a-novel-in-vitro-wound-healing-assay-to-evaluate-cell-migration

A Novel <em>In Vitro</em> Wound Healing Assay to Evaluate Cell Migration

The aim of this work is to show a novel method to evaluate the ability of some immunomodulatory molecules, such as antimicrobial peptides (AMPs), to ...

Sapienza University of Rome

In the present study, the synergism of the lipopeptide bacillomycin D in combination with the polyene amphotericin B against pathogenic Candida species is described along with their potential cytotoxicity against mammalian cells. Bacillomycin D inhibited the growth of various Candida species at minimal concentrations from 12.5 to 25 μg ml(-1). Furthermore, it showed a synergistic effect with the antifungal drug amphotericin B in inhibiting the growth of Candida strains, with fractional inhibitory concentration indices ranging from 0.28 to 0.5. Time killing studies revealed a >2-log reduction in the viability of Candida albicans ATCC 10231 cells after 3 h incubation with the combination amphotericin B plus bacillomycin D, at their subinhibitory concentration. Interestingly, when the two drugs were used together at those dosages displaying a synergism in the anti-Candida activity, no cytotoxic effect was observed against mammalian cells. Therefore, the combination bacillomycin D/amphotericin B may represent a valid alternative to conventional antifungals for topical treatment of C. albicans infections. To the best of our knowledge, this is the first report describing the in vitro interaction between the antifungal drug amphotericin B and bacillomycin D against pathogenic Candida species.

Synergistic fungicidal activity of the lipopeptide bacillomycin D with amphotericin B against pathogenic Candida species.

The 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.

Naturally Occurring Peptides from Rana temporaria: Antimicrobial Properties and More.

Naturally occurring antimicrobial peptides (AMPs) represent promising future antibiotics. We have previously isolated esculentin-1a(1-21)NH2, a short peptide derived from the frog skin AMP esculentin-1a, with a potent anti-Pseudomonal activity. Here, we investigated additional functions of the peptide and properties responsible for these activities. For that purpose, we synthesized the peptide, as well as its structurally altered analog containing two D-amino acids. The peptides were then biophysically and biologically investigated for their cytotoxicity and immunomodulating activities. The data revealed that compared to the wild-type, the diastereomer: (1) is significantly less toxic towards mammalian cells, in agreement with its lower α-helical structure, as determined by circular dichroism spectroscopy; (2) is more effective against the biofilm form of Pseudomonas aeruginosa (responsible for lung infections in cystic fibrosis sufferers), while maintaining a high activity against the free-living form of this important pathogen; (3) is more stable in serum; (4) has a higher activity in promoting migration of lung epithelial cells, and presumably in healing damaged lung tissue, and (5) disaggregates and detoxifies the bacterial lipopolysaccharide (LPS), albeit less than the wild-type. Light scattering studies revealed a correlation between anti-LPS activity and the ability to disaggregate the LPS. Besides shedding light on the multifunction properties of esculentin-1a(1-21)NH2, the D-amino acid containing isomer may serve as an attractive template for the development of new anti-Pseudomonal compounds with additional beneficial properties. Furthermore, together with other studies, incorporation of D-amino acids may serve as a general approach to optimize the future design of new AMPs.

D-Amino acids incorporation in the frog skin-derived peptide esculentin-1a(1-21)NH2 is beneficial for its multiple functions.

Cationic antimicrobial peptides (CAMPs) are very promising in the treatment of multi-drug resistant Pseudomonas aeruginosa lung infections experienced by cystic fibrosis (CF) patients. Nevertheless, there is an urgent need of inhalable formulations able to deliver the intact CAMP in conductive airways and to shield its interactions with airway mucus/bacterial biofilm, thus enhancing CAMP/bacteria interactions. Along these lines, the aim of this work was the design and development of nano-embedded microparticles (NEM) for sustained delivery of CAMPs in the lung. To this purpose, nanoparticles (NPs) made of poly(lactide-co-glycolide) (PLGA) containing a model CAMP, colistin (Col), were produced by emulsion/solvent diffusion technique. Engineering NPs with chitosan (CS) and poly(vinyl alcohol) (PVA) allowed to modulate surface properties and, in so doing, to improve NP transport through artificial CF mucus. In order to achieve a long-term stable dosage form useful for NP inhalation, NPs were spray-dried in different carriers (lactose or mannitol), thus producing NEM. The most promising NEM formulations were selected on the basis of bulk and flow properties, distribution of NPs in the carrier and aerosolization performance upon delivery through a breath-actuated dry powder inhaler. Of note, selected Col-loaded NEM were found to kill P. aeruginosa biofilm and to display a prolonged efficacy in biofilm eradication compared to the free Col. This effect was likely ascribable to the ability of NPs to penetrate into bacterial biofilm, as demonstrated by confocal analysis, and to sustain Col release inside it. Taken all together, our results indicate that adequate engineering of PLGA NPs represents an enticing technological approach to harness novel antimicrobials for P. aeruginosa lung infection, such as CAMPs, especially in CF.

Overcoming barriers in Pseudomonas aeruginosa lung infections: Engineered nanoparticles for local delivery of a cationic antimicrobial peptide.

Naturally occurring antimicrobial peptides (AMPs) hold promise as future therapeutics against multidrug resistant microorganisms. Recently, we have discovered that a derivative of the frog skin AMP esculentin-1a, Esc(1-21), is highly potent against both free living and biofilm forms of the bacterial pathogen Pseudomonas aeruginosa. However, bringing AMPs into clinics requires to overcome their low stability, high toxicity and inefficient delivery to the target site at high concentrations. Importantly, peptide conjugation to gold nanoparticles (AuNPs), which are among the most applied inorganic nanocarriers in biomedical sciences, represents a valuable strategy to solve these problems. Here we report that covalent conjugation of Esc(1-21) to soluble AuNPs [AuNPs@Esc(1-21)] via a poly(ethylene glycol) linker increased by ∼15-fold the activity of the free peptide against the motile and sessile forms of P. aeruginosa without being toxic to human keratinocytes. Furthermore, AuNPs@Esc(1-21) resulted to be significantly more resistant to proteolytic digestion and to disintegrate the bacterial membrane at very low concentration (5nM). Finally, we demonstrated for the first time the capability of peptide-coated AuNPs to display a wound healing activity on a keratinocytes monolayer. Overall, these findings suggest that our engineered AuNPs can serve as attractive novel biological-derived material for topical treatment of epithelial infections and healing of the injured tissue.

Gold-nanoparticles coated with the antimicrobial peptide esculentin-1a(1-21)NHas a reliable strategy for antipseudomonal drugs.

Antimicrobial 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)NH[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.

Effects of Aib residues insertion on the structural-functional properties of the frog skin-derived peptide esculentin-1a(1-21)NH.

Ease of access to the cornea makes antimicrobial peptides (AMPs) ideal candidates for topical drug application. However, before bringing them to the clinic, it is fundamental to evaluate in vitro: (1) the ability of AMPs to kill bacteria in the presence of human tears, by counting the number of surviving bacteria on agar plates; (2) the potential cytotoxicity of AMPs to mammalian cells by a colorimetric method based on the production of a colored formazan crystals by metabolically active cells; and (3) the ability of AMPs to neutralize the toxic effect of the bacterial cell wall component, lipopolysaccharide (LPS), by measuring the level of the pro-inflammatory cytokine, TNF-α, released from LPS-activated macrophages, using a sandwich enzyme-linked immunosorbent assay.

Methods for In Vitro Analysis of Antimicrobial Activity and Toxicity of Anti-keratitis Peptides: Bacterial Viability in Tears, MTT, and TNF-α Release Assays.

In this study we designed and synthesized a new library of antimicrobial peptides correlated to [Pro,DLeu]TL 1, a temporin L derivative devoid of cytolytic effects in vitro, and investigated the correlation between the α-helical content of the compounds and their antibacterial, cytotoxic and hemolytic activities. We systematically replaced Gly in position 10 of reference peptide with several amino acids. Structure-activity relationship studies of these analogues were performed by means of antimicrobial and cytotoxicity assays along with CD spectroscopy analyses. NMR analysis was also accomplished for compound 10. As well, the most promising peptides were additionally evaluated for their activity against some clinical strains isolated from human skin and for their mechanism of action by studying the kinetics of membrane perturbation of some representative microbial strains. We identified novel analogues with interesting properties that make them attractive lead compounds for potential topical applications.

Glycine-replaced derivatives of [Pro,DLeu]TL, a temporin L analogue: Evaluation of antimicrobial, cytotoxic and hemolytic activities.

Antimicrobial peptides (AMPs) represent new alternatives to cope with the increasing number of multi-drug resistant microbial infections. Recently, a derivative of the frog-skin AMP esculentin-1a, Esc(1-21), was found to rapidly kill both the planktonic and biofilm forms of the Gram-negative bacterium Pseudomonas aeruginosa with a membrane-perturbing activity as a plausible mode of action. Lately, its diastereomer Esc(1-21)-1c containing two d-amino acids i.e.Leu14 andSer17 revealed to be less cytotoxic, more stable to proteolytic degradation and more efficient in eradicating Pseudomonas biofilm. When tested in vitro against the free-living form of this pathogen, it displayed potent bactericidal activity, but this was weaker than that of the all-l peptide. To investigate the reason accounting for this difference, mechanistic studies were performed on Pseudomonas spheroplasts and anionic or zwitterionic membranes, mimicking the composition of microbial and mammalian membranes, respectively. Furthermore, structural studies by means of optical and nuclear magnetic resonance spectroscopies were carried out. Our results suggest that the different extent in the bactericidal activity between the two isomers is principally due to differences in their interaction with the bacterial cell wall components. Indeed, the lower ability in binding and perturbing anionic phospholipid bilayers for Esc(1-21)-1c contributes only in a small part to this difference, while the final effect of membrane thinning once the peptide is inserted into the membrane is identical to that provoked by Esc(1-21). In addition, the presence of two d-amino acids is sufficient to reduce the α-helical content of the peptide, in parallel with its lower cytotoxicity.

Membrane perturbing activities and structural properties of the frog-skin derived peptide Esculentin-1a(1-21)NHand its Diastereomer Esc(1-21)-1c: Correlation with their antipseudomonal and cytotoxic activity.

Contact lens (CL) wear is a risk factor for development of microbial keratitis, a vision threatening infection of the eye. Adverse events associated with colonization of lenses, especially by the multi-drug resistant and biofilm forming bacterium Pseudomonas aeruginosa remain a major safety issue. Therefore, novel strategies and compounds to reduce the onset of CL-associated ocular infections are needed. Recently, the activity of the frog skin-derived antimicrobial peptide Esc(1-21) and its diastereomer Esc(1-21)-1c was evaluated against both planktonic and sessile forms of this pathogen. Furthermore, Esc(1-21) was found to significantly reduce the severity of P. aeruginosa keratitis in a mouse model and preserve antipseudomonal activity in the presence of human basal tears. Here, we have analyzed the activity of the peptides on P. aeruginosa biofilm formed on soft CLs. Microbiological assays and scanning electron microscopy analysis indicated that the peptides were able to disrupt the bacterial biofilm, with the diastereomer having the greater efficacy (up to 85% killing vs no killing at 4 μM for some strains). Furthermore, upon covalent immobilization to the CL, the two peptides were found to cause more than four log reduction in the number of bacterial cells within 20 minutes and to reduce bacterial adhesion to the CL surface (77%-97% reduction) in 24 hours. Importantly, peptide immobilization was not toxic to mammalian cells and did not affect the lens characteristics. Overall, our data suggest that both peptides have great potential to be developed as novel pharmaceuticals for prevention and treatment of CL-associated P. aeruginosa keratitis.

Esculentin-1a derived peptides kill Pseudomonas aeruginosa biofilm on soft contact lenses and retain antibacterial activity upon immobilization to the lens surface.

Temporin A (FLPLIGRVLSGIL-NH), temporin F (FLPLIGKVLSGIL-NH), and temporin G (FFPVIGRILNGIL-NH), first identified in skin secretions of the frog Rana temporaria, produced concentration-dependent stimulation of insulin release from BRIN-BD11 rat clonal β-cells at concentrations ≥1 nM, without cytotoxicity at concentrations up to 3 μM. Temporin A was the most effective. The mechanism of insulinotropic action did not involve an increase in intracellular Caconcentrations. Temporins B, C, E, H, and K were either inactive or only weakly active. Temporins A, F, and G also produced a concentration-dependent stimulation of insulin release from 1.1B4 human-derived pancreatic β-cells, with temporin G being the most potent and effective, and from isolated mouse islets. The data indicate that cationicity, hydrophobicity, and the angle subtended by the charged residues in the temporin molecule are important determinants for in vitro insulinotropic activity. Temporin A and F (1 μM), but not temporin G, protected BRIN-BD11 cells against cytokine-induced apoptosis (P < 0.001) and augmented (P < 0.001) proliferation of the cells to a similar extent as glucagon-like peptide-1. Intraperitoneal injection of temporin G (75 nmol/kg body weight) together with a glucose load (18 mmol/kg body weight) in C57BL6 mice improved glucose tolerance with a concomitant increase in insulin secretion whereas temporin A and F administration was without significant effect on plasma glucose levels. The study suggests that combination therapy involving agents developed from the temporin A and G sequences may find application in Type 2 diabetes treatment.

Article	Year
Synergistic fungicidal activity of the lipopeptide bacillomycin D with amphotericin B against pathogenic Candida species. FEMS yeast research\| PubMed ID: 25956541	2015
Naturally Occurring Peptides from Rana temporaria: Antimicrobial Properties and More. Current topics in medicinal chemistry\| PubMed ID: 26139114	2016
D-Amino acids incorporation in the frog skin-derived peptide esculentin-1a(1-21)NH2 is beneficial for its multiple functions. Amino acids\| PubMed ID: 26162435	2015
Overcoming barriers in Pseudomonas aeruginosa lung infections: Engineered nanoparticles for local delivery of a cationic antimicrobial peptide. Colloids and surfaces. B, Biointerfaces\| PubMed ID: 26340361	2015
Gold-nanoparticles coated with the antimicrobial peptide esculentin-1a(1-21)NHas a reliable strategy for antipseudomonal drugs. Acta biomaterialia\| PubMed ID: 27693686	2017
Effects of Aib residues insertion on the structural-functional properties of the frog skin-derived peptide esculentin-1a(1-21)NH. Amino acids\| PubMed ID: 27726008	2017
Methods for In Vitro Analysis of Antimicrobial Activity and Toxicity of Anti-keratitis Peptides: Bacterial Viability in Tears, MTT, and TNF-α Release Assays. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 28013521	2017
Glycine-replaced derivatives of [Pro,DLeu]TL, a temporin L analogue: Evaluation of antimicrobial, cytotoxic and hemolytic activities. European journal of medicinal chemistry\| PubMed ID: 28863356	2017
Membrane perturbing activities and structural properties of the frog-skin derived peptide Esculentin-1a(1-21)NHand its Diastereomer Esc(1-21)-1c: Correlation with their antipseudomonal and c... Biochimica et biophysica acta\| PubMed ID: 28912103	2017
Esculentin-1a derived peptides kill Pseudomonas aeruginosa biofilm on soft contact lenses and retain antibacterial activity upon immobilization to the lens surface. Biopolymers\| PubMed ID: 29086910	2017
Assessment of the potential of temporin peptides from the frog Rana temporaria (Ranidae) as anti-diabetic agents. Journal of peptide science : an official publication of the European Peptide Society\| PubMed ID: 29349894	2018

Bruno Casciaro

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