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|Title: ||Studies on the function, modes of action and structure activity relationships of Pro-rich, β-defensins and helical antimicrobial peptides|
|Other Titles: ||Studio sulla funzione, meccanismo di azione e relazione struttura-attività dei peptidi antimicrobici Pro-rich, defensins e helical AMPs|
|Authors: ||Guida, Filomena|
|Supervisor/Tutor: ||Tossi, Alessandro|
|Co-supervisor: ||Scocchi, Marco|
|Issue Date: ||24-Apr-2012|
|Publisher: ||Università degli studi di Trieste|
|Abstract: ||Antimicrobial peptides (AMPs) are an important group of innate immunity effectors, needed to prevent or arrest microbial infections. In this thesis I describe an investigation on the modes of action and structure activity relationships of different types of AMPs, in particular the proline-rich bactenecins and β-defensins.
The sequence of the bovine cathelicidin Bac7, a reference Pro-rich AMP, has suggested several previous studies identifying the N-terminal region as responsible for the antimicrobial activity. To search for the minimal entry sequence into bacteria, and to investigate whether this overlaps with the minimal antimicrobial fragment a set of progressively shortened labelled N-terminal fragments of Bac7 were synthesized and tested for their antibacterial activity and internalization capacity into E. coli cells by flow cytometry and confocal microscopy. I confirmed the precise 16-residue fragment which is still fully active and is efficiently internalized into cells. Further shortening leads to a dramatic decrease of both functions. Furthermore it was found that a continuous chain is required for transmembrane transport and/or antimicrobial activity. In addition I have worked to determine the role of the two key N-terminal Arg residues of Bac7 on penetration of the outer membrane and translocation through a putative inner membrane transporter i) synthesizing Bac7 analogues with systematically altered physico-chemical properties of first two Arg residues, ii) using mutants of E.coli strains with deleted transport system or altered outer membrane characteristics iii) testing their differential potencies and internalization efficiency by flow cytometric assays and biological assay.
The results indicated that stereochemistry, charge and H-bonding all seem to be important requirements for the activity and internalization of this Pro-rich AMPs. These are relevant to both OM transit and the translocating role of inner membrane transporter, which was confirmed. These studies have helped evaluate Bac7 as a potential anti-infective agent selective for Gram-negative bacteria, as well as a possible vehicle for internalization of antibiotic cargo into these.
β-Defensins of various origins all show six highly conserved cysteines that form three S-S bridges to stabilize a β-sheet structure, suggesting a strong structure/activity relationship in their mode of action. Starting from the active and multimeric human β-defensin hBD3, I have synthesized a monomeric analogue that contains only the N-terminal α-helical stretch and one C-terminal strand from this AMP, removing most of the β-sheet core. Its activity was compared with that of hBD3 and a monomeric full sequence primate variant hcBD3. The antimicrobial activity of brevi-hBD3 was found to be comparable in potency to that of hBD3, but with significantly increased bacterial permeabilization capacity and decreased haemolysis with respect to hBD3. It appears to have switched to a different killing mechanism. It helped define the role of the long, amphipathic helical segment at the N-terminus of hBD3, a member of the -defensins which has a robust antimicrobial activity and is therefore considered a potential lead for novel anti-infective agents.
To study the interaction between antimicrobial peptides of interest to me and model membranes I have used Surface Plasmon Resonance. I observed a disparate capacity of AMPs to bind with membranes that can depend on very limited alterations in the peptides and have confirmed SPR as a valuable tool for understanding the mode-of-action of AMPs.
Throughout by PhD project, I have also actively collaborated in several studies aimed at understanding the modes of antimicrobial action and potential of different types of AMPs. In collaboration with groups at the Universities of Chieti, Udine, and Split, I have had the opportunity to study the roles of these AMPs against pathogens of cystic fibrosis or against the pathogenic alga Prototheca, causing bovine mastitis, and contributed to the identification and characterization of novel AMPs from teleosts and anurans. As these studies have resulted in publications of which I am co-author, I have included these with a brief description of methods used and the principal results.|
|PhD cycle: ||XXIV Ciclo|
|PhD programme: ||SCUOLA DI DOTTORATO DI RICERCA IN BIOMEDICINA MOLECOLARE|
|Keywords: ||Pro-rich, defensins, antimicrobial peptides|
|Main language of document: ||en|
|Type: ||Tesi di dottorato|
|Scientific-educational field: ||BIO/10 BIOCHIMICA|
|Appears in Collections:||Scienze biologiche|
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