Donatella Barra

Antimicrobial peptides from amphibian skin: What do they tell us?

Amphibian skin secretions contain many biologically active compounds, such as biogenic amines, complex alkaloids, or peptides. Within the latter class of molecules, a large number of peptide antibiotics has been isolated and characterized from different amphibian species. Antimicrobial peptides are considered the effector molecules of innate immunity, acting as a first line of defense against bacterial infections, by perturbing the phospholipid bilayer of the target cell membrane. These gene-encoded molecules are synthesized as inactive precursors and in several cases their proparts were shown to have highly conserved structures. It has also been demonstrated that the promoter regions of inducible peptide antibiotics are often regulated by the transcriptional control machinery NF-kappa B/I kappa B alpha. In amphibia of Rana and Bombina genera, inhibition of transcription of the genes encoding antimicrobial peptides has been obtained by glucocorticoid treatment, which causes an increase of I kappa B alpha synthesis. Moreover, determination of the structure of a number of genes coding for antimicrobial peptides in amphibia has actually shown that their promoter regions contain recognition sites for nuclear factors.

Amphibian skin: a promising resource for antimicrobial peptides.

Amphibian skin is a rich source of biologically active compounds that are assumed to have diverse physiological and defence functions. In addition to the range of pharmacologically active peptides present, some of which have mammalian homologues, skin secretions contain a broad spectrum of antimicrobial peptides. As yet, such peptides from only a few species have been studied, and screening of other species is expected to yield further new antimicrobial activities. Natural antimicrobial peptides isolated from amphibian skin could provide lead structures for either the chemical, or rDNA synthesis of novel antimicrobials.

A Novel Non-heme Iron-binding Ferritin Related to the DNA- binding Proteins of the Dps Family in Listeria innocua*

A multimeric protein that behaves functionally as an authentic ferritin has been isolated from the Gram-positive bacterium Listeria innocua The purified protein has a molecular mass of about 240,000 Da and is composed of a single type of subunit (18,000 Da). L. innocua ferritin is able to oxidize and sequester about 500 iron atoms inside the protein cage. The primary structure reveals a high similarity to the DNA-binding proteins designated Dps. Among the proven ferritins, the most similar sequences are those of mammalian L chains that appear to share with L. innocua ferritin the negatively charged amino acids corresponding to the iron nucleation site. In L. innocua ferritin, an additional aspartyl residue may provide a strong complexing capacity that renders the iron oxidation and incorporation processes extremely efficient. This study provides the first experimental evidence for the existence of a non-heme bacterial ferritin that is related to Dps proteins, a finding that lends support to the recent suggestion of a common evolutionary origin of these two protein families.

Bv8, a small protein from frog skin and its homologue from snake venom induce hyperalgesia in rats

From skin secretions of Bombina variegata and Bombina bombina, we isolated a small protein termed Bv8. The sequence of its 77 amino acids was established by peptide analysis and by cDNA cloning of the Bv8 precursor. Bv8 stimulates the contraction of the guinea-pig ileum at nanomolar concentrations. The contraction is not inhibited by a variety of antagonists. Injection of a few micrograms of Bv8 into the brain of rats elicits, as assessed by the tail-flick test and paw pressure threshold, a marked hyperalgesia which lasts for about 1 h. Bv8 is related to protein A, a component of the venom of the black mamba. After i.c.v. injection, protein A is even more active than Bv8 in inducing hyperalgesia.

Syringopeptins, new phytotoxic lipodepsipeptides of Pseudomonas syringae pv. syringae

The primary structure of some new lipodepsipeptides named syringopeptins, produced by plant pathogenic strains of Pseudopmonas syringae pv. syringae has been determined by a combination of chemical methods, 1H and 13C NMR spectroscopy and FAB mass spectrometry. Two syringomycin‐producing strains afforded 3‐hydroxydecanoyl‐Dhb‐Pro‐Val‐Val‐Ala‐Ala‐Val‐Val‐Dhb‐Ala‐Val‐Ala‐Ala‐Dhb‐aThr‐Ser‐Ala‐Dhb‐Ala‐Dab‐Dab‐Tyr, with Tyr acylating a Thr to form a macrolactone ring, and smaller amounts of the 3‐hydroxydodecanoyl homologue. Evidence was obtained that a third syringomycin‐producing strain and a syringotoxin‐producing strain synthesize 3‐hydroxydecanoyl‐Dhb‐Pro‐Val‐Ala‐Ala‐Val‐Leu‐Ala‐Ala‐Dhb‐Val‐Dhb‐Ala‐Val‐Ala‐Ala‐Dhb‐aThr‐Ser‐Ala‐Val‐Ala‐Dab‐Dab‐Tyr, with Tyr and aThr forming again the macrolactone ring, and smaller amounts of the 3‐hydroxydodecanoyl homologue.

Novel antimicrobial peptides from skin secretion of the European frog Rana esculenta

Three antimicrobial peptides were isolated from skin secretion of the European frog, Rana esculenta. Two of them show similarity to brevinin‐1 and brevinin‐2, respectively, two antimicrobial peptides recently isolated from a Japanese frog [Morikawa, N., Hagiwara, K. and Nakajima, T. (1992) Biochem. Biophys. Res. Commun. 189, 184‐190]. The third one, named esculentin, is 46 residues long and represents a different type of peptide. All these peptides have as a common motif an intramolecular disulfide bridge located at the COOH‐terminal end. The peptides from R. esculenta show distinctive antibacterial activity against representative Gram‐negative and Gram‐positive bacterial species. In particular, esculentin is the most active against Staphylococcus aureus, and has a much lower hemolytic activity.

Temporin L: antimicrobial, haemolytic and cytotoxic activities, and effects on membrane permeabilization in lipid vesicles

The temporins are a family of small, linear antibiotic peptides with intriguing biological properties. We investigated the antibacterial, haemolytic and cytotoxic activities of temporin L (FVQWFSKFLGRIL-NH2), isolated from the skin of the European red frog Rana temporaria. The peptide displayed the highest activity of temporins studied to date, against both human erythrocytes and bacterial and fungal strains. At variance with other known temporins, which are mainly active against Gram-positive bacteria, temporin L was also active against Gram-negative strains such as Pseudomonas aeruginosa A.T.C.C. 15692 and Escherichia coli D21 at concentrations comparable with those that are microbiocidal to Gram-positive bacteria. In addition, temporin L was cytotoxic to three different human tumour cell lines (Hut-78, K-562 and U-937), causing a necrosis-like cell death, although sensitivity to the peptide varied markedly with the specific cell line tested. A study of the interaction of temporin L with liposomes of different lipid compositions revealed that the peptide causes perturbation of bilayer integrity of both neutral and negatively charged membranes, as revealed by the release of a vesicle-encapsulated fluorescent marker, and that the action of the peptide is modulated to some extent by membrane lipid composition. In particular, the presence of negatively charged lipids in the model bilayer inhibits the lytic power of temporin L. We also show that the release of fluorescent markers caused by temporin L is size-dependent and that the peptide does not have a detergent-like effect on the membrane, suggesting that perturbation of bilayer organization takes place on a local scale, i.e. through the formation of pore-like openings.

Nociceptive sensitization by the secretory protein Bv8

The small protein Bv8, isolated from amphibian skin, belongs to a novel family of secretory proteins (Bv8‐Prokineticin family, SWISS‐PROT: Q9PW66) whose orthologues have been conserved throughout evolution, from invertebrates to humans. When injected intravenously or subcutaneously (from 0.06 to 500 pmol kg−1) or intrathecally (from 6 fmol to 250 pmol) in rats, Bv8 produced an intense systemic nociceptive sensitization to mechanical and thermal stimuli applied to the tail and paws. Topically delivered into one rat paw, 50 fmol of Bv8 decreased by 50% the nociceptive threshold to pressure in the injected paw without affecting the threshold in the contralateral paw. The two G‐protein coupled prokineticin receptors, PK‐R1 and PK‐R2, were expressed in rat dorsal root ganglia (DRG) and in dorsal quadrants of spinal cord (DSC) and bound Bv8 and the mammalian orthologue, EG‐VEGF, with high affinity. In DSC, PK‐R1 was more abundant than PK‐R2, whereas both receptors were equally expressed in DRG. IC50 of Bv8 and EG‐VEGF to inhibit [125I]‐Bv8 binding to rat DRG and DSC were 4.1±0.4 nM Bv8 and 76.4±7.6 nM EG‐VEGF, in DRG; 7.3±0.9 nM Bv8 and 330±41 nM EG‐VEGF, in DSC. In the small diameter neurons (<30 μm) of rat DRG cultures, Bv8 concentrations, ranging from 0.2 to 10 nM, raised [Ca2+]i in a dose‐dependent manner. These data suggest that Bv8, through binding to PK receptors of DSC and primary sensitive neurons, results in intense sensitization of peripheral nociceptors to thermal and mechanical stimuli.

Nitrite reductase from Pseudomonas aeruginosa: Sequence of the gene and the protein

The gene coding for nitrite reductase of Pseudomonas aeruginosa has been cloned and its sequence determined. The coding region is 1707 bp long and contains information for a polypeptide chain of 568 amino acids. The sequence of the mature protein has been confirmed independently by extensive amino acid sequencing. The amino‐terminus of the mature protein is located at Lys‐26; the preceding 25 residue long extension shows the features typical of signal peptides. Therefore the enzyme is probably secreted into the periplasmic space. The mature protein is made of 543 amino acid residues and has a molecular mass of 60204 Da. The c‐heme‐binding domain, which contains the only two Cys of the molecule, is located at the amino‐terminal region. Analysis of the protein sequence in terms of hydrophobicity profile gives results consistent with the fact that the enzyme is fully water soluble and not membrane bound; the most hydrophilic region appears to correspond to the c‐heme domain. Secondary structure predictions are in general agreement with previous analysis of circular dichroic data.

Lipopolysaccharide, a Key Molecule Involved in the Synergism between Temporins in Inhibiting Bacterial Growth and in Endotoxin Neutralization

Lipopolysaccharide (LPS) is the major structural component of the outer membrane of Gram-negative bacteria and shields them from a variety of host defense factors, including antimicrobial peptides (AMPs). LPS is also recognized by immune cells as a pathogen-associated molecular pattern and stimulates them to secrete pro-inflammatory cytokines that, in extreme cases, lead to a harmful host response known as septic shock. Previous studies have revealed that a few isoforms of the AMP temporin, produced within the same frog specimen, can synergize to overcome bacterial resistance imposed by the physical barrier of LPS. Here we found that temporins can synergize in neutralizing the LPS-induced macrophage activation. Furthermore, the synergism between temporins, to overcome the protective function of LPS as well as its endotoxic effect, depends on the length of the polysaccharide chain of LPS. Importantly, mode of action studies, using spectroscopic and thermodynamic methods, have pointed out different mechanisms underlying the synergism of temporins in antimicrobial and anti-endotoxin activities. To the best of our knowledge, such a dual synergism between isoforms of AMPs from the same species has not been observed before, and it might explain the ability of such amphibians to resist a large repertoire of microorganisms.