Vincenzo Luca

Esculentin(1-21), an amphibian skin membrane-active peptide with potent activity on both planktonic and biofilm cells of the bacterial pathogen Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that forms sessile communities, named biofilms. The non-motile forms are very difficult to eradicate and are often associated with the establishment of persistent infections, especially in patients with cystic fibrosis. The resistance of P. aeruginosa to conventional antibiotics has become a growing health concern worldwide and has prompted the search for new anti-infective agents with new modes of action. Naturally occurring antimicrobial peptides (AMPs) represent promising future template candidates. Here we report on the potent activity and membrane-perturbing effects of the amphibian AMP esculentin(1-21), on both the free-living and sessile forms of P. aeruginosa, as a possible mechanism for biofilm disruption. Furthermore, the findings that esculentin(1-21) is able to prolong survival of animals in models of sepsis and pulmonary infection indicate that this peptide can be a promising template for the generation of new antibiotic formulations to advance care of infections caused by P. aeruginosa.

Isomerization of an Antimicrobial Peptide Broadens Antimicrobial Spectrum to Gram-Positive Bacterial Pathogens

The branched M33 antimicrobial peptide was previously shown to be very active against Gram-negative bacterial pathogens, including multidrug-resistant strains. In an attempt to produce back-up molecules, we synthesized an M33 peptide isomer consisting of D-aminoacids (M33-D). This isomeric version showed 4 to 16-fold higher activity against Gram-positive pathogens, including Staphylococcus aureus and Staphylococcus epidermidis, than the original peptide, while retaining strong activity against Gram-negative bacteria. The antimicrobial activity of both peptides was influenced by their differential sensitivity to bacterial proteases. The better activity shown by M33-D against S. aureus compared to M33-L was confirmed in biofilm eradication experiments where M33-L showed 12% activity with respect to M33-D, and in vivo models where Balb-c mice infected with S. aureus showed 100% and 0% survival when treated with M33-D and M33-L, respectively. M33-D appears to be an interesting candidate for the development of novel broad-spectrum antimicrobials active against bacterial pathogens of clinical importance.

The effect of d-amino acid substitution on the selectivity of temporin L towards target cells: Identification of a potent anti-Candida peptide ☆ ☆☆

The frog skin peptide temporin L (TL, 13-residues long) has a wide and potent spectrum of antimicrobial activity, but it is also toxic on mammalian cells at its microbicidal concentrations. Previous studies have indicated that its analogue [Pro(3)]TL has a slightly reduced hemolytic activity and a stable helical conformation along residues 6-13. Here, to expand our knowledge on the relationship between the extent/position of α-helix in TL and its biological activities, we systematically replaced single amino acids within the α-helical domain of [Pro(3)]TL with the corresponding d isomers, known as helix breakers. Structure-activity relationship studies of these analogues, by means of CD and NMR spectroscopy analyses as well as antimicrobial and hemolytic assays were performed. Besides increasing our understanding on the structural elements that are responsible for cell selectivity of TL, this study revealed that a single l to d amino acid substitution can preserve strong anti-Candida activity of [Pro(3)]TL, without giving a toxic effect towards human cells.

Membrane interaction and antibacterial properties of two mildly cationic peptide diastereomers, bombinins H2 and H4, isolated from Bombina skin

Bombinins H are mildly cationic antimicrobial peptides isolated from the skin of the anuran genus Bombina, the fire-bellied toad. Some members of this peptide family coexist in skin secretions as diastereomers in which a single d-amino acid (alloisoleucine or leucine) is incorporated as a result of the post-translational modification of the respective gene-encoded l-amino acid. Here we report on the antimicrobial properties and membrane interactions of bombinins H2 and H4. The latter differs from H2 by the presence of a d-alloisoleucine at the second N-terminal position. Specifically, we have evaluated the antimicrobial activity of H2 and H4 against a large panel of reference and clinical isolates of Gram-negative and Gram-positive bacteria; performed membrane permeation assays on both intact cells and model membranes (lipid monolayers and liposomes) mimicking the composition of the plasma membrane of Gram-negative/positive bacteria; used biochemical tools, such as trypsin-encapsulated liposomes and capillary electrophoresis, to monitor the peptides’ ability to translocate through the membrane of liposomes mimicking Escherichia coli inner membrane. The results revealed interesting relationships between the presence of a single d-amino acid in the sequence of an antimicrobial peptide and its target microbial cell selectivity/membrane-perturbing activity.

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

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.

Temporins A and B Stimulate Migration of HaCaT Keratinocytes and Kill Intracellular Staphylococcus aureus

ABSTRACT The growing number of microbial pathogens resistant to available antibiotics is a serious threat to human life. Among them is the bacterium Staphylococcus aureus, which colonizes keratinocytes, the most abundant cell type in the epidermis. Its intracellular accumulation complicates treatments against resulting infections, mainly due to the limited diffusion of conventional drugs into the cells. Temporins A (Ta) and B (Tb) are short frog skin antimicrobial peptides (AMPs). Despite extensive studies regarding their antimicrobial activity, very little is known about their activity on infected cells or involvement in various immunomodulatory functions. Here we show that Tb kills both ATCC-derived and multidrug-resistant clinical isolates of S. aureus within infected HaCaT keratinocytes (80% and 40% bacterial mortality, respectively) at a nontoxic concentration, i.e., 16 μM, whereas a weaker effect is displayed by Ta. Furthermore, the peptides prevent killing of keratinocytes by the invading bacteria. Further studies revealed that both temporins promote wound healing in a monolayer of HaCaT cells, with front speed migrations of 19 μm/h and 12 μm/h for Ta and Tb, respectively. Migration is inhibited by mitomycin C and involves the epidermal growth factor receptor (EGFR) signaling pathway. Finally, confocal fluorescence microscopy indicated that the peptides diffuse into the cells. By combining antibacterial and wound-healing activities, Ta and Tb may act as multifunctional mediators of innate immunity in humans. Particularly, their nonendogenous origin may reduce microbial resistance to them as well as the risk of autoimmune diseases in mammals.

Enhanced Amphiphilic Profile of a Short β-Stranded Peptide Improves Its Antimicrobial Activity

SB056 is a novel semi-synthetic antimicrobial peptide with a dimeric dendrimer scaffold. Active against both Gram-negative and -positive bacteria, its mechanism has been attributed to a disruption of bacterial membranes. The branched peptide was shown to assume a β-stranded conformation in a lipidic environment. Here, we report on a rational modification of the original, empirically derived linear peptide sequence [WKKIRVRLSA-NH2, SB056-lin]. We interchanged the first two residues [KWKIRVRLSA-NH2, β-SB056-lin] to enhance the amphipathic profile, in the hope that a more regular β-strand would lead to a better antimicrobial performance. MIC values confirmed that an enhanced amphiphilic profile indeed significantly increases activity against both Gram-positive and -negative strains. The membrane binding affinity of both peptides, measured by tryptophan fluorescence, increased with an increasing ratio of negatively charged/zwitterionic lipids. Remarkably, β-SB056-lin showed considerable binding even to purely zwitterionic membranes, unlike the original sequence, indicating that besides electrostatic attraction also the amphipathicity of the peptide structure plays a fundamental role in binding, by stabilizing the bound state. Synchrotron radiation circular dichroism and solid-state 19F-NMR were used to characterize and compare the conformation and mobility of the membrane bound peptides. Both SB056-lin and β-SB056-lin adopt a β-stranded conformation upon binding POPC vesicles, but the former maintains an intrinsic structural disorder that also affects its aggregation tendency. Upon introducing some anionic POPG into the POPC matrix, the sequence-optimized β-SB056-lin forms well-ordered β-strands once electro-neutrality is approached, and it aggregates into more extended β-sheets as the concentration of anionic lipids in the bilayer is raised. The enhanced antimicrobial activity of the analogue correlates with the formation of these extended β-sheets, which also leads to a dramatic alteration of membrane integrity as shown by 31P-NMR. These findings are generally relevant for the design and optimization of other membrane-active antimicrobial peptides that can fold into amphipathic β-strands.

Novel α-MSH Peptide Analogues with Broad Spectrum Antimicrobial Activity

Previous investigations indicate that α-melanocyte-stimulating hormone (α-MSH) and certain synthetic analogues of it exert antimicrobial effects against bacteria and yeasts. However, these molecules have weak activity in standard microbiology conditions and this hampers a realistic clinical use. The aim in the present study was to identify novel peptides with broad-spectrum antimicrobial activity in growth medium. To this purpose, the Gly10 residue in the [DNal(2′)-7, Phe-12]-MSH(6–13) sequence was replaced with conventional and unconventional amino acids with different degrees of conformational rigidity. Two derivatives in which Gly10 was replaced by the residues Aic and Cha, respectively, had substantial activity against Candida strains, including C. albicans, C. glabrata, and C. krusei and against gram-positive and gram-negative bacteria. Conformational analysis indicated that the helical structure along residues 8–13 is a key factor in antimicrobial activity. Synthetic analogues of α-MSH can be valuable agents to treat infections in humans. The structural preferences associated with antimicrobial activity identified in this research can help further development of synthetic melanocortins with enhanced biological activity.

Alanine scanning analysis and structure–function relationships of the frog‐skin antimicrobial peptide temporin‐1Ta

The increasing resistance of bacteria and fungi to the available antibiotic/antimycotic drugs urges for a search for new anti‐infective compounds with new modes of action. In line of this, natural CAMPs represent promising and attractive candidates. Special attention has been devoted to frog‐skin temporins, because of their short size (10–14 residues long) and their unique features. In particular, temporin‐1Ta has the following properties: (i) it is mainly active on Gram‐positive bacteria; (ii) it can synergize, when combined with temporin‐1Tl, in inhibiting both gram‐negative bacterial growth and the toxic effect of LPS; (iii) it preserves biological activity in the presence of serum; and (iv) it is practically not hemolytic. Rational design of CAMPs represents a straightforward approach to obtain a peptide with a better therapeutic index. Here, we used alanine scanning analogs to elucidate the contribution of the side chains of each amino acid residue to the peptides antimicrobial and hemolytic activity. Beside providing insight into the biophysical attributes and the critical positions within the peptide sequence, which govern the antimicrobial/hemolytic activity of this temporin isoform, our studies assist in optimizing the design of temporin‐based lead structures for the production of new anti‐infective agents. Copyright

Naturally occurring peptides from Rana temporaria: Antimicrobial properties and more

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.