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Effects of two L- to D-amino acid substitutions on the structural and functional properties of the antimicrobial peptide esculentin-1a(1-21)
Full text linkDuring the last years, the excessive and improper use of commercially available antibiotics has contributed to the development of resistant microbial pathogens, which represent a serious problem for the world public health. Among these microorganisms, the Gram-negative bacterium Pseudomonas aeruginosa is one of the most difficult to eradicate due to its ability to form sessile communities, named biofilms, which cause chronic infections, especially in the lungs of cystic fibrosis (CF) patients.
Naturally occurring antimicrobial peptides (AMPs), characterized by a different mechanism of action, represent a promising alternative to the commonly used drugs. AMPs are evolutionally conserved molecules produced by almost all living organisms as a first line of immune defense and amphibian skin is one of the richest sources.
The studies carried out in this thesis focused on two peptides:
Esculentin-1a(1-21)NH2, [Esc(1-21) GIFSKLAGKKIKNLLISGLKG-NH2], derived from the N-terminal region of the frog skin AMP esculentin-1a, and its diastereomer, Esc(1-21)-1c, containing two D-amino acids at positions 14 and 17 (i.e., D-Leu and D-Ser, respectively). This latter was designed with the purpose to reduce the peptide’s cytotoxicity and to increase its stability to proteolytic enzymes.
The results achieved in this thesis have indicated that compared to Esc(1-21), the diastereomer is: i) significantly less toxic towards mammalian cells, in agreement with its lower α-helical structure, as determined by circular dichroism spectroscopy and nuclear magnetic resonance studies; ii) more effective against the biofilm form of P. aeruginosa (either reference or clinical isolates from CF patients) while maintaining high activity against the free-living form of this pathogen; iii) more effective in killing Pseudomonas cells once internalized into bronchial cells expressing either the functional or the ΔF508 mutant of the CF transmembrane conductance regulator; iv) more resistant to bacterial and human elastases, which are abundant in CF lungs. In addition, the diastereomer was found (i) to have a higher activity than the all-L peptide in promoting migration of bronchial epithelial cells and presumably in favoring re-epithelialization of damaged lung tissue; (ii) to disaggregate and detoxify the bacterial lipopolysaccharide (LPS) and to inhibit cyclooxygenase-2 (COX-2) synthesis, albeit less than the wild-type peptide.
Based on its interesting biological properties, Esc(1-21)-1c is a promising candidate for the development of a new drug that not only eliminates microbial pathogens, but also restores the integrity of a damaged tissue, such as the lung of CF patients, following Pseudomonas respiratory infections
Anti-Pseudomonal activity of the amphibian antimicrobial peptide Esculentin(1-21) and plausible mode of action
No full textThe formation of Pseudomonas biofilms is very common in the lungs of cystic fibrosis patients with chronic infections. The colonization of the respiratory tract by this pathogen usually starts with the tissue adhesion of non-mucoid and motile strains. Subsequently they evolve a mucoid phenotype forming sessile communities, with a protective layer around the cells that confers more resistance to antibiotic therapy. Conventional antibiotics are frequently ineffective mainly because of their undesiderable side effects, emergence of resistant strains or because their lacking activity against pathogenic forms. Due to these reasons, novel anti-infective agents are of great interest to the medical community. Here we report on the potent activity and membrane-perturbing effects of the amphibian antimicrobial peptide esculentin(1-21), on both the free-living and sessile forms of P. aeruginosa, as a possible mechanism for biofilm disruption. Moreover Esc(1-21) does not induce resistant strains in vitro after multiple exposure to the peptide. Overall, this peptide is a promising template for the generation of new antibiotic formulations to advance care of infections caused by P. aeruginos
A novel in vitro wound healing assay to evaluate cell migration
No full textThe aim of this work is to show a novel method to evaluate the ability of some immunomodulatory molecules, such as antimicrobial peptides (AMPs), to stimulate cell migration. Importantly, cell migration is a rate-limiting event during the wound-healing process to re-establish the integrity and normal function of tissue layers after injury. The advantage of this method over the classical assay, which is based on a manually made scratch in a cell monolayer, is the usage of special silicone culture inserts providing two compartments to create a cell-free pseudo-wound field with a well-defined width (500 μm). In addition, due to an automated image analysis platform, it is possible to rapidly obtain quantitative data on the speed of wound closure and cell migration. More precisely, the effect of two frog-skin AMPs on the migration of bronchial epithelial cells will be shown. Furthermore, pretreatment of these cells with specific inhibitors will provide information on the molecular mechanisms underlying such events
Promising Approaches to Optimize the Biological Properties of the Antimicrobial Peptide Esculentin-1a(1–21)NH2: Amino Acids Substitution and Conjugation to Nanoparticles
Full text linkAntimicrobial peptides (AMPs) represent an interesting class of molecules with expanding biological properties which make them a viable alternative for the development of future antibiotic drugs. However, for this purpose, some limitations must be overcome: (i) the poor biostability due to enzymatic degradation; (ii) the cytotoxicity at concentrations slightly higher than the therapeutic dosages; and (iii) the inefficient delivery to the target site at effective concentrations. Recently, a derivative of the frog skin AMP esculentin-1a, named esculentin-1a(1–21)NH2, [Esc(1–21): GIFSKLAGKKIKNLLISGLKG-NH2] has been found to have a potent activity against the Gram-negative bacterium Pseudomonas aeruginosa; a slightly weaker activity against Gram-positive bacteria and interesting immunomodulatory properties. With the aim to optimize the antimicrobial features of Esc(1–21) and to circumvent the limitations described above, two different approaches were followed: (i) substitutions by non-coded amino acids, i.e., α-aminoisobutyric acid or d-amino acids; and (ii) peptide conjugation to gold nanoparticles. In this mini-review, we summarized the structural and functional properties of the resulting Esc(1–21)-derived compounds. Overall, our data may assist researchers in the rational design and optimization of AMPs for the development of future drugs to fight the worldwide problem of antibiotic resistance
Esculentin-1a(1-21)NH2 and its diastereomer: antibacterial and immunomodulating activities
No full textMicrobial resistance to conventional antibiotics has become a major challenge. In this context, naturally-occurring antimicrobial peptides (AMPs) hold promise for the development of new drugsagainst microbial infections, including those caused by the bacterium Pseudomonas aeruginosa in the lungs of cystic fibrosis (CF) sufferers. Here we report on the in vitro activities of the frog-skin derived AMP Esculentin-1a(1-21)NH2 [Esc(1-21)], and its diastereomer Esc(1-21)-1c, containing two D-amino acids, on both macrophages and bronchial cells, which express either the functional or the ΔF508 mutant of the CF transmembrane conductance regulator (1). We found that the diastereomer is significantly less toxic; has significantly higher efficacy in killing intracellular Pseudomonas; has a higher activity in promoting migration of bronchial cells; disaggregates and detoxifies the bacterial lipopolysaccharide. These results support further studies towards the development of the Esc(1-21)-1c for local treatment of P. aeruginosainduced lung infections. This work was funded by Italian Cystic Fibrosis Research Foundation (FFC #11/2014)
Effects of aib residues insertion on the structural–functional properties of the frog skin-derived peptide esculentin-1a(1–21)NH2
No full textAntimicrobial peptides (AMPs) play a key role in the defence mechanism of living organisms against microbial pathogens, displaying both bactericidal and immunomodulatory properties. They are considered as a promising alternative to the conventional antibiotics towards which bacteria are becoming highly resistant. Recently, a derivative of the frog skin AMP esculentin-1a, esculentin-1a(1-21)NH2 [Esc(1-21)], showed a strong and fast membranolytic activity against Gram-negative bacteria but with a lower efficacy against Gram-positive ones. Here, with the aim to increase the α-helicity of Esc(1-21) and the expected potency against Gram-positive bacteria, we designed an analog bearing three α-aminoisobutyric acid (Aib) residues at positions 1, 10, and 18 of its primary structure. We demonstrated that the incorporation of Aib residues: (1) promoted the α-helix conformation of Esc(1-21), as confirmed by circular dichroism and two-dimensional nuclear magnetic resonance spectroscopies; (2) was sufficient to make this analog more active than the parent peptide against several Gram-positive bacterial strains without affecting its activity against Gram-negative bacteria; and (3) resulted to be devoid of toxic effect toward epithelial cells at the active antimicrobial concentrations. These results suggest that replacement of L-amino acids with Aib residues has beneficial effects on the structure and properties of the membrane-active peptide Esc(1-21), making it a better candidate for the design and development of selective drugs against Gram-positive bacteria
Naturally occurring peptides from Rana temporaria: antimicrobial properties and more
No full textThe extensive search for alternative therapeutics against microbial pathogens has led to the discovery of cationic peptides as new anti-infectives with a novel mode of action. Particular interest has been devoted to small linear peptides that can be efficiently made by chemical synthesis at competitive costs. The most promising originate from a large family of short, naturally occurring peptides found in the skin of amphibia of Rana genus, i.e. the temporins. This review is mainly focused on the recent structure-function studies of the earliest known temporin isoforms (TA, TB and TL) and their potential clinical role as novel antimicrobial agents. The development of novel antibiotics is an urgent public health concern due to the increased resistance of microorganisms to conventional antibiotics, particularly in the hospital setting
Antimicrobial peptides and their multiple effects at sub-inhibitory concentrations
No full textThe frequent occurrence of multidrug-resistant strains to conventional antimicrobials has led to a clear decline of antibiotic therapies. Therefore, new molecules with different mechanisms of action are extremely necessary. Due to their unique properties, antimicrobial peptides (AMPs) represent a valid alternative to conventional antibiotics and many of them have been characterized for their activity and cytotoxicity. However, the effects that these peptides cause at concentrations below the minimum growth inhibitory concentration (MIC) have yet to be fully analyzed along with the underlying molecular mechanism. In this mini-review, the ability of AMPs to synergize with different antibiotic classes or different natural compounds is examined. Furthermore, data on microbial resistance induction are reported to highlight the importance of antibiotic resistance in the fight against infections. Finally, the effects that sub-MIC levels of AMPs can have on the bacterial pathogenicity are summarized while showing how signaling pathways can be valid therapeutic targets for treatment of infectious diseases. All these aspects support the high potential of AMPs as lead compounds for the development of new drugs with antibacterial and immunomodulatory activities
The Antimicrobial Peptide Esculentin-1a(1-21)NH2: A Novel Promoter of Human Skin Wound Healing?
No full textAntimicrobial peptides (AMPs) are produced by all living organisms as a first line defense against invading microorganisms. In Amphibia, they are mainly present in the skin to form a protective chemical barrier between tissues and external environment. Esculentin-1a(1-21)NH2 [Esc(1-21)] is an amphibian skin-derived AMP with a broad spectrum of activity [1]. Unlike the human skin AMP LL-37, Esc(1-21) preserves its antimicrobial activity in biological fluids and is significantly less toxic at high concentrations. By using an in vitro modified scratch assay employing special cell-culture inserts, we found that Esc(1-21) significantly stimulates migration, but not proliferation, of immortalized human keratinocytes (HaCaT cells), more efficiently than LL-37. Importantly, this activity is preserved also on primary human epidermal keratinocytes [2]. Moreover, as previously shown for LL-37, we demonstrated that the Esc(1-21)-induced cell migration involves the activation of the epidermal growth factor receptor and STAT3 protein. Typical morphological changes associated with a migratory phenotype were detected in HaCaT cells upon treatment with Esc(1-21) [2]. These results focus on the wound healing-modulatory properties of Esc(1-21) and suggest it as a novel candidate promoter of skin re-epithelialisation, especially in the management of chronic human skin ulcers
Esculentin-1a(1-21)NH2: a promising peptide for prevention and eradicatin of Pseudomonas aeruginosa biofilm formation on soft contact-lenses
No full textContact lens wear is an important risk factor for microbial keratitis, a potentially vision threatening infection of the eye (1). Adverse events associated with microbial adhesion and colonization of lenses, especially by the biofilm forming Gram-negative bacterium Pseudomonas aeruginosa remain a major safety issue. This is further complicated by the increase in bacterial resistance to traditional antibiotics. Therefore, novel strategies to prevent and treat contact lens-associated keratitis are greatly needed. An important approach is the development of agents that hamper pathogen attachment and biofilm formation in the first place. Naturally occurring antimicrobial peptides (AMPs) hold particular promise in this regard. Esculentins are a family of AMPs derived from amphibian skin with a wide spectrum of antimicrobial activity. Esculentin-1a(1-21)NH2 [Esc(1-21)], that consists of the first 20 amino acids of the native Esculentin-1a, with a glycinamide residue at the C-terminus, is a novel AMP with a potent activity against both free-living and sessile forms of P. aeruginosa (2). Previous studies have shown its anti-Pseudomonal activity in the presence of human basal tears as well as a reduction of infection in a mouse model of P. aeruginosa keratitis (3). Here we investigated the peptide’s ability to inhibit and/or to disrupt biofilm formation on soft contact lenses using both reference strains and clinical isolates of P. aeruginosa. The percentage of surviving cells was evaluated by the 3(4,5-dimethylthiazol-2yl)2,5-diphenyltetrazolium bromide (MTT) assay. Our results indicate that Esc(1-21) is able to eradicate biofilm cells from contact-lenses at a concentration range of 4-16 μM, and to inhibit Pseudomonas biofilm when used at lower concentrations. The effects of Esc(1-21) on the morphology of biofilm cells on contact lenses were also visualized by scanning electron microscopy. Overall, our data suggest that Esc(1-21) has great potential for development as a novel pharmaceutical for prevention and treatment of contact lens-associated P. aeruginosa keratitis.
(1) (1) Robertson DM. Eye Contact Lens. 2013 Jan;39(1):67-72
(2) (2) Luca V et al. Cell Mol Life Sci. 2013 Aug;70(15):2773-86
(3) (3) Kolar SS et al. Cell Mol Life Sci. 2015 Feb;72(3):617-2
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