Glycine-replaced derivatives of [Pro3,DLeu9]TL, a temporin L analogue: Evaluation of antimicrobial, cytotoxic and hemolytic activities.

In this study we designed and synthesized a new library of antimicrobial peptides correlated to [Pro3,DLeu9]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.

Toward an improved structural model of the frog-skin antimicrobial peptide esculentin-1b(1-18).

Antimicrobial peptides (AMPs) are found in various classes of organisms as part of the innate immune system. Despite high sequence variability, they share common features such as net positive charge and an amphipathic fold when interacting with biologic membranes. Esculentin-1b is a 46-mer frog-skin peptide, which shows an outstanding antimicrobial activity. Experimental studies revealed that the N-terminal fragment encompassing the first 18 residues, Esc(1-18), is responsible for the antimicrobial activity of the whole peptide, with a negligible toxicity toward eukaryotic cells, thus representing an excellent candidate for future pharmaceutical applications. Similarly to most of the known AMPs, Esc(1-18) is expected to act by destroying/permeating the bacterial plasma-membrane but, to date, its 3D structure and the detailed mode of action remains unexplored. Before an in-depth investigation on peptide/membranes interactions could be undertaken, it is necessary to characterize peptide's folding propensity in solution, to understand what is intrinsically due to the peptide sequence, and what is actually driven by the membrane interaction. Circular dichroism and nuclear magnetic resonance spectroscopy were used to determine the structure adopted by the peptide, moving from water to increasing amounts of trifluoroethanol. The results showed that Esc(1-18) has a clear tendency to fold in a helical conformation as hydrophobicity of the environment increases, revealing an intriguing amphipathic structure. The helical folding is adopted only by the N-terminal portion of the peptide, while the rest is unstructured. The presence of a hydrophobic cluster of residues in the C-terminal portion suggests its possible membrane-anchoring role.

NMR structures and interactions of temporin-1Tl and temporin-1Tb with lipopolysaccharide micelles: mechanistic insights into outer membrane permeabilization and synergistic activity.

Temporins are a group of closely related short antimicrobial peptides from frog skin. Lipopolysaccharide (LPS), the major constituent of the outer membrane of gram-negative bacteria, plays important roles in the activity of temporins. Earlier studies have found that LPS induces oligomerization of temporin-1Tb (TB) thus preventing its translocation across the outer membrane and, as a result, reduces its activity on gram-negative bacteria. On the other hand, temporin-1Tl (TL) exhibits higher activity, presumably because of lack of such oligomerization. A synergistic mechanism was proposed, involving TL and TB in overcoming the LPS-mediated barrier. Here, to gain insights into interactions of TL and TB within LPS, we investigated the structures and interactions of TL, TB, and TL+TB in LPS micelles, using NMR and fluorescence spectroscopy. In the context of LPS, TL assumes a novel antiparallel dimeric helical structure sustained by intimate packing between aromatic-aromatic and aromatic-aliphatic residues. By contrast, independent TB has populations of helical and aggregated conformations in LPS. The LPS-induced aggregated states of TB are largely destabilized in the presence of TL. Saturation transfer difference NMR studies have delineated residues of TL and TB in close contact with LPS and enhanced interactions of these two peptides with LPS, when combined together. Fluorescence resonance energy transfer and (31)P NMR have pointed out the proximity of TL and TB in LPS and conformational changes of LPS, respectively. Importantly, these results provide the first structural insights into the mode of action and synergism of antimicrobial peptides at the level of the LPS-outer membrane.

An integrated analysis of the effects of Esculentin 1-21 on Saccharomyces cerevisiae.

The antimicrobial peptide esculentin 1-21 (Esc 1-21) is a shorter synthetic version of the 46-residue peptide occurring in the Rana esculenta skin secretion. Here we propose an integrated proteomic and transcriptomic approach to interpret the biological effects of this peptide on Saccharomyces cerevisiae. We further investigated the response to this peptide by correlating the results of the transcriptome and proteome analysis with phenotypic effects. The results show that S. cerevisiae adapts to Esc 1-21 using the High Osmolarity Glycerol (HOG) pathway involved in osmotic tolerance and cell wall maintenance. Comparative proteomics reveals that Esc 1-21 causes downregulation of enzymes of the lower glycolytic pathway and in genes involved in spindle body formation and remodelling of cell-wall synthesis. Moreover the peptide induces downexpression of protein actin within 45 min and cells pre-treated with peptide show less sensitivity to osmotic stress and increased sensitivity to heat shock stress. The results obtained with the two different methodologies are in agreement at the cellular process levels. A combined approach may help elucidate the main aspects related to the effects of this peptide on the eukaryotic cell. The employment of different technologies may reveal the potential and limitations of each adapted approach in a prospective application for drug screening.