Insight into the mechanism of action of temporin-SHa, a new broad-spectrum antiparasitic and antibacterial agent.

Antimicrobial peptides (AMPs) are promising drugs to kill resistant pathogens. In contrast to bacteria, protozoan parasites, such as Leishmania, were little studied. Therefore, the antiparasitic mechanism of AMPs is still unclear. In this study, we sought to get further insight into this mechanism by focusing our attention on temporin-SHa (SHa), a small broad-spectrum AMP previously shown to be active against Leishmania infantum. To improve activity, we designed analogs of SHa and compared the antibacterial and antiparasitic mechanisms. [K3]SHa emerged as a highly potent compound active against a wide range of bacteria, yeasts/fungi, and trypanosomatids (Leishmania and Trypanosoma), with leishmanicidal intramacrophagic activity and efficiency toward antibiotic-resistant strains of S. aureus and antimony-resistant L. infantum. Multipassage resistance selection demonstrated that temporins-SH, particularly [K3]SHa, are not prone to induce resistance in Escherichia coli. Analysis of the mode of action revealed that bacterial and parasite killing occur through a similar membranolytic mechanism involving rapid membrane permeabilization and depolarization. This was confirmed by high-resolution imaging (atomic force microscopy and field emission gun-scanning electron microscopy). Multiple combined techniques (nuclear magnetic resonance, surface plasmon resonance, differential scanning calorimetry) allowed us to detail peptide-membrane interactions. [K3]SHa was shown to interact selectively with anionic model membranes with a 4-fold higher affinity (KD = 3 x 10-8 M) than SHa. The amphipathic α-helical peptide inserts in-plane in the hydrophobic lipid bilayer and disrupts the acyl chain packing via a detergent-like effect. Interestingly, cellular events, such as mitochondrial membrane depolarization or DNA fragmentation, were observed in L. infantum promastigotes after exposure to SHa and [K3]SHa at concentrations above IC50. Our results indicate that these temporins exert leishmanicidal activity via a primary membranolytic mechanism but can also trigger apoptotis-like death. The many assets demonstrated for [K3]SHa make this small analog an attractive template to develop new antibacterial/antiparasitic drugs.

Fatty acids, essential oil and phenolics composition of Silybum marianum seeds and their antioxidant activities.

The presentstudydescribes the biochemical evaluation of Silybum marianum seed. The analysis of essential oil composition of Silybum marianum seed by Gas Chromatography-Mass Spectrometry GC-MS showed the presence of14 volatile components with the predominance of γ-cadinene (49.8%) and α-pinene (24.5%). Whereas, the analysis of fatty acids composition, showed the predominance of linoleic (50.5%) and oleic (30.2%) acids. Silybum marainum presented also an important polyphenol contents with 29mgGAE/g DW, a good antiradical activity (CI(50)=39µg/ml) but a lower reducing power ability. Flavonoid and condensed tannin contents were about 3.39mg EC/g DW and 1.8mg EC/gDW, respectively. The main phenolic compounds identified by RP-HPLC, were silybin A (12.2%), silybin B (17.67%), isosilybin A (21.9%), isosilybin B (12.8%), silychristin (7.9%) andsilydianin (7.5%).

Chemical composition, antioxidant and antimicrobial activities of the edible medicinal Ononis natrix growing wild in Tunisia.

Results showed that leaf methanolic extract of Ononis natrix has important total phenol (51 mg GAE/g DW) and flavonoid (14.76 CE/g DW) contents. The chemical composition of O. natrix leaf revealed the presence of quercitine (24.5%), amentoflavone (14.1%), flavones (11.3%) and kaempferol (10.5%). The leaf extract showed a high total antioxidant activity with 60.94 mg of GAE/g DW, displayed a high 2,2-diphenyl-1-picrylhydrazyl scavenging ability with low IC50 value (29 µg/mL) and a great reducing power (EC50 = 100 µg/mL). O. natrix leaf extract exhibited a significant broad spectrum activity against all tested microorganisms with bacterial inhibition zone sizes ranging from 8.5 to 17 mm in diameter.

Phenolic contents and antimicrobial activity of squirting cucumber (Ecballium elaterium) extracts against food-borne pathogens.

Squirting cucumber (Ecballium elaterium), is an indigenous species of the south of Europe and the Mediterranean basin, sometimes cultivated for its use as a medicinal plant. This study compared phenolic contents in Ecballium elaterium organs and their antimicrobial activities against some foodborne pathogens. Our results indicated that the plant organs had different total polyphenol contents (ranging from 6.744 to 46.848 mg GAE g(-1) DW) the leaves and fruits contained about 6-fold higher phenol contents than the root. The same tendency was observed for flavonoid and tannin levels. An interesting antimicrobial activity was also observed against the food pathogens at concentrations ranging from 0.004 to 2.5 mg ml(-1). Ecballium elaterium extracts might therefore be a potential source of preservative candidates for use in food or pharmaceutical industries.

Effects of residue 5-point mutation and N-terminus hydrophobic residues on temporin-SHc physicochemical and biological properties.

Temporin-SHc (FLSHIAGFLSNLFamide) first isolated from skin extraction of the Tunisian frog Pelophylax saharica, which shows potent antimicrobial activity against Gram-positive bacteria and is highly active against yeasts and fungi without hemolytic activity at antimicrobial concentrations. The peptide adopts well-defined α-helical conformation when bound to SDS micelles. In this study, we explored the effects of residue at position 5 and the N-terminus hydrophobic character on the hydrophilic/polar face of temp-SHc, on its biological activities (antimicrobial and hemolytic) and biophysical properties (hydrophobicity, amphipathicity and helicity). Antibacterial and hemolytic properties of temporin-SHc derivatives depend strongly on physicochemical properties. Therefore, slight decreasing amphipathicity together with hydrophobicity and helicity by the substitution Ile(5) → Leu decreased antimicrobial potency approximately twofold without changing of hemolytic activity. It is noteworthy that a conservative amino acid substitution decreases the antimicrobial activity, underlining the differences between Leu/Ile side chains insertion into the lipid bilayer. While the modification of N-terminal hydrophobic character by four residue inversion decreased amphipathicity (twofold) of (4-1)L5temp-SHc and resulted in an increase in antibacterial activity against E. coli, E. faecalis and C. parapsilosis of at least fourfold, its therapeutic potential is limited by its drastic increase of hemolysis (LC50 = 2 µM). We found that the percentage of helicity of temp-SHc analog is directly correlated to its hemolytic activity. Last, the hydrophobic N-terminal character is an important determinant of antimicrobial activity.

Antibacterial and leishmanicidal activities of temporin-SHd, a 17-residue long membrane-damaging peptide.

Temporins are a family of short antimicrobial peptides (8-17 residues) that mostly show potent activity against Gram-positive bacteria. Herein, we demonstrate that temporin-SHd, a 17-residue peptide with a net charge of +2 (FLPAALAGIGGILGKLF(amide)), expressed a broad spectrum of antimicrobial activity. This peptide displayed potent antibacterial activities against Gram-negative and Gram-positive bacteria, including multi-drug resistant Staphylococcus aureus strains, as well as antiparasitic activity against promastigote and the intracellular stage (amastigote) of Leishmania infantum, at concentration not toxic for the macrophages. Temporin-SHd that is structured in a non-amphipathic α-helix in anionic membrane-mimetic environments, strongly and selectively perturbs anionic bilayer membranes by interacting with the polar head groups and acyl region of the phospholipids, with formation of regions of two coexisting phases: one phase rich in peptide and the other lipid-rich. The disruption of lipid packing within the bilayer may lead to the formation of transient pores and membrane permeation/disruption once a threshold peptide accumulation is reached. To our knowledge, Temporin-SHd represents the first known 17-residue long temporin expressing such broad spectrum of antimicrobial activity including members of the trypanosomatidae family. Additionally, since only a few shorter members (13 residues) of the temporin family are known to display antileishmanial activity (temporins-TA, -TB and -SHa), SHd is an interesting tool to analyze the antiparasitic mechanism of action of temporins.

Characterization of Deinococcus sahariens sp. nov., a radiation-resistant bacterium isolated from a Saharan hot spring.

An ultraviolet-radiation-resistant, Gram-positive, orange-pigmented, thermophilic and strictly aerobic cocci was isolated from Saharan water hot spring in Tunisia. The newly isolated bacterium, designated HAN-23(T), was identified based on polyphasic taxonomy including genotypic, phenotypic and chemotaxonomic characterization. Phylogenetic analysis based on 16S rRNA gene sequences placed this strain within Deinococcus genus. However, strain HAN-23(T) is different from recognized species of the genus Deinococcus, showing less than 94.0% similarity values to its closest relatives. The predominant cellular fatty acids determined by gas chromatography were iso-C(15:0), iso-C(17:0) and iso C(17:1) ω9c. The major respiratory quinone was MK-8. The DNA G + C content was 66.9 mol%. DNA-DNA hybridization measurements revealed low DNA relatedness (6%) between the novel isolate and its closest neighbor, the type strain Deinococcus geothermalis DSM 11300. On the basis of the phenotypic, chemotaxonomic and phylogenetic data, strain HAN-23(T) represents a novel species of the genus Deinococcus, for which the name Deinococcus sahariens sp. nov. is proposed, the type strain being HAN-23(T) (=DSM 18496(T); LMG 23756(T)).

In vitro antibacterial and antifungal activities of Rhus tripartitum used as antidiarrhoeal in Tunisian folk medicine.

Rhus tripartitum (African sumac) is a plant commonly used in Tunisian traditional medicine to treat diarrhoea and dysentery. In this study, we have demonstrated that R. tripartitum extracts exhibited a significant broad spectrum activity against one or more of the test microorganisms with a zone size ranging from 8 to 28 mm in diameter. These diameters are much higher than those obtained with standard antibiotics. The chloroformic extracts were found to be effective against bacterial and fungal strains tested, with MIC values ranging between 0.07 and 0.62 mg mL(-1) against Staphylococcus aureus and Candida albicans. However, ethyl-acetate/methanol fractions showed a selective activity only against bacterial microorganisms with low MIC values between 0.07 and 0.15 mg mL(-1). The overall results suggested that the traditional use of R. tripartitum for the treatment of diarrhoea tract infections was attributed to the presence of antibacterial agents.

Temporin-SHf, a new type of phe-rich and hydrophobic ultrashort antimicrobial peptide.

Because issues of cost and bioavailability have hampered the development of gene-encoded antimicrobial peptides to combat infectious diseases, short linear peptides with high microbial cell selectivity have been recently considered as antibiotic substitutes. A new type of short antimicrobial peptide, designated temporin-SHf, was isolated and cloned from the skin of the frog Pelophylax saharica. Temporin-SHf has a highly hydrophobic sequence (FFFLSRIFa) and possesses the highest percentage of Phe residues of any known peptide or protein. Moreover, it is the smallest natural linear antimicrobial peptide found to date, with only eight residues. Despite its small size and hydrophobicity, temporin-SHf has broad-spectrum microbicidal activity against Gram-positive and Gram-negative bacteria and yeasts, with no hemolytic activity. CD and NMR spectroscopy combined with restrained molecular dynamics calculations showed that the peptide adopts a well defined non-amphipathic alpha-helical structure from residue 3 to 8, when bound to zwitterionic dodecyl phosphocholine or anionic SDS micelles. Relaxation enhancement caused by paramagnetic probes showed that the peptide adopts nearly parallel orientations to the micelle surface and that the helical structure is stabilized by a compact hydrophobic core on one face that penetrates into the micelle interior. Differential scanning calorimetry on multilamellar vesicles combined with membrane permeabilization assays on bacterial cells indicated that temporin-SHf disrupts the acyl chain packing of anionic lipid bilayers, thereby triggering local cracks and microbial membrane disintegration through a detergent-like effect probably via the carpet mechanism. The short length, compositional simplicity, and broad-spectrum activity of temporin-SHf make it an attractive candidate to develop new antibiotic agents.

Mechanism of antibacterial action of dermaseptin B2: interplay between helix-hinge-helix structure and membrane curvature strain.

Dermaseptin B2 (Drs B2) is a 33-residue-long cationic, alpha-helical antimicrobial peptide endowed with membrane-damaging activity against a broad spectrum of microorganisms, including bacteria, yeasts, fungi, and protozoa, but its precise mechanism of action remained ill-defined. A detailed characterization of peptide-membrane interactions of Drs B2 was undertaken in comparison with a C-terminal truncated analogue, [1-23]-Drs B2, that was virtually inactive on bacteria despite retaining the cationic charge of the full-length peptide. Both peptides were tested on living cells using membrane permeabilization assays and on large unilamellar and multilamellar phospholipid vesicles composed of binary lipid mixtures by dye leakage assay, fluorescence spectroscopy, circular dichroism, and differential scanning calorimetry and also on SDS micelles using NMR spectroscopy. The results indicate that Drs B2 induces a strong perturbation of anionic lipid bilayers, resides at the hydrocarbon core-water interface, parallel to the plane of the membrane, and interacts preferentially with the polar head groups and glycerol backbone region of the anionic phospholipids, as well as the region of the lipid acyl chain near the bilayer surface. The interfacial location of Drs B2 induces a positive curvature of the bilayer and clustering of anionic lipids, consistent with a carpet mechanism, that may lead to the formation of mixed peptide-phospholipid toroidal, transient pores and membrane permeation/disruption once a threshold peptide accumulation is reached. In constrast, the truncated [1-23]-Drs B2 analogue interacts at the head group level without penetrating and perturbing the hydrophobic core of the bilayer. NMR study in SDS micelles showed that [1-23]-Drs B2 adopts a well-defined helix encompassing residues 2-20, whereas Drs B2 was previously found to adopt helical structures interrupted around the Val(9)-Gly(10) segment. Thus the antibacterial activity of Drs B2 depends markedly on a threshold number of hydrophobic residues to be present on both extremities of the helix. In a membrane environment with a strong positive curvature strain, Drs B2 can adopt a flexible helix-hinge-helix structure that facilitates the concomitant insertion of the strongly hydrophobic N- and C-termini of the peptide into the acyl core of the membrane.

Solution structure and model membrane interactions of temporins-SH, antimicrobial peptides from amphibian skin. A NMR spectroscopy and differential scanning calorimetry study.

Temporin-SHa and temporin-SHc are 13 residue long antimicrobial peptides from frog skin that have similar sequences but differ markedly in their membrane-damaging properties. Temporin-SHa contains a single basic lysine residue and has a unique antimicrobial spectrum of action among temporins, being very potent against Gram-positive and Gram-negative bacteria, yeasts, fungi, and protozoa. Temporin-SHc, which contains a single basic histidine residue, is inactive against Gram-negative bacteria, has a reduced efficacy against Gram-positive bacteria, but is still active against yeasts and fungi. Temporin-SHb, with no basic residue, has no antimicrobial activity. The three-dimensional structures of the peptides bound to SDS micelles were analyzed by CD and NMR spectroscopy combined with restrained molecular dynamics calculations. The peptides adopt well-defined amphipathic alpha-helical structures extending from residue 3 to residue 12, when bound to SDS micelles. The structures are stabilized by extensive interactions between aliphatic and aromatic side chains on the nonpolar face. Relaxation enhancements caused by paramagnetic probes showed that the peptides adopt nearly parallel orientations to the micelle surface and do not deeply penetrate into the micelle. The interaction of the peptides with model membranes was investigated by differential scanning calorimetry on anionic and zwitterionic multilamellar vesicles and membrane-permeabilization assays on calcein-loaded large unilamellar vesicles. Calorimetric data indicated that both temporin-SHa and -SHc reside at the hydrocarbon core-water interface of the anionic lipid bilayer but interact with anionic bilayers in a very different manner. This suggests that the charge-induced activity of temporins-SH for bacterial cells is due to changes in the membrane-disturbing mechanism of the bound peptides.

Isolation, characterization and molecular cloning of new temporins from the skin of the North African ranid Pelophylax saharica.

Temporins are small antimicrobial peptides isolated from North American and Eurasian ranid frogs that are particularly active against Gram-positive bacteria. To date, no temporins have been characterized from North African frog species. We isolated three novel members of the temporin family, named temporin-1Sa (FLSGIVGMLGKLF(amide)), -1Sb (FLPIVTNLLSGLL(amide)), and -1Sc (FLSHIAGFLSNLF(amide)), from the skin of the Sahara frog Pelophylax (Rana) saharica originating from Tunisia. These temporins were identified by a combined mass spectrometry/molecular cloning approach. Temporin-1Sa was found to be highly active against Gram-positive and Gram-negative bacteria, yeasts and fungi (MIC=2-30 microM). To our knowledge, this is the first 13-residue member of the temporin family with a net charge of +2 that shows such broad-spectrum activity with particularly high potency on the clinically relevant Gram-negative strains, Escherichia coli (MIC=10 microM) and Pseudomonas aeruginosa (MIC=31 microM). Moreover, temporin-1Sa displays significant antiparasitic activity (IC50 approximately 20 microM) against the promastigote and amastigote stages of Leishmania infantum.