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- Materials and methods
Temporins, antimicrobial peptides of 10–13 residues, were isolated from secretions of Rana temporaria[Simmaco, M., Mignogna, G., Canofeni, S., Miele, R., Mangoni, M.L. & Barra, D. (1996) Eur. J. Biochem.242, 788–792]. These molecules are specific to this amphibian species, which is also able to secrete on its skin other antimicrobial peptides similar to those found in different Rana species. The effect of temporins A, B and D (13 residues, net charge +2), and H (10 residues, net charge +1 and +2, respectively) against both artificial membranes of differing lipid composition and bacteria has been investigated in order to gain insight into their mechanisms of action. The results indicate that: the lytic activity of temporins is not greatly affected by the membrane composition; temporins A and B allow the leakage of large-size molecules from the bacterial cells; temporin H renders both the outer and inner membrane of bacteria permeable to hydrophobic substances of low molecular mass; and temporin D, although devoid of antibacterial activity, has a cytotoxic effect on erythrocytes. The results allow important conclusions to be drawn about the minimal structural requirements for lytic efficiency and specificity of temporins.
Skin secretions of several amphibian species have been analysed in detail and found to contain a large number of different antimicrobial peptides, which represent the effector molecules of innate immunity [1,2]. Recent experiments on Rana esculenta or Bombina orientalis have also demonstrated that in amphibia genes for antimicrobial peptides are controlled by NF-κB-regulated transcription [3–6].
All anti-microbial peptides studied so far have a cationic character that allows their preferential interaction with the anionic phospholipids of the target bacterial membranes. Some of them have been shown to lyse, with a certain extent of selectivity, cancer cells, which also contain negatively charged phospholipids, whereas normal cells do not . Others are able to interact with erythrocytes causing a rapid hemolysis. Most of the antimicrobial peptides can adopt an amphipathic α-helical structure in hydrophobic environments, thus perturbing the phospholipid bilayer of the target membrane [8,9]. Inhibition of growth as well as cell death may then be a consequence of the disturbance of membrane functions.
A large variety of peptides of 20–46 amino acid residues has been isolated from frogs belonging to the Rana genus and found to be active against bacteria and fungi. These are the brevinins, esculentins and their related forms, ranalexin, gaegurins and rugosins: all these peptides share a peculiar structural motif, i.e. an intramolecular disulfide bridge forming a heptapeptide ring at the C-terminal end . From Rana temporaria, a family of small (10–13 residues) antibacterial peptides called the temporins have also been isolated; so far they have been detected only in this amphibian species .
Temporins are among the smallest antimicrobial peptides so far described, together with the 13-residue peptide indolicidin  and the cyclic dodecapeptide bactenecin , both from bovine neutrophils. The last two are highly hydrophobic cationic peptides with a net charge of +4. Bactenecin contains a disulfide bridge, which confers a looped structure on the peptide, while indolicidin is amidated at the C-terminus. Both peptides are mainly active against Gram-negative bacteria [13,14]. Temporins are all amidated at the C-terminus; those containing one basic residue, either lysine or arginine, in the sequence (net charge +2) were found to be active specifically against Gram-positive bacteria and Candida albicans. When assayed against laboratory bacterial strains, temporins lacking a basic residue were inactive, and the 10-residue members of this family, although containing a basic residue, were inactive also .
To gain furher insight into the mechanism of action of these small antimicrobial peptides, we have investigated their effects against both artificial membranes of differing lipid composition and bacteria.
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- Materials and methods
Skin secretions of R. temporaria contain antimicrobial peptides belonging to the brevinin-1 and brevinin-2 families . Characteristic of this amphibian species are temporins, recovered from secretions in the range of 14–40 nmol·mg−1 dry weight . Temporin D is the least abundant molecule in the secretion (1 nmol·mg−1), although its identity with the most abundant temporin C is about 84%. On the basis of the retention time in RP-HPLC as well as from the calculated mean residue hydrophobicity, temporin H is the least hydrophobic peptide, but the calculated value for the hydrophobic moment is very similar to that of magainin 2.
Although temporins contain only 13 residues, with the exception of temporin H which is 10 residues long, CD studies indicate that these molecules adopt an α-helical structure in a hydrophobic environment. Moreover, the helical-wheel projections  of these peptides show an amphiphilic nature (Fig. 1).
Lipid vesicle permeabilization studies demonstrate that all the peptides are able to lyse artificial membranes, although at different concentrations. The lytic activity of temporins is not affected by the membrane composition, differing from magainins  and other antimicrobial peptides , which present a lower affinity towards zwitterionic (PtdCho) phospholipid vesicles than towards acidic ones. The lack of selectivity of temporins may be related to the low number of positive charges. In this case, binding to the membrane is mostly due to hydrophobic interactions; such behaviour suggests the occurrence of a barrel-stave mechanism for temporins .
The antibacterial assays suggest that both the amphiphilic α-helical conformation and the net positive charge of the peptide are determinant for the bacterial membrane-perturbing ability. In fact, temporin D, which has a hydrophobic moment similar to that of the other temporins but a net charge of +1, is not able to change the permeability properties of the bacterial membrane. The assay performed using LPS-defective strains of E. coli confirms that the low activity of temporins A and B on Gram-negative bacteria is due to their net charge. Once the peptide has reached the bacterial inner membrane, targeted through electrostatic interactions, the hydrophobic interactions play a crucial role in bacterial lysis. Thus, the length of the helix could play an important role in determining the extent of the membrane lesion, as demonstrated by temporin H, which appears to be unable to kill bacteria. In effect, temporin H alters the outer membrane of E. coli D21, inducing a modification in its permeability, as demonstrated by the results obtained following exposure of bacteria to sublethal concentration of rifampicin (Fig. 8). Moreover, the fact that treatment of E. coli D22 with temporin H does not cause the release of β-galactosidase (Fig. 9), although the enzyme substrate Gal-ONp is efficiently hydrolysed inside the bacterial cell (Fig. 10), demonstrates that the inner membrane permeability is also affected.
In conclusion, temporins A, B and H change the permeability properties of the bacterial membranes. According to the results illustrated in Figs 8–10, temporin H renders both the outer and inner membrane permeable to hydrophobic substances of low molecular mass, such as rifampicin and the substrate Gal-ONp, whereas temporins A and B allow the leakage from the cells of larger size molecules, such as β-galactosidase. Temporin D, although devoid of antibacterial activity, has a defined role in the animal defence, being specifically active against eukaryotic cells. Its cytotoxicity against erythrocytes confirms that the hydrophobicity and the relatively low positive charge correlate well with the capacity to perturb the eukaryotic membrane.
The study of the effect of temporins on bacterial cells and artificial vesicles allows important conclusions to be drawn about the minimal structural requirements for lytic efficiency and specificity. This aspect is crucial in economical terms both for the animal, which is challenged with the problem of rapidly synthesizing its own innate immune system against invading microorganisms, and for the opening of new perspectives in the industrial production of new antibiotics.