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 towards Escherichia coli. BTP-001 is a ß-clamp targeting antimicrobial peptide containing an AlkB homologu 2 PCNA-interacting motif (APIM) linked to a cell penetrating peptide composed of 11 arginine residues (R11). Our data indicates that R11 mediates 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 affects the bacterial membrane, inducing increased expression and activation of the Cpx, ?E and Rcs cell envelope stress responses, which trigger the production of reaction oxygen species (ROS). This contributes to a rapid bactericidal effect as evidenced by increased short-term survival by addition of a ROS scavenger. Furthermore, we show that BTP-001 reduces the development of resistance to ciprofloxacin likely by binding to the ß-clamp via APIM, thereby inhibiting translesion synthesis. In addition, our multi-omics data suggests that the ?-clamp is part of ribosomal complexes, and that regulation of translation is targeted by BTP-001.  In conclusion, BTP-001 exhibits a multifaceted mode of action which strengthens its potential as a novel therapeutic drug against antibiotic resistant bacteria Overall design: To elucidate BTP-001's mode of action, we conducted RNA-sequencing (RNA-seq) of E. coli MG1655 treated with 2 doses of the novel antibiotic peptide BTP-001 and 1 dose of the cell penetrating peptide R11. We chose a low dose of BTP-001 (1 µM) with no impact on growth and a high dose (3 µM; equivalent to the minimum inhibitory concentration [MIC]) that affected the growth but did not yield extensive lethality during batch cultivation in bioreactors. The R11 dose (1 µM) reflects the BTP-001 low dose. The high dose (3 µM) stalled growth for around 10 min before the culture recovered and eventually reached the same growth rate as the control. All experiments were conducted with continuous dissolved oxygen and off-gas analysis. All treatments were given at OD600 = 0.2 to ensure exponential growth during the entire sampling period. To investigate the temporal effects of BTP-001 on E. coli, the low dose of BTP-001 (1 µM) culture was sampled for transcriptomics  over a time course between 1 to 120 min after treatment.  The high dose of BTP-001 (3 µM) and R11 (1 µM) were sampled for transcriptomics 10 min after treatment.