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Vasostatin-I, the natural fragment of chromogranin A-(1-76), is a neuropeptide able to kill a large variety of fungi and yeast cells in the micromolar range. We have examined the antifungal properties of synthetic vasostatin-I-related peptides. The most active shortest peptide, named chromofungin, corresponds to the sequence Arg(47)-Leu(66). Extensive (1)H NMR analysis revealed that it adopts a helical structure. The biophysical mechanism implicated in the interaction of chromofungin with fungi and yeast cells was studied, showing the penetration of this peptide with different lipid monolayers. In order to examine thoroughly the antifungal activity of chromofungin, confocal laser microscopy was used to demonstrate the ability of the rhodamine-labeled peptide to interact with the fungal cell wall, to cross the plasma membrane, and to accumulate in Aspergillus fumigatus, Alternaria brassicola, and Candida albicans. Our present data reveal that chromofungin inhibits calcineurin activity, extending a previous observation that the N-terminal region of chromogranin A interacts with calmodulin in the presence of calcium. Therefore, the destabilization of fungal wall and plasma membrane, together with the possible intracellular inhibition of calmodulin-dependent enzymes, is likely to represent the mechanism by which vasostatin-I and chromofungin exert antifungal activity.

Iturins are a family of lipopeptides extracted from the culture media of various strains of Bacillus subtilis. These amphiphilic compounds are characterized by a peptide ring of seven amino acid residues including an invariable D-Tyr2, with the constant chiral sequence LDDLLDL closed by a C14-C17 aliphatic beta-amino acid. They exhibit strong antifungal activities against a wide variety of pathogenic yeasts and fungi but their antibacterial activities are restricted to some bacteria such as Micrococcus luteus. The biological activity of the iturin lipopeptides is modulated by the primary structure of the peptide cycle as illustrated by the methylation of the D-Tyr2 residue which dramatically decreases the activity or by the inversion of the two adjacent Ser6-Asn7 residues which makes mycosubtilin more active than iturin A. The antifungal activity is related to the interaction of the iturin lipopeptides with the cytoplasmic membrane of target cells, the K+ permeability of which is greatly increased. The ability of iturin compounds to increase the membrane cell permeability is due to the formation of ion-conducting pores, the characteristics of which depend both on the lipid composition of the membrane and on the structure of the peptide cycle. From monolayer experiments it has been suggested that these ionic pores are the consequence of the presence of aggregates (lipopeptide aggregates or lipopeptide/phospholipid complex aggregates) in the phospholipid membrane. It has also been shown that, when active, iturins interact strongly with sterols, forming lipopeptide/cholesterol complexes. Therefore, the biologically efficient structure might be a ternary structure: iturin/phospholipid/sterol.

Iturin A and surfactin are two lipopeptides extracted from a same strain of Bacillus subtilis. Iturin A possesses antibiotic and antifungal activities and surfactin is a strong surfactant. The presence of surfactin, at a concentration at which, alone, it is inactive, increases to a very large extent the haemolysis percent induced by iturin A. This synergistic effect seems to be in relation with interactions between iturin A and surfactin. Iturin A adsorbs to and penetrates into surfactin monolayers. Iturin A and surfactin are miscible and interact specifically in mixed monolayers.

The antifungal activity of iturin A and its interaction with erythrocyte membranes were enhanced in the presence of surfactin. The modification of the properties of iturin A was explained by the formation of mixed iturin A-surfactin micelles. Such mixed micelles were easily generated when both lipopeptides were in aqueous solutions in the absence of mineral salts but the formation of these micelles did not occur when the solutions contained a high molarity of mineral cations.