides to achieve the desired enhancement of activity.

Exploration of the Structure-Function Relationships of a Novel Frog Skin Secretion-Derived Bioactive Peptide, t-DPH1, through Use of Rational Design, Cationicity Enhancement and In Vitro Studies.

Amphibian skin-derived antimicrobial peptides (AMPs) have attracted increasing attention from scientists because of their excellent bioactivity and low drug resistance. In addition to being the alternative choice of antibiotics or anticancer agents, natural AMPs can also be modified as templates to optimise their bioactivities further. Here, a novel dermaseptin peptide, t-DPH1, with extensive antimicrobial activity and antiproliferative activity, was isolated from the skin secretion of Phyllomedusa hypochondrialis through ‘shotgun’ cloning. A series of cationicity-enhanced analogues of t-DPH1 were designed to further improve its bioactivities and explore the charge threshold of enhancing the bioactivity of t-DPH1. The present data suggest that improving the net charge can enhance the bioactivities to some extent. However, when the charge exceeds a specific limit, the bioactivities decrease or remain the same. When the net charge achieves the limit, improving the hydrophobicity makes no sense to enhance bioactivity. For t-DPH1, the upper limit of the net charge was +7. All the designed cationicity-enhanced analogues produced no drug resistance in the Gram-negative bacterium, Escherichia coli. These findings provide creative insights into the role of natural drug discovery in providing templates for structural modification for activity enhancement.

Temporin G, an amphibian antimicrobial peptide against influenza and parainfluenza respiratory viruses: Insights into biological activity and mechanism of action.

Treatment of respiratory viral infections remains a global health concern, mainly due to the inefficacy of available drugs. Therefore, the discovery of novel antiviral compounds is needed; in this context, antimicrobial peptides (AMPs) like temporins hold great promise. Here, we discovered that the harmless temporin G (TG) significantly inhibited the early life-cycle phases of influenza virus. The in vitro hemagglutinating test revealed the existence of TG interaction with the viral hemagglutinin (HA) protein. Furthermore, the hemolysis inhibition assay and the molecular docking studies confirmed a TG/HA complex formation at the level of the conserved hydrophobic stem groove of HA. Remarkably, these findings highlight the ability of TG to block the conformational rearrangements of HA2 subunit, which are essential for the viral envelope fusion with intracellular endocytic vesicles, thereby neutralizing the virus entry into the host cell. In comparison, in the case of parainfluenza virus, which penetrates host cells upon a membrane-fusion process, addition of TG to infected cells provoked~1.2 log reduction of viral titer released in the supernatant. Nevertheless, at the same condition, an immunofluorescent assay showed that the expression of viral hemagglutinin/neuraminidase protein was not significantly reduced. This suggested a peptide-mediated block of some late steps of viral replication and therefore the impairment of the extracellular release of viral particles. Overall, our results are the first demonstration of the ability of an AMP to interfere with the replication of respiratory viruses with a different mechanism of cell entry and will open a new avenue for the development of novel therapeutic approaches against a large variety of respiratory viruses, including the recent SARS-CoV2.

The amphibian antimicrobial peptide uperin 3.5 is a cross-α/cross-β chameleon functional amyloid

Antimicrobial activity is being increasingly linked to amyloid fibril formation, suggesting physiological roles for some human amyloids, which have historically been viewed as strictly pathological agents. This work reports on formation of functional cross-α amyloid fibrils of the amphibian antimicrobial peptide uperin 3.5 at atomic resolution, an architecture initially discovered in the bacterial PSMα3 cytotoxin. The fibrils of uperin 3.5 and PSMα3 comprised antiparallel and parallel helical sheets, respectively, recapitulating properties of β-sheets. Uperin 3.5 demonstrated chameleon properties of a secondary structure switch, forming mostly cross-β fibrils in the absence of lipids. Uperin 3.5 helical fibril formation was largely induced by, and formed on, bacterial cells or membrane mimetics, and led to membrane damage and cell death. These findings suggest a regulation mechanism, which includes storage of inactive peptides as well as environmentally induced activation of uperin 3.5, via chameleon cross-α/β amyloid fibrils.

In Silico Discovery of Antimicrobial Peptides as an Alternative to Control SARS-CoV-2.

A serious pandemic has been caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The interaction between spike surface viral protein (Sgp) and the angiotensin-converting enzyme 2 (ACE2) cellular receptor is essential to understand the SARS-CoV-2 infectivity and pathogenicity. Currently, no drugs are available to treat the infection caused by this coronavirus and the use of antimicrobial peptides (AMPs) may be a promising alternative therapeutic strategy to control SARS-CoV-2. In this study, we investigated the in silico interaction of AMPs with viral structural proteins and host cell receptors. We screened the antimicrobial peptide database (APD3) and selected 15 peptides based on their physicochemical and antiviral properties. The interactions of AMPs with Sgp and ACE2 were performed by docking analysis. The results revealed that two amphibian AMPs, caerin 1.6 and caerin 1.10, had the highest affinity for Sgp proteins while interaction with the ACE2 receptor was reduced. The effective AMPs interacted particularly with Arg995 located in the S2 subunits of Sgp, which is key subunit that plays an essential role in viral fusion and entry into the host cell through ACE2. Given these computational findings, new potentially effective AMPs with antiviral properties for SARS-CoV-2 were identified, but they need experimental validation for their therapeutic effectiveness.

Review: Examining the Natural Role of Amphibian Antimicrobial Peptide Magainin.

Host defense peptides (HDPs) are a group of antimicrobial peptides (AMPs) that are crucial components of the innate immune system of many different organisms. These small peptides actively kill microbes and prevent infection. Despite the presence of AMPs in the amphibian immune system, populations of these organisms are in decline globally. Magainin is an AMP derived from the African clawed frog (Xenopus laevis) and has displayed potent antimicrobial effects against a wide variety of microbes. Included in this group of microbes are known pathogens of the African clawed frog and other amphibian species. Arguably, the most deleterious amphibious pathogen is Batrachochytrium dendrobatidis, a chytrid fungus. Investigating the mechanism of action of magainin can help understand how to effectively fight off infection. By understanding amphibian AMPs’ role in the frog, a potential conservation strategy can be developed for other species of amphibians that are susceptible to infections, such as the North American green frog (Rana clamitans). Considering that population declines of these organisms are occurring globally, this effort is crucial to protect not only these organisms but the ecosystems they inhabit as well.

Bioinformatic Analysis of 1000 Amphibian Antimicrobial Peptides Uncovers Multiple Length-Dependent Correlations for Peptide Design and Prediction

Amphibians are widely distributed on different continents, except for the polar regions. They are important sources for the isolation, purification and characterization of natural compounds, including peptides with various functions. Innate immune antimicrobial peptides (AMPs) play a critical role in warding off invading pathogens, such as bacteria, fungi, parasites, and viruses. They may also have other biological functions such as endotoxin neutralization, chemotaxis, anti-inflammation, and wound healing. This article documents a bioinformatic analysis of over 1000 amphibian antimicrobial peptides registered in the Antimicrobial Peptide Database (APD) in the past 18 years. These anuran peptides were discovered in Africa, Asia, Australia, Europe, and America from 1985 to 2019. Genomic and peptidomic studies accelerated the discovery pace and underscored the necessity in establishing criteria for peptide entry into the APD. A total of 99.9% of the anuran antimicrobial peptides are less than 50 amino acids with an average length of 24 and a net charge of +2.5. Interestingly, the various amphibian peptide families (e.g., temporins, brevinins, esculentins) can be connected through multiple length-dependent relationships. With an increase in length, peptide net charge increases, while the hydrophobic content decreases. In addition, glycine, leucine, lysine, and proline all show linear correlations with peptide length. These correlations improve our understanding of amphibian peptides and may be useful for prediction and design of new linear peptides with potential applications in treating infectious diseases, cancer and diabetes.

Separation of D-amino acid-containing peptide phenylseptin using 3,3′-phenyl-1,1′-binaphthyl-18-crown-6-ether columns

Several D-amino acid-containing peptides (DAACPs) with antimicrobial, cardio-excitatory, or neuronal activities have been found in several species. Here, we demonstrated the chiral separation of the antimicrobial peptide diastereomers, D-phenylseptin and L-phenylseptin using (S) and (R) 3,3′-phenyl-1,1′-binaphthyl-18-crown-6-ether columns (CR-I (+) and CR-I (−), respectively) and also investigated the underlying mechanism. First, using D-amino acid-containing tripeptide Phe-Phe-Phe-OH, we found that CR-I (+) could be used to recognize diastereomeric tripeptides containing an L-amino acid as the first residue. On the contrary, CR-I (−) enabled separation of a series of diastereomers with D-amino acid as the first residue. Therefore, we achieved separation of the stereoisomers using the chiral columns depending on the position of the D- amino acid in the peptide and demonstrated the orthogonality of separations of the chiral columns. Then, using CR-I (+), we separated amphibian antimicrobial peptide diastereomers, L- and D-phenylseptin, which have the sequences, L-Phe-L-Phe-L-Phe and L-Phe-D-Phe-L-Phe at their N-termini, respectively. In order to understand the host-guest interactions, we performed molecular dynamics simulations for L-Phe-L-Phe-L-Phe tripeptide-CR-I molecule complex systems. Three hydrogen bonds between the N-terminal amine group -NH3+ and the crown ether oxygens were the dominant interactions. The hydrophobic interactions between phenyl-rings in the chiral selector unit of CR-I (+) and the side chains of 2nd and 3rd residues of the peptide also contributed to the affinity. Our results show that the CR-I (+)-column can be applied for the separation of endogenous DAACPs generated by the post-translational modification.

Antimicrobial Peptides: Amphibian Host Defense Peptides.

Antimicrobial Peptides (AMPs) are one of the most common components of the innate immune system that protect multicellular organisms against microbial invasion. The vast majority of AMPs are isolated from the frog skin. Anuran (frogs and toads) skin contains abundant AMPs that can be developed therapeutically. Such peptides are a unique but diverse group of molecules. In general, more than 50% of the amino acid residues form the hydrophobic part of the molecule. Normally, there are no conserved structural motifs responsible for activity, although the vast majority of the AMPs are cationic due to the presence of multiple lysine residues; this cationicity has a close relationship with antibacterial activity. Notably, recent evidence suggests that synthesis of AMPs in frog skin may confer an advantage on a particular species, although they are not essential for survival. Frog skin AMPs exert potent activity against antibiotic-resistant bacteria, protozoa, yeasts, and fungi by permeating and destroying the plasma membrane and inactivating intracellular targets. Importantly, since they do not bind to a specific receptor, AMPs are less likely to induce resistance mechanisms. Currently, the best known amphibian AMPs are esculentins, brevinins, ranacyclins, ranatuerins, nigrocin-2, magainins, dermaseptins, bombinins, temporins, and japonicins-1 and -2, and palustrin-2. This review focuses on these frog skin AMPs and the mechanisms underlying their antimicrobial activity. We hope that this review will provide further information that will facilitate further study of AMPs and cast new light on novel and safer microbicides.

A Comparative Study on Interactions of Antimicrobial Peptides L- and D-phenylseptin with 1,2-dimyristoyl-sn-glycero-3-phosphocholine

L-phenylseptin (L-Phes) and D-phenylseptin (D-Phes) are amphibian antimicrobial peptides isolated from the skin secretion of Hypsiboas punctatus. In the N-termini, L-Phes and D-Phes contain three consecutive phenylalanine residues, l-Phe-l-Phe-l-Phe and l-Phe-d-Phe-l-Phe, respectively. They are known to exhibit antimicrobial activity against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Xanthomonas axonopodis pv. Glycines. However, their mechanism of action and the role of the D-amino acid residue have not been elucidated yet. In this study, the interactions of both peptides with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) were investigated by means of quartz crystal microbalance, circular dichroism, vibrational circular dichroism, 31P solid-state NMR, and molecular dynamics simulation. Both peptides have similar binding constants to the DMPC lipid bilayers, in the order of 106 M−1, and form an α-helix structure in the DMPC lipid bilayers. Both the peptides induce similar changes in the dynamics of DMPC lipids. Thus, in spite of the difference in the conformations caused by the chirality at the N-terminus, the peptides showed similar behavior in the membrane-bound state, experimentally and computationally.

Effects of C-terminal amidation and heptapeptide ring on the biological activities and advanced structure of amurin-9KY, a novel antimicrobial peptide identified from the brown frog, Rana kunyuensis.

Rana kunyuensis is a species of brown frog that lives exclusively on Kunyu Mountain, Yantai, China. In the current study, a 279-bp cDNA sequence encoding a novel antimicrobial peptide (AMP), designated as amurin-9KY, was cloned from synthesized double-strand skin cDNA of R. kunyuensis. The amurin-9KY precursor was composed of 62 amino acid (aa) residues, whereas the mature peptide was composed of 14 aa and contained two cysteines forming a C-terminal heptapeptide ring (Rana box domain) and an amidated C-terminus. These structural characters represent a novel amphibian AMP family. Although amurin-9KY exhibited high similarity to the already identified amurin-9AM from R. amurensis, little is known about the structures and activities of amurin-9 family AMPs so far. Therefore, amurin-9KY and its three derivatives (amurin-9KY1–3) were designed and synthesized. The structures and activities were examined to evaluate the influence of C-terminal amidation and the heptapeptide ring on the activities and structure of amurin-9KY. Results indicated that C-terminal amidation was essential for antimicrobial activity, whereas both C-terminal amidation and the heptapeptide ring played roles in the low hemolytic activity. Circular dichroism (CD) spectra showed that the four peptides adopted an α-helical conformation in THF/H2O (v/v 1:1) solution, but a random coil in aqueous solution. Elimination of the C-terminal heptapeptide ring generated two free cysteine residues with unpaired thiol groups, which greatly increased the concentration-dependent anti-oxidant activity. Scanning electron microscopy (SEM) was also performed to determine the possible bactericidal mechanisms.

Caerin1.1 Suppresses the Growth of Porcine Epidemic Diarrhea Virus In Vitro via Direct Binding to the Virus.

Porcine epidemic diarrhea (PED) has re-emerged in recent years and has already caused huge economic losses to the porcine industry all over the world. Therefore, it is urgent for us to find out efficient ways to prevent and control this disease. In this study, the antiviral activity of a cationic amphibian antimicrobial peptide Caerin1.1 against porcine epidemic diarrhea virus (PEDV) was evaluated by an in vitro system using Vero cells. We found that even at a very low concentration, Caerin1.1 has the ability to destroy the integrity of the virus particles to block the release of the viruses, resulting in a considerable decrease in PEDV infections. In addition, Caerin1.1 showed powerful antiviral activity without interfering with the binding progress between PEDV and the receptor of the cells, therefore, it could be used as a potential antiviral drug or as a microbicide compound for prevention and control of PEDV.

The Amphibian Antimicrobial Peptide Temporin B Inhibits In Vitro Herpes Simplex Virus 1 Infection.

ABSTRACTThe herpes simplex virus 1 (HSV-1) is widespread in the population, and in most cases its infection is asymptomatic. The currently available anti-HSV-1 drugs are acyclovir and its derivatives, although long-term therapy with these agents can lead to drug resistance. Thus, the discovery of novel antiherpetic compounds deserves additional effort. Naturally occurring antimicrobial peptides (AMPs) represent an interesting class of molecules with potential antiviral properties. To the best of our knowledge, this study is the first demonstration of the in vitro anti-HSV-1 activity of temporin B (TB), a short membrane-active amphibian AMP. In particular, when HSV-1 was preincubated with 20 μg/ml TB, significant antiviral activity was observed (a 5-log reduction of the virus titer). Such an effect was due to the disruption of the viral envelope, as demonstrated by transmission electron microscopy. Moreover, TB partially affected different stages of the HSV-1 life cycle, including the attachment and the entry of the virus into the host cell, as well as the subsequent postinfection phase. Furthermore, its efficacy was confirmed on human epithelial cells, suggesting TB as a novel approach for the prevention and/or treatment of HSV-1 infections.

A Review of the Mechanism of Action of Amphibian Antimicrobial Peptides Focusing on Peptide-Membrane Interaction and Membrane Curvature.

Research interests on amphibian antimicrobial peptides (AMPs) are currently increasing because of their capability to combat microorganisms from both terrestrial and aquatic environments, which are the warehouses of human pathogens. The most remarkable feature of AMPs are their mechanism of action, primarily targeted to anionic membranes. Researchers have postulated many models to describe peptide- membrane interaction, which leads to membrane permeation/intracellular targeting. Despite these models information regarding the relationship between membrane curvature and peptidemembrane interaction is scarce. This relationship could be clearly depicted using the two-state model and interfacial activity model. In the review, we discuss in detail the two state and interfacial activity models and explain the influence of membrane curvature on peptide binding and the membrane interaction of curvature-sensitive peptides. In addition, the models proposed to explain the mechanism of action of membrane lytic and non-lytic AMPs are also reviewed.

Targeted Modification of a Novel Amphibian Antimicrobial Peptide from Phyllomedusa tarsius to Enhance Its Activity against MRSA and Microbial Biofilm.

Antimicrobial peptides (AMPs) in the skin secretions of amphibians are fundamental components of a unique defense system that has evolved to protect these hosts from microbial invasion. Medusins constitute a recently-discovered AMP family from phyllomedusine leaf frog skin and exhibit highly-conserved structural characteristics. Here, we report a novel medusin, medusin-PT, from the skin secretion of the Tarsier Leaf Frog, Phyllomedusa tarsius. The mature peptide was initially identified from its cloned biosynthetic precursor-encoding cDNA as obtained by the rapid amplification of cDNA ends (RACE) method. Reverse-phase HPLC and tandem mass spectrometry confirmed both the presence of medusin-PT in the skin secretion and its primary structure. In a range of bioassays, medusin-PT exhibited antimicrobial activity against only the Gram-positive bacterium Staphylococcus aureus at 64 μg/ml. However, after directed changes to enhance the cationicity and amphipathicity of the peptide structure, three analog showed more potent antimicrobial activity against several additional bacteria including the antibiotic-resistant bacterium MRSA. In addition, these analog exhibited activity against microbial biofilm (minimum biofilm inhibitory and eradication concentrations of 32 μg/ml and over 64 μg/ml, respectively). These data provide evidence that medusins might be promising candidates as novel antibiotic leads and that the targeted modification of a natural AMP can both improve its efficacy so as to provide new insights into antibiotic design and development.