In vivo Display Technology for the Isolation of Antimicrobial Peptides
Professor Philip R. Cunningham, Ph.D.
Department of Biological Sciences @ Wayne State University
Interest in peptides as antimicrobial agents has increased dramatically in recent years, driven primarily by the development of new technologies for producing, screening and analyzing peptides coupled with a greater understanding of the nature of peptide-ligand interactions. The Cunningham lab has invented a new genetic method called In vivo Display (IVD) technology that has fostered the discovery of over one hundred novel peptides with potent broad-spectrum antimicrobial activity.
IVD was developed to identify peptides that inhibit critical processes in the cytoplasm and/or the periplasm and may therefore be used to identify novel antimicrobials. This technology fills a critical gap in the identification of novel peptides with antimicrobial activity:
1) No target bias; any peptide that inhibits bacterial growth for any reason may be identified without prior knowledge of the target.
2) The screen is performed in vivo; inhibitory peptides identified using IVD are by definition, active under physiological conditions, providing assurance that peptides isolated using this technology will be effective within their target cells.
3) Expression of the peptide occurs within the cell; peptides do not need to cross the outer membrane to reach their targets during the initial screen.
4) Expression of the peptides may be monitored in situ.
IVD was specifically developed to address the limitations of in vitro methods like phage display and ribosome display.
BRIEF DESCRIPTION OF IN VITRO DISPLAY TECHNOLOGY
Peptides are isolated using one of two systems: A cytoplasmic system for isolation of peptides that specifically disrupt cytoplasmic processes and/or structures and a periplasmic system for the isolation of antimicrobial peptides that specifically target components of the periplasm. The Cunningham lab has isolated peptides that fall into all four phenotypic categories: bacteriolytic, bacteriocidal, bacteriostatic, and inhibitory.
Following the initial screens, the antimicrobial peptides are characterized using a variety of in vivo and in vitro assays and this information is used to optimize their antimicrobial activity. Optimization involves changing the amino acid sequence and using peptidomimetic and medicinal chemistry to increase the antimicrobial activity, permeability, bioavailability, etc. of the peptide leads isolated with IVD. Over 100 hundred antimicrobial peptides have been identified and sequenced using these two systems.