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

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 peptide-based 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.