Cathelicidins are a family of innate immunity effectors present in most vertebrates. Cathelicidin-derived antimicrobial peptides play an essential role in host defence, with a direct antimicrobial action as part of the innate immune responses and the capacity to modulate cellular immunity, affecting both innate and adaptive responses. Some can also have protective roles connected with wound healing, angiogenesis or sepsis. Cathelicidins are characterized by a conserved pro-region, known as 'cathelin-like' domain, carrying a highly variable, C-terminal antimicrobial domain. Many mammalian species express only one cathelicidin, but artiodactyles are known to express up to a dozen, bearing structurally and functionally very diverse AMPs. These including small disulfide-stabilized cyclic peptides, extended linear peptides rich in specific residues (e.g. proline, arginine, phenylalanine, tryptophan) and helical peptides of different sizes. Cetaceans are phylogenetic with artiodactyls, so are also expected to express this wide repertoire of cathelicidin-derived AMPs, whose functional characteristics may however have been shaped by the particular pathogenic microbiota they face in their aquatic lifestyle. With the known artiodactyl peptides, these might provide interesting leads for biomedical application. My Ph.D. followed to two parallel lines of research: i) the study of cathelicidin AMPs identified in the dolphin Tursiops truncatus (bottlenose dolphin), identified by searching genomic databases, to define their antimicrobial activities and obtain information on the modes-of-action, using different types of functional assays. These studies were carried out in parallel with selected orthologues from other artiodactyls, such as cow, sheep or pig, to screen for differences among them, and revealed both convergences and some interesting differences, especially relating to a proline-rich (PR-AMP) and an -helical one. These studies established a potent antimicrobial activity for the helical peptide, with a remarkable capacity too inhibit bacterial growth also at sub-lethal concentrations. The PR-AMP instead showed an increased internalization capacity, due to its apparent efficient use of alternative transporters. These data provided interesting insights into specific aspects of Cetaceans cathelicidins in relation to those of other mammals, and lay the groundwork for the possible development of novel antimicrobial agents. ii) the possible application of the bovine cathelicidin-derived PR-AMP Bac7(1-35) as a vehicle for delivering antibiotic cargo into susceptible bacterial cells. This peptide acts intracellularly, entering cells using specific transport systems. The aim was to conjugate it to the antibiotic tobramycin and expand its capacity to penetrate into Gram-negative pathogens, possibly overcoming resistance mechanisms. I began to develop synthetic strategies for modifying the antibiotic and then linking it to the peptide vehicle. Functional assays showed that one type of construct had a broader spectrum of activity than the individual components, and in some cases was active against resistant strains. This new strategy may potentially be applied to other types or classes of currently available antibiotics, as long as they can be modified for conjugation without affecting activity.

Cathelicidin host defence peptides in cetartiodactyls – identification and characterization of novel members and modification for exploitation as anti-infective agents

GAMBATO, STEFANO
2017-05-09

Abstract

Cathelicidins are a family of innate immunity effectors present in most vertebrates. Cathelicidin-derived antimicrobial peptides play an essential role in host defence, with a direct antimicrobial action as part of the innate immune responses and the capacity to modulate cellular immunity, affecting both innate and adaptive responses. Some can also have protective roles connected with wound healing, angiogenesis or sepsis. Cathelicidins are characterized by a conserved pro-region, known as 'cathelin-like' domain, carrying a highly variable, C-terminal antimicrobial domain. Many mammalian species express only one cathelicidin, but artiodactyles are known to express up to a dozen, bearing structurally and functionally very diverse AMPs. These including small disulfide-stabilized cyclic peptides, extended linear peptides rich in specific residues (e.g. proline, arginine, phenylalanine, tryptophan) and helical peptides of different sizes. Cetaceans are phylogenetic with artiodactyls, so are also expected to express this wide repertoire of cathelicidin-derived AMPs, whose functional characteristics may however have been shaped by the particular pathogenic microbiota they face in their aquatic lifestyle. With the known artiodactyl peptides, these might provide interesting leads for biomedical application. My Ph.D. followed to two parallel lines of research: i) the study of cathelicidin AMPs identified in the dolphin Tursiops truncatus (bottlenose dolphin), identified by searching genomic databases, to define their antimicrobial activities and obtain information on the modes-of-action, using different types of functional assays. These studies were carried out in parallel with selected orthologues from other artiodactyls, such as cow, sheep or pig, to screen for differences among them, and revealed both convergences and some interesting differences, especially relating to a proline-rich (PR-AMP) and an -helical one. These studies established a potent antimicrobial activity for the helical peptide, with a remarkable capacity too inhibit bacterial growth also at sub-lethal concentrations. The PR-AMP instead showed an increased internalization capacity, due to its apparent efficient use of alternative transporters. These data provided interesting insights into specific aspects of Cetaceans cathelicidins in relation to those of other mammals, and lay the groundwork for the possible development of novel antimicrobial agents. ii) the possible application of the bovine cathelicidin-derived PR-AMP Bac7(1-35) as a vehicle for delivering antibiotic cargo into susceptible bacterial cells. This peptide acts intracellularly, entering cells using specific transport systems. The aim was to conjugate it to the antibiotic tobramycin and expand its capacity to penetrate into Gram-negative pathogens, possibly overcoming resistance mechanisms. I began to develop synthetic strategies for modifying the antibiotic and then linking it to the peptide vehicle. Functional assays showed that one type of construct had a broader spectrum of activity than the individual components, and in some cases was active against resistant strains. This new strategy may potentially be applied to other types or classes of currently available antibiotics, as long as they can be modified for conjugation without affecting activity.
TOSSI, ALESSANDRO
29
2015/2016
Settore BIO/10 - Biochimica
Università degli Studi di Trieste
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11368/2908183
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