“Host defence peptides” (HDPs) are an important component of innate immune system in animals, where they participate in the prevention and eradication of infections by killing pathogens and/or stimulating other components of the immune system.  The group’s research program focuses on the study of how evolved features in their sequences or structures determine their modes of action on both bacterial and host cells. This knowledge can then be used to help design HDP-based molecules for biomedical and biotechnological applications. The objectives of the group’s research are:

• - To find and characterize mammalian, anuran and marine organism HDPs (especially cathelicidins and defensins) and identify significant evolved features.
• - To investigate the relationship between these features (e.g. sequence, physical characteristics, conformation, self-interaction) and modes-of-action on host or bacterial cells.
• - To use the acquired knowledge to rationally modify HDPs (including attachment to other anti-infective agents, biocompatible surfaces or nanoparticles) or de-novo design of artificial AMPs for biomedical or biotechnological applications.

Project 1: Identification, characterization and evolution of host defence peptides

All vertebrate animals express at least one cathelicidin, and some express several. We have been scanning genome, transcript and protein databases using bioinformatics tools to identify many novel members of the cathelicidin family. Although cathelicidin HDPs show a considerable sequence diversity at the HDP level, they have conserved pre-prosequences and gene organizations that allow efficient sequence mining. These novel sequences are then inserted into a dedicated database, which apart from allowing a detailed study of their phylogenetic relationships and evolution, allows to probe the HDP domains for specific sequence and physico-chemical hallmarks that predict the antimicrobial or immunomodulatory potential. Chemical synthesis of selected peptides then allows characterization of functional features, such as capacity to interact with membranes or internalize into bacteria, the extent and type of lesions they can cause to biological membranes, intracellular activities, ecc. This then allows to validate and refine the original prediction used for selection, and to relate particular structural features that have been naturally selected for during their evolution, with specific functional features of the peptides. 

This information leads to i) a better understanding of the roles of cathelicidins in the immune responses of man and other animals, and ii) assessing what can be altered or needs be maintained in developing or redesigning the peptides (or de novo designing artificial molecules based on them), for biotechnological or biomedical applications.

(A. Tossi,  with Drs. Dennis Prickett and Pietro Decarli as external collaborators)

 

Project 2:  Structural factors defining the effects of host defence peptides on microbial and host cells.

HDPs can act either at cellular membranes or intracellularly on their bacterial targets, and either impair the membrane at micromolar concentrations (via formation of pores or other types of lesions) or internalize and inactivate essential cytoplasmic machineries, and can sometimes do both. At higher concentrations, they may also damage the membranes of animal cells (cytotoxic effects), whereas at the lower antimicrobial concentrations they can stimulate host cells by interacting with membrane or internal receptors (stimulating effects such as proliferation, chemotaxis and/or degranulation).  A better understanding of these multiple different functions will lead to a better understanding of the role of HDPs in immune responses and potentially to their therapeutic exploitation. We are probing the biological roles of selected HDPs (mainly human LL-37 and its primate or bovine orthologues, and the Pro-rich artiodactyl bactenecins) by rationally modifying their sequence and/or identifying active fragments and then studying their capacity to interact with and alter biological membranes, or their capacity to internalize and hit internal targets. We use a minimalist approach, where rational and knowledge-based modifications are effected so as to independently vary functionally important attributes one at a time and probe how this affects the mode of action.

To this end, we make use of a wide range of synthetic, biophysical and biochemical methods, such as solid phase peptide synthesis to prepare analogues, fluorescence and CD spectroscopy in different environments to study intermolecular interactions and functionally important structural changes, surface plasmon resonance to probe interactions with model membranes, or flow cytometry and various types of activity assays to study effects on host or bacterial cells.

(A.  Tossi and S. Pacor in collaboration with  M. Scocchi, M. Mardirossian, P. Cescutti  and C. Lagatolla)

 

Project 3. Rational redesign and optimization of HDPs

HDPs have a mode of action different to that of most clinically used antibiotics, and are considered to have a high potential as lead compounds for development of novel anti-infective agents  that could help overcome the current resistance crisis. This is also due to their potent activity against multi-drug resistant bacterial strains and relatively low tendency to elicit resistance themselves. However, they are not suitable for systemic use due to low bioavailability and often insufficient therapeutic indices. Rational modification of their structures and sequences can however increase their potential for specific uses:

• Optimization of size and physico-chemical characteristics can allow a cost-effective application for topical uses, or to prevent contamination of biomedical devices.
• Functionalization of the  relatively non-toxic, internally acting Pro-rich class of HDPs could not only provide a means for probing their mode of internalization and bacterial inactivation, but also allow for their use as vehicles to carry poorly permeant antimicrobial agents into bacteria.
• Anchoring of HDPs or their derivatives to nano-constructs of different type could increase their stability and in vivo lifetimes, reduce cytotoxicity and allow for a better distribution to the site of infection.

The implementation of these modifications benefits from knowledge gathered on HDP characteristics and SAR obtained in the other projects of the group, and from a long-standing experience with solid phase peptide synthesis, modification and conjugation.

(A. Tossi, S. Pacor in collaboration with  Andrea Caporale, CNR guest scientist)