Welcome to the jungle: insights into the innate defence peptides
Understanding innate defence mechanisms is not only an exercise of pure science, but can give the scientific community new tools in fighting pathogens. In particular, the emergence of strains multi resistant to common antibiotics gave a rush to the identifi cation of new bioactive molecules. Possible candidates are a variety of endogenous gene encoded antibiotics, evolved together with other humoral effectors to protect hosts from infective agents, beyond adaptive immunity. The main target of the thesis is to get further insights in the actual "jungle" of AMPs, in particular on their mechanism of action, with the final goal of improving their perfor mance against so called ESKAPE pathogens. Thus, I've developed new specific methods, starting from in silico predictions to high throughput characterisation of mutants, and characterized new molecules, in particular peptides derived from bivalves, as lead com pounds for anti infective drugs. To achieve these goals, I worked with different labora tories expert in peptide synthesis, structural bioinformatics or recombinant protein pro duction. The first chapter gives an overview on antimicrobial peptides, with particular attention to CSab peptides. Such peptides display features useful for the development of new antibiotics, such as high stability and immunomodulatory properties. The second chapter presents the work done in collaboration with the Chemical sci ences Department of the University of Padua on the structural characterization of CSab peptides. Considering the abundance of putative AMP sequences and the scarcity of ef fectively produced and market oriented peptides, we developed a cysteine guided mo lecular dynamics protocol for CSab structure refinement. This could support the research of active fragments before the recombinant production or chemical synthesis of the whole peptide. The proposed protocol is now published in the Journal of Biomolecular Structure and Dynamics, issue of Sep 2016. As proof of application of chapter 2 protocol, the third chapter illustrates the exper imental work on myticin C, a polymorphic AMP from M. galloprovincialis. Such molecule displays interesting immuno related properties. Starting from a structural model, we were able to design MytC derived active fragments showing properties similar to the whole peptide. Part of results presented in this thesis are now published in the Journal of Agricultural and Food Chemistry, issue of Oct. 2015. Aiming to increase the repertoire of available bioactive AMPs, we selected five putative antimicrobial peptide sequences from M. galloprovincialis transcriptomic data and, after preliminary trials in E. coli, the yeast Pichia pastoris was selected for a more advantageous recombinant production. This work, described in the fourth chapter, was done in collaboration with ETHZ (Swiss) namely in the laboratory of Prof. Markus Aebi, where I spent six months under the supervision of Doctor Andreas Essig. Actually two proteins have been produced, one of them showing an interesting range of antimicrobial activity. During my staying at ETHZ, I've worked on copsin, a defensin like peptide previously identified in the fungus Coprinopsis cinerea and the fifth chapter describes the high throughput production of site directed copsin mutants and the in silico characterization of membrane interaction of copsin and of one copsin mutant with improved features, named k copsin. In the appendix are reported experimental inputs supporting other research lines carried on in the Dott. Paola Venier laboratory.