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Insights into the mechanism(s) of action and therapeutic applications of Esculentin-1a-derived antimicrobial peptides
Full text linkCationic α-helical antimicrobial peptides (AMPs) hold promise for treatment of the raising multi-drug resistant microbial infections, due to their broad spectrum of activity and membrane-perturbing mechanism of action. Compared to conventional antibiotics, these features make them newsworthy molecules that hardly induce microorganisms to acquire resistance to them. Among these pathogens, Pseudomonas aeruginosa is the most clinically relevant Gram-negative bacterium known to cause serious human infections, e.g. pneumoniae, especially in immune-compromised patients, such as cystic fibrosis (CF) sufferers and keratitis, associated to contact lens (CL) wear. This is due to the unique ability of this pathogen to adhere to different types of inert materials or biological tissues, and to grow in a more resistant and dangerous sessile life form, called biofilm. Recently, two Esculentin-1a-derived antimicrobial peptides i.e. Esc(1-21) and its D-amino acids containing Esc(1-21)-1c, [Esc peptides], have been fully characterized for their powerful antipseudomonal activity against both planktonic and biofilm forms. The diastereomer showed a higher bactericidal activity than the all-L isomer against the more dangerous Pseudomonas biofilm phenotype; a lower cytotoxicity and higher biostability. However, when tested in vitro against the free-living form of this pathogen, it displayed a weaker bactericidal effect. Here, to investigate the reason accounting for this discrepancy, mechanistic studies on intact bacterial cells were initially carried out. Then to further understand the effect of packing parameters, i.e. composition, charge, shape and negative intrinsic curvature of membrane phospholipids in the membrane-permeabilizing activity of Esc peptides, leakage assays and circular dichroism spectroscopy analysis were carried out. Our results have suggested that the weaker in vitro antibacterial activity of Esc(1-21)-1c on the planktonic phenotype of the Gram-negative bacterium P. aeruginosa is mainly correlated to a slighter ability in permeabilizing both outer and inner bacterial membranes. Notably, experiments with lipid vesicles have suggested that if electrostatic interactions between negatively-charged membrane phospholipids and positively-charged peptide molecules do play a crucial role in the peptides’ membrane perturbing activity, this latter is hampered by the bilayer structure packing parameters including hydrogen bonding and intrinsic curvature, associated to phosphatidylserine (PE), especially for the diastereomer compared to all-L parent peptide. In parallel, we explored the molecular mechanism underlying the biofilm inhibition activity of Esc peptides when used at dosages below the minimal growth inhibitory concentration (1/8 MIC), by studying the peptides’ effect on the expression of key genes involved in the bacterial virulence and motility, as well as the peptide’ interaction with the bacterial signaling nucleotide ppGpp. Our findings revealed that the two D-amino acids containing Esc(1-21)-1c, confer the peptide the ability to downregulate the expression of biofilm-associated genes, likely as a result of increased peptide stability and prolonged binding to ppGpp compared to the all-L peptide. Furthermore, we reported two different applicative strategies to ameliorate the biological properties of these two AMPs: (i) encapsulation in poly(lactide-co-glycolide) (PLGA) nanoparticles; and (ii) covalent conjugation to soft CLs. In the first case, to enhance the peptides’ bioavailability and to optimize their translocation to the target infectious site, Esc peptides were loaded into PLGA nanoparticles (NPs) engineered with polyvinyl alcohol (PVA). The peptides-loaded NPs were found to be more efficient in diffusing through artificial CF mucus and simulated bacterial extracellular matrix compared to the free peptides. Moreover, they were more efficient in inhibiting P. aeruginosa growth under both in vitro and in vivo conditions at long term. In the second case, Esc peptides were covalently immobilized to hydrogel soft CLs and tested for their ability to reduce bacterial colonization. The antimicrobial CLs were able to cause more than four log reduction in the number of bacterial cells within 20 min and to reduce bacterial adhesion to their surface in 24 hours. Finally, the ability of both peptides to limit the onset of microbial resistance was also evaluated by exposing Pseudomonas strains to multiple cycles of treatment at sub-MIC dosages. Interestingly, in contrast with conventional antibiotics, Esc peptides did not induce resistance in P. aeruginosa cells. Overall, besides providing knowledges on the molecular mode(s) of action the two esculentin-derived AMPs, our data suggest that Esc peptides, particularly Esc(1-21)-1c, have great potential to be developed as novel drugs for treatment and prevention of P. aeruginosa pneumonia and keratitis
Derivatives of esculentin-1a: promising frog-skin peptides for the development of new antipseudomonal drugs with expanding properties
No full textNaturally-occurring antimicrobial peptides (AMPs) represent an interesting class of molecules for the development of new anti-infective agents with multiple properties. The discovery of new antimicrobials is highly demanded, due to the increasing number of microorganisms that are resistant to the currently-used drugs. Remarkably, one of the most feared opportunistic pathogens, especially in the lungs of cystic fibrosis (CF) sufferers is the gram-negative bacterium Pseudomonasaeruginosa,becauseofitsintrinsiclowsusceptibilitytoantibioticsandabilitytoformbiofilm-communities that are resistant to a variety of environmental insults. Recently, we discovered that a derivative of the frog-skin AMP esculentin-1a, esculentin-1a(1-21)NH2 [Esc(1-21) GIFSKLAGKKIKNLLISGLKG-NH2 had potent antimicrobial activity against both free-living and biofilm forms ofP. aeruginosa with minimal concentrations inhibiting microbial growth (MICs) ranging from 4 to 8 μM (1). Subsequently, a diastereomer of Esc(1-21), Esc(1-21)-1c, carrying two D-amino acids was designed. It resulted to be slightly weaker than the all-L peptide against the planktonic form ofP. aeruginosa (with a concentration causing 99.9% killing of bacterial cells equal to 4 μMversus 1 μM of the parent peptide). However, it showed a higher bactericidal activity against the more dangerous biofilm phenotype; a lower cytotoxicity and higher biostability (2). In this work, further biological properties were investigated. Among them: (i) the ability of Esc(1-21) and its diastereomer to induce bacterial resistance after multiple cycles of treatment at sub-MICs in comparison with conventional antibiotics; (ii) the peptides' effect on the motility of the planktonic form of CF clinical isolates of P. aeruginosa and (iii) the effect of the diastereomer on preformed Pseudomonas biofilm in the presence of artificial sputum medium (ASM) that better simulates the lung environment (i.e. sputum composition) of CF patients. The results of these experiments have indicated that in contrast with aztreonam, colistin, tobramycin and ciprofloxacin, both peptides do not induce resistance after a prolonged exposure (~ 1 month) of the bacterial culture to them, as pointed out by the invariant MIC. In addition, the diastereomer was found to have a higher ability than the all-L Esc(1-21) to inhibit the bacterium's motility and presumably its ability to reach and colonize a biological surface (e.g. airways epithelium) switching to the biofilm form. Finally, when the diastereomer was added to Pseudomonas biofilm in ASM it was found to cause 20 % killing of the biofilm after 6 h treatment at the MIC, while only 2% reduction of viable biofilm was found for the conventional antibiotic aztreonam. All these data suggest that the two frog-skin derived AMPs represent very interesting candidates for the development of new antipseudomonal drugs for treatment of lung infections
Antimicrobial peptides for novel antiviral strategies in the current post-COVID-19 pandemic
Full text linkThe recent pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has highlighted how urgent and necessary the discovery of new antiviral compounds is for novel therapeutic approaches. Among the various classes of molecules with antiviral activity, antimicrobial peptides (AMPs) of innate immunity are among the most promising ones, mainly due to their different mechanisms of action against viruses and additional biological properties. In this review, the main physicochemical characteristics of AMPs are described, with particular interest toward peptides derived from amphibian skin. Living in aquatic and terrestrial environments, amphibians are one of the richest sources of AMPs with different primary and secondary structures. Besides describing the various antiviral activities of these peptides and the underlying mechanism, this review aims at emphasizing the high potential of these small molecules for the development of new antiviral agents that likely reduce the selection of resistant strains
Effects of the conjugation of the antimicrobial peptide Esc(1-21) to gold-nanoparticles
No full textIt has been recently demonstrated that the derivative of the frog skin antimicrobial peptide (AMP) esculentin-1a, Esc(1-21), has a strong microbicidal activity against both the free living and the biofilm forms of the bacterial pathogen Pseudomonas aeruginosa with a membrane-perturbing activity as a plausible mode of action.[1] With the aim to overcome the principal limits for the development of AMPs as new therapeutic agents, such as a low biostability, high toxicity and inefficient delivery to the target site, the conjugation of AMPs to inorganic nanoparticles (NPs), e.g. gold NPs (AuNPs), could represent a valuable solution. We demonstrated that the conjugation of Esc(1-21) to AuNPs via a polyethyleneglycol linker significantly increases the activity of the free peptide against the motile and sessile form of P. aeruginosa, respectively, without being harmful to mammalian cells. Furthermore, our engineered NPs were found to preserve the peptide’s ability to disrupt the bacterial membrane and to retain the peptide’s wound healing activity on a keratinocytes monolayer.
Our findings suggest that the conjugation of a linear AMP to inorganic nanocarriers, i.e. AuNPs represents an innovative nanoscale formulation for topical treatment of epithelial infections
Esculentin-1 aderived antipseudomonal peptides. limited induction of resistance and synergy with aztreonam
No full textBACKGROUND: The massive use of antibiotics has led to the selection of resistant bacterial strains that are difficult to eradicate. Among these, Pseudomonas aeruginosa most frequently colonizes and infects the airways of cystic fibrosis patients. Cationic Antimicrobial Peptides (AMPs) represent interesting molecules for the development of new antimicrobial agents. Thanks to their mechanism of action that involves the permeabilization of the bacterial cytoplasmic membrane, the induction of resistance is quite limited. OBJECTIVE: The evaluation of the capability of two frog-skin derived AMPs, i.e. Esc(1-21) and its diastereomer Esc(1-21)-1c, to induce resistance in P. aeruginosa and synergize with aztreonam. METHOD: The induction of resistance was evaluated after 15 cycles of exposure to non-inhibitory growth concentrations of antibiotics and peptides. Subsequently, the Minimal Inhibitory Concentration (MIC) was calculated and compared to that obtained before drug exposure. Furthermore, MICs of AMPs and antibiotics were evaluated in Artificial Sputum Medium (ASM). Finally, the ability of the two peptides to synergize with aztreonam was determined by the checkerboard titration method. RESULTS: Pseudomonas aeruginosa acquired resistance to antibiotics, as evidenced by the increased MICs compared to the initial ones (from 8 to 128-fold higher), while no change in MICs was observed after multiple treatments with the Esc-peptides. In addition, both peptides showed significantly lower MICs than aztreonam in ASM. Finally, the diastereomer Esc(1-21)-1c had the ability to synergize with aztreonam in inhibiting growth and in killing Pseudomonas cells. CONCLUSION: Both peptides represent promising candidates for the development of new antipseudomonal compounds, which do not induce resistance
In vitro effect of the acidic pH on the susceptibility of the antimicrobial peptide Esc(1-21) against respiratory pathogens in cystic fibrosis lung environment
No full textIn healthy individuals, to maintain lung function and sterility, a thin sheet of fluid that covers the
airway epithelium (airways surface liquid, ASL) is present. ASL contains several innate defenses,
including a complex mixture of antimicrobials that kill bacteria, and contributes to the mucociliary
clearance system that carries pathogens out of the lungs. Recently, in vivo studies on airway epithelia
have shown that defective Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), as found in
cystic fibrosis (CF) patients, reduces ASL pH, compromising its antimicrobial activity. This causes a
pathologic process characterized by chronic infection, inflammation, mucus obstruction, and
bronchiectasis [1].
Antimicrobial peptides (AMPs) are components of the innate immunity in all life forms and represent
promising molecules for the development of new antibacterial drugs. Recently, a frog skin derived
AMP, i.e. Esc(1-21), has been characterized for its bactericidal activity against the planktonic and
biofilm forms of the Gram-negative bacterium Pseudomonas aeruginosa, which is responsible for
more than 80% of drug-resistant infections in CF patients [2]. We carried out studies to evaluate the
in vitro antimicrobial efficacy of Esc(1-21), compared to a conventional antibiotic, i.e. tobramycin,
when used in CF-like acidic lung environment against the planktonic and biofilm forms of a panel of
multidrug resistant Gram-negative clinical isolates. In parallel, the effect of Esc(1-21) on the viability
of eukaryotic cells, under acidic conditions, was also investigated.
Our results have shown that Esc(1-21) is able to preserve its antimicrobial activity when used in acidic
pH conditions. In addition, its ability to kill bacterial biofilm is maintained at low pH and appears to
be even more effective than when tested under non-CF conditions. On the contrary, tobramycin
showed a significant weaker activity when tested in acidic environment at a wide range of
concentrations against most of the selected strains. Finally, Esc(1-21) did not markedly reduce the
percentage of viable eukaryotic cells in acidic conditions.
Altogether, these results highlight Esc(1-21) as an attractive candidate for the production of new
antimicrobial compounds against Gram-negative respiratory infections, especially in CF patients.
[1] Pezzulo AA et al. (2012) Nature 487, 109
[2] Luca V et al. (2013) Cell Mol Life Sci 70, 27732
This research was supported by EU funding within the NextGeneration EU-MUR PNRR Extended
Partnership initiative on Emerging Infectious Diseases (Project no. PE00000007, INF-ACT) to M.L.M
and Fondazione Italiana per la Ricerca Cistica (Project FFC#4/2022) Delegazione FFC Ricerca di Roma
e della Franciacorta e Val Camonic
The potential of frog skin peptides for anti-infective therapies: the case of esculentin-1a(1-21)nh2
No full textAntimicrobial peptides (AMPs) are key effectors of the innate immunity and represent promising molecules for the development of new antibacterial drugs. However, to achieve this goal, some problems need to be overcome: (i) the cytotoxic effects at high concentrations; (ii) the poor biostability and (iii) the difficulty in reaching the target site. Frog skin is one of the richest natural storehouses of AMPs and over the years, many peptides have been isolated from it, characterized and classified into several families encompassing temporins, brevinins, nigrocins and esculentins. In this review, we summarized how the isolation/characterization of peptides belonging to the esculentin-1 family drove us to the design of an analogue, i.e. esculentin-1a(1-21)NH2, with a powerful antimicrobial action and immunomodulatory properties. The peptide had a wide spectrum of activity, especially against the opportunistic Gram-negative bacterium Pseudomonas aeruginosa. We described the structural features and the in vitro/in vivo biological characterization of this peptide as well as the strategies used to improve its biological properties. Among them: (i) the design of a diastereomer carrying D-amino acids in order to reduce the peptide's cytotoxicity and improve its half-life; (ii) the covalent conjugation of the peptide to gold nanoparticles or its encapsulation into poly(lactide- co-glycolide) nanoparticles; and (iii) the peptide immobilization to biomedical devices (such as silicon hydrogel contact lenses) to obtain an antibacterial surface able to reduce microbial growth and attachment. Summing up the best results obtained so far, this review traces all the steps that led these frog-skin AMPsto the direction of peptide-based drugs for clinical use
Effetti della coniugazione del peptide antimicrobico Esc(1-21) a nanoparticelle di oro
No full textE’ stato recentemente dimostrato che il peptide antimicrobico (AMP) d’anfibio esculentin-1a(1-21)NH2 (Esc(1-21)), ha una forte azione microbicida contro la forma planctonica e sessile del batterio patogeno Pseudomonas aeruginosa con un plausibile meccanismo d’azione che prevede la perturbazione della membrana cellulare. Con lo scopo di superare i principali limiti che ostacolano lo sviluppo degli AMP come potenziali nuovi farmaci, come la loro bassa biostabilità, alta tossicità e la difficoltà di raggiungere il sito dell’infezione, la coniugazione a nanoparticelle (NPs) inorganiche, come quelle di oro (AuNPs), potrebbe risultare una soluzione vantaggiosa. In questo studio abbiamo dimostrato che la coniugazione del peptide antimicrobico Esc(1-21) a nanoparticelle di oro tramite un polyethyleneglycol linker aumenta significativamente l’attività battericida del peptide libero contro entrambe le forme del batterio Pseudomonas aeruginosa, senza risultare tossico per le cellule di mammifero. Inoltre, le nanoparticelle coniugate hanno dimostrato di preservare sia il meccanismo di perturbazione di membrana del peptide che le sue proprietà di “wound healing” su una “pseudo-ferita” in un monostrato di cheratoniciti.
I nostri risultati suggeriscono che la coniugazione di un peptide antimicrobico lineare come l’Esc(1-21) a nanoparticelle di oro potrebbe rappresentare un’innovativa formulazione per il trattamento topico di infezioni epiteliali.It has been recently demonstrated that the derivative of the frog skin antimicrobial peptide (AMP) esculentin-1a, Esc(1-21), has a strong microbicidal activity against both the free living and the biofilm forms of the bacterial pathogen Pseudomonas aeruginosa with a membrane-perturbing activity as a plausible mode of action.[1] With the aim to overcome the principal limits for the development of AMPs as new therapeutic agents, such as a low biostability, high toxicity and inefficient delivery to the target site, the conjugation of AMPs to inorganic nanoparticles (NPs), e.g. gold NPs (AuNPs), could represent a valuable solution. We demonstrated that the conjugation of Esc(1-21) to AuNPs via a polyethyleneglycol linker significantly increases the activity of the free peptide against the motile and sessile form of P. aeruginosa, without being harmful to mammalian cells. Furthermore, our engineered NPs were found to preserve the peptide’s ability to disrupt the bacterial membrane and to retain the peptide’s wound healing activity on a keratinocytes monolayer.
Our findings suggest that the conjugation of a linear AMP to inorganic nanocarriers, i.e. AuNPs represents an innovative nanoscale formulation for topical treatment of epithelial infections
An Overview of Frog Skin-Derived Esc Peptides: Promising Multifunctional Weapons against Pseudomonas aeruginosa-Induced Pulmonary and Ocular Surface Infections
No full textAntimicrobial resistance is a silent pandemic harming human health, and Pseudomonas aeruginosa is the most common bacterium responsible for chronic pulmonary and eye infections. Antimicrobial peptides (AMPs) represent promising alternatives to conventional antibiotics. In this review, the in vitro/in vivo activities of the frog skin-derived AMP Esc(1-21) are shown. Esc(1-21) rapidly kills both the planktonic and sessile forms of P. aeruginosa and stimulates migration of epithelial cells, likely favoring repair of damaged tissue. However, to undertake preclinical studies, some drawbacks of AMPs (cytotoxicity, poor biostability, and limited delivery to the target site) must be overcome. For this purpose, the stereochemistry of two amino acids of Esc(1-21) was changed to obtain the diastereomer Esc(1-21)-1c, which is more stable, less cytotoxic, and more efficient in treating P. aeruginosa-induced lung and cornea infections in mouse models. Incorporation of these peptides (Esc peptides) into nanoparticles or immobilization to a medical device (contact lens) was revealed to be an effective strategy to ameliorate and/or to prolong the peptides' antimicrobial efficacy. Overall, these data make Esc peptides encouraging candidates for novel multifunctional drugs to treat lung pathology especially in patients with cystic fibrosis and eye dysfunctions, characterized by both tissue injury and bacterial infection
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