Dottorato in BIOCHIMICA

Titolo della tesi: Insights into the mechanism(s) of action of the membrane-active host-defence Esc peptides on model and living systems

The increase in bacterial strains resistant to conventional antibiotics is an alarming problem for human health and could cause future pandemics. Among such bacterial pathogens responsible for a large variety of severe infections there is P. aeruginosa. In this scenario, the Antimicrobial Peptides (AMPs) hold great promise for the near future. Recently, a derivative of the frog-skin AMP esculentin-1a, namely Esc(1-21), was found to display a fast and potent killing activity against both the planktonic and biofilm forms of P. aeruginosa with a membrane perturbing activity as a plausible mode of action. However, no studies have been carried out so far to explore the mechanism employed by this peptide to perturb bacterial membranes, in real time. Furthermore, a diastereomer of Esc(1-21), named Esc(1-21)-1c, containing two D amino acids, i.e. D-Leu14 and D-Ser17, revealed to be more resistant to proteases, less cytotoxic and with a higher antibiofilm efficacy than the all-L parental peptide. Esc(1-21)-1c was also discovered to display a synergistic effect in inhibiting the growth of P. aeruginosa when combined with the antibiotic aztreonam. However, studies dealing with the combinatorial effect of this peptide with a panel of antibiotics belonging to different classes, on the growth of P. aeruginosa cells, along with the underlying molecular mechanism are still missing. Note that P. aeruginosa is the main bacterial pathogen responsible for chronic lung infections leading to mortality in CF patients. Considering (i) the ability of Esc peptides to stimulate migration of bronchial epithelial cells, which is expected to restore the integrity of a damaged lung tissue and (ii) the role of CFTR in maintaining lung function and wound repair, it is reasonable to investigate the effect of Esc peptides on the ion currents controlled by this channel. Esc peptides would give particular benefit to CF patients by combining their capability to eradicate lung infections and to act as promoters of airway wound repair with their ability to ameliorate the activity of the channel with conductance defects. Based on these premises, the aims of this work thesis were the following: -To Study the interaction of Esc(1-21) on lipid bilayers mimicking bacterial membranes by Atomic force microscopy (AFM) and Molecular dynamics simulations ( MD); -To investigate the ability of Esc(1-21)-1c to potentiate the effect of antibiotics in inhibiting the growth of P. aeruginosa cells and the plausible mode of action, by differential proteomic analysis; -To study the effect of Esc peptides on the activity of CFTR, by electrophysiology and computational methods.