Biological characterization and modes of action of temporins and bombinins h, multiple forms of short and mildly cationic antimicrobial peptides from amphibian skin

Genetically encoded cationic anti-microbial peptides (AMPs) are essential components of the ancient and non-specific innate immune system, which is the principal defence mechanism of all species of life, with the primary role to kill infectious microorganisms. Amphibian skin is one of the richest natural sources of such molecules, which are produced by holocrine-type dermal glands and released upon stimulation. This review highlights the attractive and unique structural/functional properties of temporins and bombinins H, two families of short and mildly cationic peptides, isolated from the skin of frogs belonging to Rana and Bombina genera, respectively. Beside improving our knowledge on the role of AMPs in the regulation of the innate immunity, the biological significance of the existence of multiple forms of a prototypic peptide sequence within the same organism and the implication of short peptides in the endotoxin neutralization, these two classes of AMPs can be also considered as valid candidates for the design of novel anti-infective and anti-sepsis drug

Anti-pseudomonas activity of frog skin antimicrobial peptide in Caenorhabditis elegans infection model.

The emergence of multidrug-resistant (MDR) microorganisms makes it increasingly difficult to treat infections. These infections include those associated with Pseudomonas aeruginosa, which is hard to eradicate, especially in patients with a compromised immune system. Naturally occurring membrane-active cationic antimicrobial peptides (CAMPs) serve as attractive candidates for the development of new therapeutic agents. Amphibian skin is one of the richest sources for such peptides, but only a few studies on their in vivo activity and mode of action were reported. Here we investigated: (i) the activity and mechanism underlying the killing of short CAMPs from frog skin (e.g., temporins and esculentin fragments) on a MDR clinical isolate of P. aeruginosa; (ii) their in vivo antimicrobial activity and mode of action, using the mini-host model of Caenorhabditis elegans. Our data revealed that in vivo, both temporin-1Tb and esculentin(1-18) were highly active in promoting the survival of pseudomonas-infected nematodes, although temporin-1Tb did not show significant activity in vitro, under the experimental conditions used. Importantly, esculentin(1-18) permeated the membrane of Pseudomonas cells inside the gut of the infected nematode. To the best of our knowledge, this is the first report showing the ability of a CAMP to permeate the microbial membrane within a living organism. Besides shedding light on a plausible mode of action in vivo of frog skin CAMPs, our data suggest that temporins and esculentins would be attractive molecules as templates for the development of new therapeutics against life-threatening infections

The effect of d-amino acids in the target cell selectivity of the frog skin peptide temporin L.

Temporin L (TL, 13-residues long) is a frog-skin peptide with a wide and potent spectrum of antimicrobial activity, but with a toxic effect on mammalian cells at its microbicidal concentration. Previous studies indicated [Pro3]TL as an analog with a slightly lower hemolytic activity than the native TL. Here, a systematic replacement of single residues within the alpha-helix domain of Pro3 TL (Lys7 to Leu13) with D aminoacids, known as helix breakers, has been carried out. Structure-activity relationship studies, by means of CD/NMR spectroscopy analysis and antimicrobial/hemolytic assays have been performed leading to a better understanding on the structural elements that are responsible for the cell selectivity of TL. Most importantly, we have found how a single L-to D aminoacid substitution can preserve the strong anti-candida activity of [Pro3]TL, making it completely harmless towards human cells

Comparative Analysis of the Bactericidal Activities of Amphibian Peptide Analogues against Multidrug-Resistant Nosocomial Bacterial Strains▿

Due to the widespread resistance of bacteria to the available drugs, the discovery of new classes of antibiotics is urgently needed, and naturally occurring antimicrobial peptides (AMPs) are considered promising candidates for future therapeutic use. Amphibian skin is one of the richest sources of such AMPs. In the present study we compared the in vitro bactericidal activities of five AMPs from three different species of anurans against multidrug-resistant clinical isolates belonging to species often involved in nosocomial infections (Staphylococcus aureus, Enterococcus faecium, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, and Acinetobacter baumannii). The peptides tested were temporins A, B, and G from Rana temporaria; the fragment from positions 1 to 18 of esculentin 1b [Esc(1-18)] from Rana esculenta; and bombinin H2 from Bombina variegata. When they were tested in buffer, all the peptides were bactericidal against all bacterial species tested (three strains of each species) at concentrations ranging from 0.5 to 48 μΜ, with only a few exceptions. The temporins were found to be more active against gram-positive bacteria, especially when they were assayed in human serum; Esc(1-18) showed fast and strong bactericidal activity, within 2 to 20 min, especially against the gram-negative species, which were killed by Esc(1-18) at concentrations ranging from 0.5 to 1 μΜ; bombinin H2 displayed similar bactericidal activity toward all isolates. Interestingly, while the activities of the temporins and bombinin H2 were almost completely inhibited in the presence of 20% human serum, the activity of Esc(1-18) against the gram-negative species was partially preserved in the presence of 40% serum. This property renders this peptide an attractive molecule for use in the development of new compounds for the treatment of infectious diseases

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.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