Peptide-Based Drug Design

9
Investigating the Mode of Action of Proline-Rich
Antimicrobial Peptides Using a Genetic Approach:
A Tool to Identify New Bacterial Targets Amenable
to the Design of Novel Antibiotics
Marco Scocchi, Maura Mattiuzzo, Monica Benincasa,
Nikolinka Antcheva, Alessandro Tossi, and Renato Gennaro
Summary
The proline-rich antimicrobial peptides (PRPs) are considered to act by crossing
bacterial membranes without altering them and then binding to, and functionally modifying,
one or more specific targets. This implies that they can be used as molecular hooks to
identify the intracellular or membrane proteins that are involved in their mechanism of
action and that may be subsequently used as targets for the design of novel antibiotics with
mechanisms different from those now in use. The targets can be identified by using peptidebased
affinity columns or via the genetic approach described here. This approach depends
on chemical mutagenesis of a PRP-susceptible bacterial strain to select mutants that are
either more resistant or more susceptible to the relevant peptide. The genes conferring the
mutated phenotype can then be isolated and identified by subcloning and sequencing. In this
manner, we have currently identified several genes that are involved in the mechanism of
action of these peptides, including peptide-transport systems or potential resistance factors,
which can be used or taken into account in drug design efforts.
Key Words: Antimicrobial peptide; proline-rich peptide; Bac7; antibiotic resistant
mutant; chemical mutagenesis; fluorescence quenching; cell uptake.
1. Introduction
Host defense peptides are ancient effectors of innate immunity found in
all organisms, which have evolved several different mechanisms of action
(1–3). Many of these peptides display amphipathic, cationic scaffolds that allow
From: Methods in Molecular Biology, vol. 494: Peptide-Based Drug Design
Edited by: L. Otvos, DOI: 10.1007/978-1-59745-419-3 9, © Humana Press, New York, NY
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