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

Abstract

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.

FIGURE 1.

A, predicted amino acid sequence (single-letter code) of precursors of temporin-SHd to -SHf from the skin of P. saharica. The open reading frame contains a part of the signal peptide (gray) followed by the acidic propiece, and the mature peptide (underlined). The G residue (italics) at the C-terminal of the mature peptide sequence serves as an amide donor after removal of the Lys residue (italics) with a carboxypeptidase. Deduced amino acid sequence of brevinin-1 precursor cDNAs from P. saharica are also indicated with the predicted mature brevinins-1(underlined). B, multiple sequence alignment of the amino acid sequences of polypeptide precursors representative of the esculentin-1 (E-1), esculentin-2 (E-2), brevinin-2 (B-2), ranatuerin-2 (Rt-2), palustrin-1 (P-1), brevinin-1 (B-1), nigrocin-2 (N-2), ranacyclin (Rc), and temporin (T) families of antimicrobial peptides from ranid frogs. Gaps (−) have been introduced to maximize sequence similarities. Identical (grey background) amino acid residues are highlighted. Cysteine residues that form disulfide bonds are indicated (black background). Residues G or GK (italics) at the end of the mature peptide sequence serve for C-terminal amidation.

FIGURE 2.

Identification of temporin-SHf in the skin of P. saharica frog. A, fractionation of an acidic extract of P. saharica skin by reversed-phase HPLC on an analytical Lichrospher 5-μm C-18 column. The solvent system used was composed of H₂O-0.1% trifluoroacetic acid (solvent A) and ACN containing 0.07% trifluoroacetic acid (solvent B). After 3-min equilibration with solvent A, separation was performed with a 0–60% linear gradient of solvent B (1%/min) at a flow rate of 0.75 ml/min. Absorbance was monitored at 220 nm. B, MALDI-TOF mass spectrometry analysis of semi-preparative HPLC fractions. HPLC fractions (4 ml) were obtained after separation of the acidic extract of P. saharica skin on a reversed-phase Nucleosil 5-μm C-18 column eluted with a 0–60% linear gradient of solvent B (1%/min) at a flow rate of 4 ml/min. The mass spectrometry spectrum of fraction at 52 min (∼49% ACN) is shown and reveals a mass product ([M+H]⁺, 1075.6 m/z) corresponding to temporin-SHf indicated by a circle.

FIGURE 3.

Molecular phylogram of antimicrobial peptide precursors from ranid frogs constructed with the maximum likelihood method. Maximum parsimony and LogDet analyses yielded the same ordinal phylogeny. Numbers at nodes are bootstrap values based on 100 replicates. Preprodermaseptin B2 from Phyllomedusa bicolor was assigned as the out-group (16). Frogs belonging to the Ranidae family are described by the species name set out in research.amnh.org/herpetology/amphibia/index.php (D. R. Frost, 2007, Amphibian Species of the World: an Online Reference. Version 5.1, American Museum of Natural History, New York). Nomenclature adopted for antimicrobial peptides follows recent guidelines (36). Note that the names lividin-1, chensirin-2, ranalexin, and gaegurin-5 are synonymous with brevinin-1. AMA polypeptide-1, -2, and -3, amurin-2, chensirin-1, and pelophylaxin-4 are synonymous with temporin. The name peptide LR is synonymous with ranacyclin.

FIGURE 4.

Kinetics of the cytoplasmic membrane leakage of E. coli ML-35p after treatment with increasing concentrations (2, 4, 10, 15, 30, and 100 μm) of temporin-SHf (top) and with 25 μm melittin or 50 μm dermaseptin B2 as positive controls (bottom). The negative control (without peptide) is also shown. The membrane leakage was followed by measuring the hydrolysis of ONPG at 405 nm by the cytoplasmic bacterial β-galactosidase after incubation with the peptides (see “Experimental Procedures”). Data are expressed as the mean ± S.E.

FIGURE 5.

High sensitivity DSC heating scans illustrating the effect of temporin-SHf on anionic DMPG (left panel) and zwitterionic DMPC (right panel) MLVs. The corresponding pretransition regions are shown on a larger scale (insets). Scans were acquired without peptide (blank) and at different peptide/lipid molar ratios (1:100, 1:50, and 1:20).

FIGURE 6.

CD spectra of temporin-SHf (100 μm) in H₂O (thin solid line), 80 mm SDS solution (dotted line), and 120 mm DPC solution (thick solid line) at 20 °C.

FIGURE 7.

¹H^α and ¹³C^α CSDs of temporin-SHf in water (white bars), in 80 mm SDS (gray bars), and in 120 mm DPC (black bars).

FIGURE 8.

NOE connectivities of temporin-SHf (2 mm) in 120 mm DPC. The relative intensity of NOEs is indicated by horizontal bars of varying thickness.

FIGURE 9.

NMR structure family of temporin-SHf in micellar DPC. A, all backbone atoms; B, all backbone atoms and heavy atoms of residues 3–8. Structures were superimposed by best fitting of N, C^α, and C′ atoms of residues 3–8. Atoms are shown with different colors (amide hydrogen in white, carbon in green, nitrogen in blue, and oxygen in red). N and C, N and C termini.

FIGURE 10.

Attenuations of temporin-SHf resonances induced by paramagnetic probes. The relaxation enhancements induced by Mn²⁺ (0.7 mm) or 16-doxylstearic acid (3.2 mm) are shown in blue and red, respectively. The volumes of CH^α and CH^β cross-peaks on two-dimensional ¹H-¹³C heteronuclear single quantum coherence spectra were compared with corresponding correlations on a reference spectrum in the absence of paramagnetic agent. For diastereotopic methylene groups, the average of both peaks is given. Missing attenuations are due to peak overlap.