Temporin-1TGa

The temporins are a family of hydrophobic, C-terminally alpha-amidated antimicrobial peptides that are synthesized in the skins of a wide range of species of frogs belonging to the genus Rana. In the present study, we investigated using RT-PCR the expression of preprotemporin mRNAs in extradermal tissues of Tago's brown frog Rana tagoi. cDNAs encoding temporin-1TGa (FLPILGKLLS(10)GIL.NH2), previously isolated from an extract of the skin of R. tagoi skin, were amplified and cloned from the stomach, liver, kidney, skeletal muscle. However, a net insertion of 10 nucleotides resulted in the presence of a premature stop codon in the open reading frame that was not present in the corresponding region of preprotemporin-1TGa from skin. The preprotemporin cDNA obtained from small intestine contained an additional 12 nucleotide insertion in the region that encodes the temporin sequence so that a novel peptide (FLPVILPVIG(10)KLLSGIL.NH2), termed temporin-1TGc, is specified. This cDNA also contained a premature stop codon in the open reading frame. Although it is unclear whether temporin-1TGc is produced in R. tagoi tissues, a synthetic replicate of the peptide of was biologically active, inhibiting the growth of Staphylococcus aureus (minimal inhibitory concentration = 37.5 microM) and producing hemolysis of human erythrocytes (LD50 = 50 microM).

The increasing resistance of bacteria and fungi to the available antibiotic/antimycotic drugs urges for a search for new anti-infective compounds with new modes of action. In line of this, natural CAMPs represent promising and attractive candidates. Special attention has been devoted to frog-skin temporins, because of their short size (10-14 residues long) and their unique features. In particular, temporin-1Ta has the following properties: (i) it is mainly active on Gram-positive bacteria; (ii) it can synergize, when combined with temporin-1Tl, in inhibiting both gram-negative bacterial growth and the toxic effect of LPS; (iii) it preserves biological activity in the presence of serum; and (iv) it is practically not hemolytic. Rational design of CAMPs represents a straightforward approach to obtain a peptide with a better therapeutic index. Here, we used alanine scanning analogs to elucidate the contribution of the side chains of each amino acid residue to the peptide's antimicrobial and hemolytic activity. Beside providing insight into the biophysical attributes and the critical positions within the peptide sequence, which govern the antimicrobial/hemolytic activity of this temporin isoform, our studies assist in optimizing the design of temporin-based lead structures for the production of new anti-infective agents.

Using RT-PCR, two cDNAs encoding preprotemporins were cloned from a total RNA preparation of the skin of Tago's brown frog Rana tagoi. Preprotemporin-1TGa cDNA directs the synthesis of temporin-1TGa (FLPILGKLLSGIL.NH2) previously isolated from R. tagoi skin. Preprotemporin-1TGb cDNA directs the synthesis of a novel 16-amino-acid-residue peptide (AVDLAKIANKVLSSLF.NH2) that, atypically for members of the temporin family, inhibits the growth of Gram-negative bacteria more effectively than Gram-positive bacteria. Preprotemporin-1TGa mRNA and preprotemporin-1TGb mRNA were not detected in skin prior to the onset of metamorphosis (stage 35) but the levels of the transcripts increased markedly during metamorphosis reaching a maximum at stage 38. Exposure of adult animals to 10(-8) M triiodothyronine (T3) for 72 h enhanced expression of the preprotemporin-1TGb gene (approximately threefold) but did not significantly change the level of expression of the preprotemporin-1TGa gene. Exposure of the animals to 10(-8) M T3 and 10(-6) M bisphenol A, an endocrine disrupting chemical that potently inhibits the action of thyroid hormones (THs), reduced expression of the preprotemporin-1TGb gene by 10-fold and the preprotemporin-1TGa gene by threefold. We propose that T3-stimulated synthesis of antimicrobial peptides is important in protecting the animal against microorganisms, particularly at metamorphosis and during skin moulting, but environmental pollutants can inhibit peptide synthesis and render the animal susceptible to invasion by pathogens.