Proline-rich peptides are a unique group of antimicrobial peptides that exert their activity selectively against Gram-negative bacteria through an
apparently non-membranolytic mode of action that is not yet well understood. We have investigated the mechanism underlying the antibacterial
activity of the proline-rich cathelicidin Bac7 against Salmonella enterica and Escherichia coli. The killing and membrane permeabilization
kinetics as well as the cellular localization were assessed for the fully active N-terminal fragment Bac7(1–35), its all-D enantiomer and for
differentially active shortened fragments. At sub-micromolar concentrations, Bac7(1–35) rapidly killed bacteria by a non-lytic, energy-dependent
mechanism, whereas its D-enantiomer was inactive. Furthermore, while the L-enantiomer was rapidly internalized into bacterial cells, the Denantiomer
was virtually excluded. At higher concentrations (≥64 μM), both L- and D-Bac7(1–35) were instead able to kill bacteria also via a
lytic mechanism. Overall, these results suggest that Bac7 may inactivate bacteria via two different modes of action depending on its concentration:
(i) at near-MIC concentrations via a mechanism based on a stereospecificity-dependent uptake that is likely followed by its binding to an
intracellular target, and (ii) at concentrations several times the MIC value, via a non-stereoselective, membranolytic mechanism.
