Analisi funzionale e strutturale di peptidi antimicrobici prodotti da Pseudomonas spp. e loro potenzialità in applicazioni biotecnologiche

Lipodepsipeptides (LDP) are a group of cyclic secondary metabolites produced by many Pseudomonas species. On the basis of their amino acid chain length, they are usually divided into two groups: the mycins and the peptins. The first group consists of acylated peptides of 9 amino acids closed in a cyclic structure between the first and the last residue. The peptins are bigger and more complex than the former, being composed of 18 to 25 amino acids, again cyclized (usually in an 8 or in a 5 residues ring) and acylated. Both kind of peptides are expressed in vivo, they significantly contribute to bacterial pathogenesis and have the plasma membrane of plants as their primary site of action, but may have also different target organisms. Both classes of LDPs induce hemolysis and leakage from liposomes by the formation of pores. The peptins inhibit the growth of Gram positive bacteria and have stronger phytotoxic activity than the mycins. The mycins displays a prominent antifungal activity. The antifungal property is potentially useful for the development of biocontrol agents and also appears interesting in the perspective of biomedical applications. For these strategies is very important to clarify the mechanism of action of LDPs. We investigated the importance of LDPs structural characteristics on their ability to create a lesion on liposomes and RBC. Some structural analogues of tol I, produced by Pseudomonas tolaasii, and WLIP, by P. reactans, were tested. The results have suggested the importance of the lactone ring and the N-terminus acyl moiety. Furthermore, the importance of some residues in the peptide chain was observed. In particular structural changes in these amino acids may modify the LDP conformation and consequently their permeabilization ability. The analysis individuated some WLIP derivatives completely non active. Hydrolyzed WLIP resulted particularly interesting. The study of the interaction of the LDP with lipid monolayer showed that the opening of the ring decreases the ability to bind the membrane. Moreover, hydrolized WLIP is able to inhibit the permeabilizing ability of its natural form, WLIP, and also of tol I on vesicles. Experiments on monolayer suggested that hydrolyzed WLIP does not affect the insertion in the membrane of WLIP while it decreases the tol I interaction with the monolayer. We hypothesized that the interaction between WLIP and hydrolyzed WLIP can happen at the membrane level, blocking the oligomerization process which is necessary for the pore formation. Tol I and hydrolyzed WLIP instead can interact in solution, forming an aggregate that subtract the active LDP to the interaction with the membrane. Furthermore, the nature of the interaction of the different LDPs with lipids by determining the transmembrane movement of a fluorescent lipid asimetrically included in the bilayer was also investigated. The results suggested that peptins promoted the lipid transfer between the inner and the outer membrane layer. This was a clear indication that a mixed lipid/peptide pore with "toroidal" structure is formed. This lesion permits the contact of the inner and outer leaflets of the membrane bilayer. Finally we prepared vesicles from different fungal pathogen lipids to test peptins and mycins activity on systems more similar to natural targets. On these new model systems peptins displayed more activity than the mycins, supporting the strong antifungal role of the peptins when the cell wall is removed.