Masino, Antonio (2022) In silico identification, production and characterization of novel bioactive peptides with antimicrobial, anti-biofilm and anti-inflammatory activity. [Tesi di dottorato]

Preview

Text Masino_Antonio_34.pdf Download (17MB) | Preview

Collection description

The rapid increase in drug-resistant infections emphasizes the urgent need to develop new antimicrobials. Promising candidates are antimicrobial peptides (AMPs), also known as host defense peptides (HDPs), essential components of innate immunity in eukaryotes. Among them, cationic AMPs (CAMPs) are particularly intriguing: due to a peculiar amino acidic composition, they damage selectively bacterial membranes rich in anionic lipids bypassing bacterial resistance. The activities of this PhD thesis were framed in the context of a wider project whose goal is the development and validation of a computational/experimental platform for the identification, preparation and characterization of CAMPs with the final aim to develop biotechnological and biomedical CAMP-based applications. Using a previously developed in silico tool, human secretome was screened to identify “cryptic” CAMPs hidden inside human proteins. 34 new promising CAMPs from 17 proteins were selected, synthesized and characterized thus allowing both to confirm the validity of the tool and to obtain a panel of new potential antimicrobials. Six peptides derived from the three subunits of fibrinogen were selected for a deeper characterization and prepared in the recombinant form using another tool of the platform for the scalable, cheap production of CAMPs in E. coli. These peptides proved to be wide spectrum bactericidal antimicrobials with synergic activity when combined with each other, with a thrombin-derived CAMP – thus suggesting that cryptic CAMPs from the same district can cooperate – and with the antibiotics colistin and tobramycin. Some of the peptides also showed a significant antibiofilm activity. In parallel, a new tool for the characterization of CAMPs and, more in general, of any bioactive peptide has been developed exploiting the environment-dependent fluorescence of luciferin and aminoluciferin. These fluorophores allow the monitoring of even small variations in the local microenvironment, acting as reporters of conformational changes and binding events. The two probes allowed to study the interaction of the peptides with model membranes, SDS, LPS and E. coli cells. Using the new labelling strategy, the interaction with LPS of some CAMPs, including one of the newly identified fibrinogen-derived CAMPs, has been studied quantitatively. The high affinity of this peptide for micellar and non-micellar LPS suggests that it might work also as an LPS-scavenger, a property very intriguing from the pharmacological point of view.

Downloads

Actions (login required)

View Item