Insights into structural features that affect the biological activities and mode of action of the human antimicrobial cathelicidin LL-37
Year: 2014
Type: dissertation
Abstract: The human peptide LL-37 is an important innate immune effector that contributes to defending the organism against infection in different scenarios, ranging from direct bacterial killing to the modulation of immune responses and acting as a signal molecule for different cell types involved in defence or healing. All these functions are possible despite its relatively simple structure, due to its capacity to interact with different types of biological membranes. This cationic peptide is able to switch from a random coil in aqueous solutions into an amphipathic helical structure in presence of salt ions or in the presence of membranes. Intra- and inter-molecular hydrophobic interactions in the hydrophobic sector of the amphipathic helix, together with ionic interactions in the polar sector, make LL-37 prone to self-association, which strongly affects its multiple biological roles, and these therefore need to be explored in detail. \nIn this thesis I have investigated how folding occurs when LL-37 passes from bulk solution to the surface of membranes, and which structural features could be relevant for its in vitro antimicrobial and host cell modulating activities. For this reason comparative biophysical studies were performed on LL-37 and its structurally diverse orthologue in macaque, RL-37. Fluorescent analogues of LL-37 and RL-37, obtained from the systematic substitution of Phe residues with the non-invasive fluorescent probe, p-cyanophenylalanine, contributed to elucidate differential conformational variations in these peptides. Steady-state and time-resolved fluorescence studies confirmed that unlike RL-37, which is monomeric in physiological solution or in presence of neutral model membranes, LL-37 is in an oligomeric form in solution, with a hydrophobic core likely forming near the central region. Furthermore LL-37 appears to interact with both anionic and neutral membranes in an oligomeric form. Cytofluorimetric studies, performed on bacterial cells treated at toxic peptide concentrations, confirmed that the different conformations of LL- and RL-37 in solution give rise to quite different modes of bacterial permeabilization. Lesions of a larger size were observed for LL-37, consistent with a toroidal pore-forming mechanism, while smaller lesions were detected for RL-37 and may indicate an incipient detergent-like mechanism of bacterial inactivation. \nI further investigated how many molecules are involved in the self-associated form of LL-37, either in solution or in presence of membranes, replacing the Phe5 residue with an analogue bearing Benzoyl-phenylalanine on the side-chain, which is able to crosslink to adjacent molecules when irradiated with UV light. Photo-crosslinking studies revealed that LL-37 is in equilibrium between forms ranging from monomeric to hexameric in physiological solution, and dissociates to lower order oligomers in the presence of membranes, but does not become completely monomeric. The effects of oligomerization on the biological activities of LL-37 were further explored through testing its antiparallel and parallel covalently bound dimers, linked at either end, designed to mimic an obligatory dimeric form of the peptide. Both types of dimerization reduced the antibacterial potency of LL-37 and its capacity to permeabilize the bacterial membrane. Cytotoxic effect against the host cells was instead different for the three forms of dimers tested, and was related to the degree of freedom of the C- or N-terminal region. \nFrom the information obtained on the role of oligomerization on the antibacterial and cytotoxic activity of LL-37, I then rationally designed four different analogues of LL-37, which bear minimal primary structure alterations from the native peptide, such as the replacement of one, or exchange of two adjacent residues, in order to modify inter- and intra-molecular salt-bridging. I attempted in this way to transfer some of the RL-37 characteristics to LL-37 in order to improve its antimicrobial properties through reducing its propensity to self-assemble. I confirmed that variations in the equilibrium between attractions or repulsions in LL-37 significantly affect its antibacterial activity and mode of action.
Author: Daniela Xhindoli
Language: English
Subfield: Microbiology
Field: Immunology and Microbiology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W1500658831