A dual-mechanism antimicrobial peptide with antimutagenic activity targets the replisome and induces cell envelope stress.

To combat the growing threat of multidrug-resistant bacteria, we need to develop novel antibiotics with unique modes of action. This study investigates the antibacterial properties of BTP-001 toward Escherichia coli. BTP-001 targets the β-clamp subunit of the DNA Pol III holoenzyme and is composed of the binding motif APIM linked to a cell-penetrating part composed of 11 arginine residues (R11). Our data indicate that R11 facilitates energy-dependent transport of BTP-001 across the cell membrane, possibly via iron transport systems such as the TonB system. The full-length BTP-001 peptide rapidly disturbs membrane integrity, inducing expression and activation of the Cpx cell envelope stress response. This response likely triggers the production of reactive oxygen species (ROS), contributing to the rapid bactericidal effect, as evidenced by increased short-term survival by addition of a ROS scavenger. Furthermore, this study confirms that BTP-001 targets DNA replication and reduces resistance development by inhibiting translesion synthesis. In addition, our data suggest that the β-clamp is associated with ribosomal complexes and that BTP-001 disrupts translational processes. In conclusion, BTP-001 exhibits a multifaceted mode of action, which strengthens its potential as a novel therapeutic drug against antibiotic-resistant bacteria.IMPORTANCEAs antimicrobial resistance (AMR) increases, the world needs new antibiotics with new modes of action to avoid cross-resistance. In this study, we investigated how BTP-001, a novel cell-penetrating peptide that contains a protein-binding motif for the essential DNA replication protein β-clamp, kills bacteria. We demonstrate that BTP-001 has a dual mode of action in which it i) targets the β-clamp and inhibits replication and mutagenesis and ii) disrupts the bacterial cell envelope, causing ROS accumulation and rapid cell death. In addition, our data indicate that BTP-001 affects translation, suggesting that the β-clamp may have unknown roles beyond replication. Our data also suggest that the bacterial import of BTP-001, via the cell-penetrating part of the peptide, is dependent on active transport and involves iron uptake mechanisms. BTP-001 has many properties that could be useful for further development as a new antibiotic.