During the last years, the excessive and improper use of commercially available antibiotics has contributed to the development of resistant microbial pathogens, which represent a serious problem for the world public health. Among these microorganisms, the Gram-negative bacterium Pseudomonas aeruginosa is one of the most difficult to eradicate due to its ability to form sessile communities, named biofilms, which cause chronic infections, especially in the lungs of cystic fibrosis (CF) patients. Naturally occurring antimicrobial peptides (AMPs), characterized by a different mechanism of action, represent a promising alternative to the commonly used drugs. AMPs are evolutionally conserved molecules produced by almost all living organisms as a first line of immune defense and amphibian skin is one of the richest sources. The studies carried out in this thesis focused on two peptides: Esculentin-1a(1-21)NH2, [Esc(1-21) GIFSKLAGKKIKNLLISGLKG-NH2], derived from the N-terminal region of the frog skin AMP esculentin-1a, and its diastereomer, Esc(1-21)-1c, containing two D-amino acids at positions 14 and 17 (i.e., D-Leu and D-Ser, respectively). This latter was designed with the purpose to reduce the peptide’s cytotoxicity and to increase its stability to proteolytic enzymes. The results achieved in this thesis have indicated that compared to Esc(1-21), the diastereomer is: i) significantly less toxic towards mammalian cells, in agreement with its lower α-helical structure, as determined by circular dichroism spectroscopy and nuclear magnetic resonance studies; ii) more effective against the biofilm form of P. aeruginosa (either reference or clinical isolates from CF patients) while maintaining high activity against the free-living form of this pathogen; iii) more effective in killing Pseudomonas cells once internalized into bronchial cells expressing either the functional or the ΔF508 mutant of the CF transmembrane conductance regulator; iv) more resistant to bacterial and human elastases, which are abundant in CF lungs. In addition, the diastereomer was found (i) to have a higher activity than the all-L peptide in promoting migration of bronchial epithelial cells and presumably in favoring re-epithelialization of damaged lung tissue; (ii) to disaggregate and detoxify the bacterial lipopolysaccharide (LPS) and to inhibit cyclooxygenase-2 (COX-2) synthesis, albeit less than the wild-type peptide. Based on its interesting biological properties, Esc(1-21)-1c is a promising candidate for the development of a new drug that not only eliminates microbial pathogens, but also restores the integrity of a damaged tissue, such as the lung of CF patients, following Pseudomonas respiratory infections.
|Titolo:||Effects of two L- to D-amino acid substitutions on the structural and functional properties of the antimicrobial peptide esculentin-1a(1-21)|
|Data di discussione:||20-dic-2017|
|Appartiene alla tipologia:||07a Tesi di Dottorato|