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Hepcidin 25 (hep-25) is a peptide primarily produced by human liver with a central role in iron homeostasis. Its isoform, hepcidin 20 (hep-20), has an unknown function and lacks the first five aminoacids of the amino-terminal portion. This sequence is crucial for iron regulation by hep-25 and contains a molecular motif able to bind metals. Aim of this study, was to evaluate the antibacterial properties of both peptides in vitro, against a wide range of bacterial clinical isolates and in different experimental conditions. Although both peptides were found to be bactericidal against a variety of clinical isolates with different antibiotic resistance profiles, hep-20 was active at lower concentrations than hep-25, in most of the cases. Killing kinetics, carried on in sodium-phosphate buffer at pH 7.4, demonstrated that bactericidal activity occurred not earlier than 30-90 min of incubation. Bactericidal activity of hep-25 was slightly enhanced in the presence of copper, while the same metal did not affect the activity of hep-20. Interestingly, bactericidal activity of both hepcidins was highly enhanced at acidic pH. Acidic pH (pH 5.0 and 6.6) not only reduced the microbicidal concentrations of hepcidins, but also shortened the killing times of both peptides, as compared to pH 7.4. Combining hep-20 and hep-25 at pH 5.0 a bactericidal effect could be obtained at very low concentrations of both peptides. These results render hepcidins interesting for the design of new drugs for the treatment of infections occurring in body districts with physiologic acidic pH.

Hepcidin is a new peptide hormone secreted by the liver and widely distributed among animals, from fish to human. It acts as a signal molecule in iron metabolism and plays an important role in iron overload diseases. Recently, it has been reported that hepcidin is able to regulate the iron efflux by binding to the ferroportin protein and inducing its internalization. This activity is specific only for bioactive hepcidin-25 and not for the truncated hepcidin-20, lacking the five amino terminal residues. We have characterized the N-terminal region of the hepcidin-25 as a metal-binding site by several spectroscopic techniques and chemical studies. This region, specific for the coordination of Cu(II) and Ni(II), is known as ATCUN motif. Moreover, we have demonstrated that the metal binding inhibits the aggregation propensity of both whole hepcidin-25 and its N-terminal fragments, probably inducing a conformational change leading to the stabilization of the N-terminal region. The identification of a metal-binding site at the N-terminus of hepcidin-25, which is necessary for its biological activity as iron-regulatory hormone, will help us to cast light both on the role of the divalent metals and of the hepcidin-25 in the iron homeostatis, and to better understand structure-function relationships of this hormonepeptide.

Thermodynamics of the binding of Ni2+, Cu2+ and Zn2+ to bacitracin A(1) was studied by capillary electrophoresis measuring the peptide effective mobility at different pH in the presence of increasing concentration of the three ligands. The affinity follows the order Ni2+ > Cu2+ > Zn2+, with association constant values of (2.3+/- 0.1) X 10(4), (4.9+/-0.2) x 10(3), and (1.5 +/- 0.1) x 10(3) m(-1), respectively. The only model able to rationalize mobility data implies that metal ion binds to the P-O peptide form. Moreover, mobility values indicated a change of bacitracin A(1) acidic properties on Ni2+ and Cu2+ binding, with a shift of the pK(a), of N-terminal Ile-1 from 7.6 to about 5 and of the pK(a) of the delta-amino group Of D-Orn-7 from 9.7 to about 7. Even though on Zn2+ binding a shift of the N-terminal Ile-1 pK(a) was observed, restrictions in the pH range suitable for investigation, due to precipitation phenomena, did not allow establish if the shift Of D-Orn-7 lateral chain pK(a) also occurred. Nonetheless, if present, the shift should be limited to the 7.8-9.7 range. Mobility data indicated that the Stokes radius of the complexes is ca. 3 Angstrom lower than that of the free peptide. The present results indicate that metal-ion binding to bacitracin A(1) is more complex than previously assumed.

Intracellular pathogens have evolved effective mechanisms in order to survive in an intracellular environment, thus avoiding destruction by phagocytic cells. In this regard, a correlation between resistance to phagocytic killing and expression of pathogenic potency has been established. In this report, we have studied the interaction between human polymorphonuclear cells (PMN) and two gram-negative microorganisms, Afipia felis and Rochalimaea henselae, which belong to the alpha-2 subgroup of the class Proteobacteria. A. falis has been previously proposed as the causative agent of Cat Scratch Disease (CSD), but several recent lines of evidence attribute a major role to R. henselae. Of note, CSD is a syndrome characterized by a chronic lymphoadenopathy, involving macrophages and endothelial cells with a progression towards a granulomatous process and/or angiogenesis. Since members of the alpha-2 subgroup of Proteobacteria have the property to survive intracellularly, we have evaluated the effects exerted by A. felis and R. henselae on human PMN in terms of chemotaxis locomotion, degranulation and oxidative metabolism. Results will show an impairment of PMN activities as a consequence of the challenge with both microrganisms. In particular, inhibition of PMN oxidative function occurred either as result of a direct exposure to both A. felis and R. henselae or when PMN were primed by bacteria for the N-formyl-methionyl-leucyl-phenylalanine enhancement of the oxidative burst. These findings may account for the ability of A. felis and R. henselae to survive within PMN as expression of a further mechanism of pathogenic potency, influencing also the nature and the evolution of inflammatory response in the lesion sites.