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.

Structure-activity relationship, conformational and biological studies of temporin L analogues.

Temporins are naturally occurring peptides with promising features, which could lead to the development of new drugs. Temporin-1Tl (TL) is the strongest antimicrobial peptide, but it is toxic on human erythrocytes and this fact makes the design of synthetic analogues with a higher therapeutic index vital.We studied the structure-activity relationships of a library of TL derivatives focusing on the correlation between the α-helix content of the peptides, the nature of their cationic residues, and their antibacterial/antiyeast/hemolytic activities. We found that the percentage of helicity of TL analogues is directly correlated to their hemolytic activity but not to their antimicrobial activity. In addition, we found that the nature of positively charged residues can affect the biological properties of TL without changing the peptide's helicity. It is noteworthy that a single amino acid substitution can prevent the antimicrobial activity of TL, making it a lytic peptide presumably due to its self-association. Last, we identified a novel analogue with properties that make it an attractive topic for future research.

Anti-Pseudomonas activity of frog skin antimicrobial peptides in a Caenorhabditis elegans infection model: a plausible mode of action in vitro and in vivo.

The emergence of multidrug-resistant (MDR) microorganisms makes it increasingly difficult to treat infections. These infections include those associated with Pseudomonas aeruginosa, which are 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 activities and modes of action have been reported. We investigated (i) the activity and mechanism underlying the killing of short CAMPs from frog skin (e.g., temporins and esculentin fragments) on an MDR clinical isolate of P. aeruginosa and (ii) their in vivo antibacterial activities and modes of action, using the minihost 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 within 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 of frog skin CAMPs in vivo, our data suggest that temporins and esculentins would be attractive molecules as templates for the development of new therapeutics against life-threatening infections.

Fluorescence and electron microscopy methods for exploring antimicrobial peptides mode(s) of action.

Due to the increasing resistance of microbial pathogens to the available drugs, the identification of new antimicrobial agents with a new mechanism of action is urgently needed. In this context, cationic antimicrobial peptides (AMPs) are considered promising candidates. Although there is evidence that, in contrast to conventional antibiotics, microbial membranes are the principal target of a large number of AMPs, thus making it difficult for the pathogen to acquire resistance, their mode(s) of action is not yet completely clear. Intense research is currently devoted to understand the effect(s) of AMPs on intact cells, either at sub-lethal or at lethal peptide concentrations, and fluorescence/electron microscopy techniques represent a valid tool to get insight into the damage caused by these molecules on the morphology and membrane structure of the target cell. We here present an overview of some microscopic methodologies to address this issue.

Esculentin-1b(1-18)--a membrane-active antimicrobial peptide that synergizes with antibiotics and modifies the expression level of a limited number of proteins in Escherichia coli.

Antimicrobial peptides constitute one of the main classes of molecular weapons deployed by the innate immune system of all multicellular organisms to resist microbial invasion. A good proportion of all antimicrobial peptides currently known, numbering hundreds of molecules, have been isolated from frog skin. Nevertheless, very little is known about the effect(s) and the mode(s) of action of amphibian antimicrobial peptides on intact bacteria, especially when they are used at subinhibitory concentrations and under conditions closer to those encountered in vivo. Here we show that esculentin-1b(1-18) [Esc(1-18)] (GIFSKLAGKKLKNLLISG-NH(2)), a linear peptide encompassing the first 18 residues of the full-length esculentin-1b, rapidly kills Escherichia coli at the minimal inhibitory concentration. The lethal event is concomitant with the permeation of the outer and inner bacterial membranes. This is in contrast to what is found for many host defense peptides, which do not destabilize membranes at their minimal inhibitory concentrations. Importantly, proteomic analysis revealed that Esc(1-18) has a limited ability to modify the bacterium's protein expression profile, at either bactericidal or sublethal concentrations. To the best of our knowledge, this is the first report on the effects of an antimicrobial peptide from frog skin on the proteome of its bacterial target, and underscores the fact that the bacterial membrane is the major target for the killing mechanism of Esc(1-18), rather than intracellular processes.

Esculentin 1-21: a linear antimicrobial peptide from frog skin with inhibitory effect on bovine mastitis-causing bacteria.

Mastitis, or inflammation of the mammary gland, is the most common and expensive illness of dairy cows throughout the world. Although stress and physical injuries may give rise to inflammation of the udders, infections by bacteria or other microorganisms remain the major cause, and infusion of antibiotics is the main treatment approach. However, the increased emergence of multidrug-resistant pathogens and the production of milk contaminated with antibiotics has become a serious threat in the livestock. Hence, there is an urgent need for the discovery of new therapeutic agents with a new mode of action. Gene-encoded AMPs, which represent the first line of defence in all living organisms, are considered as promising candidates for the development of new anti-infective agents. This paper reports on the antibacterial activities in vitro and in an animal model, of the frog skin AMP esculentin 1-21 [Esc(1-21)], along with a plausible mode of action. Our data revealed that this peptide (i) is highly potent against the most common mastitis-causing microbes (e.g. Streptococcus agalactiae); and (ii) is active in vivo, causing a visible regression of the clinical stage of mastitis in dairy cows, after 1 week of peptide treatment. Biophysical characterisation revealed that the peptide adopts an alpha-helical structure in microbial mimicking membranes and is able to permeate the membrane of S. agalactiae in a dose-dependent manner. Overall, these data suggest Esc(1-21) as an attractive AMP for the future design of new antibiotics to cure mastitis in cattle.