Caractérisation structurale et fonctionnelle des temporines-SH (de nouveaux peptides antimicrobiens isolés chez l'espèce de grenouille nord-africaine Pelophylax saharica)

Durant cette thèse, nous avons étudié le répertoire peptidique antimicrobien de la grenouille Pelophylax saharica. Nous avons identifié trois nouvelles temporines : Temp-SHa, -SHb et -SHc. Les temporines sont de petits peptides antimicrobiens (13-14 résidus) surtout actifs contre les bactéries Gram+. Seule la Temp-SHa s est révélée très active, particulièrement contre les bactéries Gram- et contre le parasite Leishmania infantum. Parmi les temporines (13 résidus, charge nette +2), la Temp-SHa présente la meilleure activité et le plus large spectre antimicrobien. L analogue [K3]Temp-SHa plus cationique que le peptide parent est, quant à lui, beaucoup plus antimicrobien. Il perturbe plus efficacement et sélectivement les modèles membranaires anioniques et perméabilise la membrane de bactéries Gram- (E. coli). La petite taille de l analogue [K3]Temp-SHa, son fort pouvoir antimicrobien et son large spectre d action en font un bon candidat pour une utilisation potentielle en thérapeutiquePARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceTunisiaFRT

Engineering of Antimicrobial Surfaces by Using Temporin Analogs to Tune the Biocidal/antiadhesive Effect

Proliferation of resistant bacteria on biomaterials is a major problem leading to nosocomial infections. Due to their broad-spectrum activity and their ability to disrupt bacterial membranes through a rapid membranolytic mechanism, antimicrobial peptides (AMPs) are less susceptible to the development of bacterial resistance and therefore represent good candidates for surface coating strategies to prevent biofilm formation. In this study, we report on the covalent immobilization of temporin-SHa, a small hydrophobic and low cationic antimicrobial peptide exhibiting broad-spectrum activity, and (SHa) analogs on modified gold surfaces. Several analogs derived from SHa with either a carboxamidated ([K3]SHa, d-[K3]SHa) or a carboxylated C-terminus ([K3]SHa-COOH) were used to achieve peptide grafting on gold surfaces modified by a thiolated self-assembled monolayer (SAM). Surface functionalization was characterized by polarization modulation infrared reflection absorption spectroscopy (PM-RAIRS) and X-ray photoemission spectroscopy (XPS). The antibacterial properties of the temporin-functionalized surfaces were tested against the Gram-positive Listeria ivanovii. Direct visualization of the peptide effects on the bacterial membrane was investigated by scanning electron microscopy equipped with a field emission gun (SEM-FEG). All active temporin analogs were successfully grafted and display significant antibacterial activity (from 80 to 90% killing efficiency) in addition to a 2-fold decrease of bacterial adhesion when all d-SHa analogs were used

The plasticins: Membrane adsorption, lipid disorders, and biological activity

International audienceThe present study investigates the relationships between structural polymorphism, adsorption onto membrane mimetic support, lipid disturbance, and biological activity of bactericidal 23-residue, glycine-leucine-rich dermaseptin orthologues from the Phyllomedusinae frog skin, the “plasticins”. Biological activities were evaluated using the membrane models DMPG (1,2-dimyristoyl-sn-glycero-3-phosphatidylglycerol) for prokaryotic membranes and DMPC (1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine) for eukaryotic membranes. We performed a conformational analysis of plasticins by molecular simulations and spectroscopic methods and analyzed phospholipid perturbations by infrared spectroscopy. Adsorption onto synthetic model membranes was quantified by surface plasmon resonance. Biological assays including antimicrobial and membrane potential-dissipating activities, together with hemolytic tests and imaging analysis of cytotoxicity, were carried out to clarify the peptide−membrane interactions. Two major groups were distinguished: (i) Neutral plasticins revealed the presence of strong β-structures with the zwitterionic or anionic phospholipid vesicles. They were weakly adsorbed in the range of antibacterial activity concentrations (micromolar). Nevertheless, for millimolar concentrations, they caused perturbations at the interface peptide−DMPG vesicles and in the bilayer alkyl chains, suggesting insertion into bacterial membranes. (ii) Cationic plasticins revealed multiple conformational transitions, including destabilized helix states, β-structures, and disordered states. Peptide−lipid complex densities depended on hydrophobic bond strengths. The most soluble cationic plasticins were strongly adsorbed, with stable peptide−lipid interactions inducing noticeable perturbations of bilayer alkyl chains, pointing out possible insertion into bacterial membranes. In contrast, cytotoxic plasticins were less adsorbed, with less stable peptide−lipid interactions causing membrane dehydration, formation of peptide−membrane hydrogen bonds, and little disturbances of lipid alkyl chains. These characteristics could be compatible with their putative action on intracellular targets leading to apoptosis

Improving the Selectivity of Antimicrobial Peptides from Anuran Skin

International audienceAnuran skin is known to be a rich source of antimicrobial peptides although their therapeutic potential is often limited due to their toxicity against mammalian cells. The analysis of structure-activity relationships among anuran antimicrobial peptides provided the parameters to construct the Mutator tool for improving their selectivity for bacterial cells, by suggesting appropriate point substitutions. Double substitution analogues [K2, K16] of the Xenopus tropicalis peptide XT-7 and [I2, K19] of the Ascaphus truei peptide ascaphin-8 were predicted by this tool to have an increased `therapeutic index' (TI = HC50/MIC for erythrocytes with respect to bacteria) > 80. The mutated peptides were synthesized and respectively found to have experimental TI values > 130 for S. aureus or E. coli, a considerable improvement with respect to TI 270, also due to a significant improvement in minimal inhibitory concentration. We have shown that the Mutator tool is capable of suggesting limited variations in natural anuran peptides capable of increasing peptide selectivity, by decreasing toxicity against mammalian erythrocytes, in general without compromising antibacterial activity. The tool is freely available on the Mutator Web server at http://split4.pmfst.hr/mutator/

Functional Characterization of Temporin-SHe, a New Broad-Spectrum Antibacterial and Leishmanicidal Temporin-SH Paralog from the Sahara Frog (Pelophylax saharicus)

International audienceAmphibian skin is a promising natural resource for antimicrobial peptides (AMPs), key effectors of innate immunity with attractive therapeutic potential to fight antibiotic-resistant pathogens. Our previous studies showed that the skin of the Sahara Frog (Pelophylax saharicus) contains broad-spectrum AMPs of the temporin family, named temporins-SH. Here, we focused our study on temporin-SHe, a temporin-SHd paralog that we have previously identified in this frog but was never structurally and functionally characterized. We synthesized and determined the structure of temporin-SHe. This non-amphipathic α-helical peptide was demonstrated to strongly destabilize the lipid chain packing of anionic multilamellar vesicles mimicking bacterial membranes. Investigation of the antimicrobial activity revealed that temporin-SHe targets Gram-negative and Gram-positive bacteria, including clinical isolates of multi-resistant Staphylococcus aureus strains. Temporin-SHe exhibited also antiparasitic activity toward different Leishmania species responsible for visceral leishmaniasis, as well as cutaneous and mucocutaneous forms. Functional assays revealed that temporin-SHe exerts bactericidal effects with membrane depolarization and permeabilization, via a membranolytic mechanism observed by scanning electron microscopy. Temporin-SHe represents a new member of the very limited group of antiparasitic temporins/AMPs. Despite its cytotoxicity, it is nevertheless an interesting tool to study the AMP antiparasitic mechanism and design new antibacterial/antiparasitic agent