Cationic α-helical antimicrobial peptides (AMPs) hold promise for treatment of the raising multi-drug resistant microbial infections, due to their broad spectrum of activity and membrane-perturbing mechanism of action. Compared to conventional antibiotics, these features make them newsworthy molecules that hardly induce microorganisms to acquire resistance to them. Among these pathogens, Pseudomonas aeruginosa is the most clinically relevant Gram-negative bacterium known to cause serious human infections, e.g. pneumoniae, especially in immune-compromised patients, such as cystic fibrosis (CF) sufferers and keratitis, associated to contact lens (CL) wear. This is due to the unique ability of this pathogen to adhere to different types of inert materials or biological tissues, and to grow in a more resistant and dangerous sessile life form, called biofilm. Recently, two Esculentin-1a-derived antimicrobial peptides i.e. Esc(1-21) and its D-amino acids containing Esc(1-21)-1c, [Esc peptides], have been fully characterized for their powerful antipseudomonal activity against both planktonic and biofilm forms. The diastereomer showed a higher bactericidal activity than the all-L isomer against the more dangerous Pseudomonas biofilm phenotype; a lower cytotoxicity and higher biostability. However, when tested in vitro against the free-living form of this pathogen, it displayed a weaker bactericidal effect. Here, to investigate the reason accounting for this discrepancy, mechanistic studies on intact bacterial cells were initially carried out. Then to further understand the effect of packing parameters, i.e. composition, charge, shape and negative intrinsic curvature of membrane phospholipids in the membrane-permeabilizing activity of Esc peptides, leakage assays and circular dichroism spectroscopy analysis were carried out. Our results have suggested that the weaker in vitro antibacterial activity of Esc(1-21)-1c on the planktonic phenotype of the Gram-negative bacterium P. aeruginosa is mainly correlated to a slighter ability in permeabilizing both outer and inner bacterial membranes. Notably, experiments with lipid vesicles have suggested that if electrostatic interactions between negatively-charged membrane phospholipids and positively-charged peptide molecules do play a crucial role in the peptides’ membrane perturbing activity, this latter is hampered by the bilayer structure packing parameters including hydrogen bonding and intrinsic curvature, associated to phosphatidylserine (PE), especially for the diastereomer compared to all-L parent peptide. In parallel, we explored the molecular mechanism underlying the biofilm inhibition activity of Esc peptides when used at dosages below the minimal growth inhibitory concentration (1/8 MIC), by studying the peptides’ effect on the expression of key genes involved in the bacterial virulence and motility, as well as the peptide’ interaction with the bacterial signaling nucleotide ppGpp. Our findings revealed that the two D-amino acids containing Esc(1-21)-1c, confer the peptide the ability to downregulate the expression of biofilm-associated genes, likely as a result of increased peptide stability and prolonged binding to ppGpp compared to the all-L peptide. Furthermore, we reported two different applicative strategies to ameliorate the biological properties of these two AMPs: (i) encapsulation in poly(lactide-co-glycolide) (PLGA) nanoparticles; and (ii) covalent conjugation to soft CLs. In the first case, to enhance the peptides’ bioavailability and to optimize their translocation to the target infectious site, Esc peptides were loaded into PLGA nanoparticles (NPs) engineered with polyvinyl alcohol (PVA). The peptides-loaded NPs were found to be more efficient in diffusing through artificial CF mucus and simulated bacterial extracellular matrix compared to the free peptides. Moreover, they were more efficient in inhibiting P. aeruginosa growth under both in vitro and in vivo conditions at long term. In the second case, Esc peptides were covalently immobilized to hydrogel soft CLs and tested for their ability to reduce bacterial colonization. The antimicrobial CLs were able to cause more than four log reduction in the number of bacterial cells within 20 min and to reduce bacterial adhesion to their surface in 24 hours. Finally, the ability of both peptides to limit the onset of microbial resistance was also evaluated by exposing Pseudomonas strains to multiple cycles of treatment at sub-MIC dosages. Interestingly, in contrast with conventional antibiotics, Esc peptides did not induce resistance in P. aeruginosa cells. Overall, besides providing knowledges on the molecular mode(s) of action the two esculentin-derived AMPs, our data suggest that Esc peptides, particularly Esc(1-21)-1c, have great potential to be developed as novel drugs for treatment and prevention of P. aeruginosa pneumonia and keratitis.
Insights into the mechanism(s) of action and therapeutic applications of Esculentin-1a-derived antimicrobial peptides / Loffredo, MARIA ROSA. - (2019 Dec 19).
|Titolo:||Insights into the mechanism(s) of action and therapeutic applications of Esculentin-1a-derived antimicrobial peptides|
|Data di discussione:||19-dic-2019|
|Appartiene alla tipologia:||07a Tesi di Dottorato|