Abstract
Antimicrobial peptides (AMPs) are promising alternatives to classical antibiotics against antibiotic-resistant pathogens. TAT-RasGAP(317-326) is an AMP with broad range antibacterial activity, but its mechanism of action is unknown. In this study, we analyzed a strain of Escherichia coli with extensive resistance to TAT-RasGAP(317-326) but not to other AMPs that we obtained after twenty passages during an in vitro resistance selection experiment. This strain accumulated four mutations. One of these is a point mutation in bamA, which encodes an essential protein involved in the folding and proper insertion of outer membrane proteins. The mutation resulted in a change of charge in a surface-exposed negatively charged loop of the BamA protein. Using CRISPR-Cas9-based targeted mutagenesis, we showed that mutations lowering the negative charge of this loop decreased sensitivity of E. coli to TAT-RasGAP(317-326). In silico simulations unveiled the molecular driving forces responsible for the interaction between TAT-RasGAP(317-326) and BamA. These results indicated that electrostatic interactions, particularly hydrogen bonds, are involved in the stability of the molecular complex, representing a predictive fingerprint of the TAT-RasGAP(317-326) - BamA interaction strength. Interestingly, BamA activity was only partially affected by TAT-RasGAP(317-326), indicating that BamA may function as a specific receptor for this AMP. Our results indicate that binding and entry of TAT-RasGAP(317-326) may involve different mechanisms compared to other AMPs, which is in line with limited cross-resistance observed between different AMPs. This limited cross-resistance is important for the clinical application of AMPs towards drug-resistant pathogens.