Understanding the Mechanism of Action of Tryptophan- and Arginine-rich Host-Defence Peptides

Abstract

In multicellular organisms antimicrobial peptides (AMPs) are a key component of the host innate immune system, hence they are also known as host defence peptides. AMPs demonstrate a direct antimicrobial activity against a broad range of pathogens and in some cases they can modulate the host immune system. Among the large number of distinct AMPs, the work described in this thesis is focused on a subfamily of peptides characterized by a high content of tryptophan and arginine residues. The tritrpticin and lactoferricin peptides are two important examples of this subfamily. For tritrpticin our work has focused on three consecutive tryptophan residues presented at the center of this peptide. An extensive and systematic substitution approach established that not all Trp residues were crucial for the antimicrobial activity of tritrpticin. However, subtle modifications of the sidechains of all three Trp residues could lead to changes in the mechanism of action of the peptide from a strong membrano-lytic to an intracellular perturbation mechanism. These results established the importance of the Trp residues in modulating tritrpticin’s bactericidal activity and its mode of action. Additionally, a new and cost-effective methodology for the incorporation of fluorinated Trp residues and fluorine-NMR analysis has opened the door for future studies of individual Trp residues in other Trp-rich AMPs. With regards to AMPs derived from the N-terminal region of the antimicrobial protein lactoferrin several contributions emerged from this thesis. First, the combination of the N-terminal region of the bovine lactoferricin peptide (LFcin) with the C-terminal region of the human LFcin resulted in a chimera peptide that showed the same activity as the original bovine LFcin. Secondly, the use of different methods of peptide cyclization proved to be useful in designing peptide analogs, which exhibited similar antimicrobial activity as bovine LFcin. Finally, the study of peptides derived from the first 11 residues of bovine and human LF (hLF11 and bLF10) established that hLF11 was not directly inhibiting the immune-related enzyme myeloperoxidase, as had been reported. Instead the Cys residue in the peptide was directly reacting with the enzyme reaction product.