Identification of intracellular target of antimicrobial peptide EWAMP-R in bacteria

Antimicrobial peptides (AMPs) exhibit diverse antibacterial mechanisms. To elucidate the intracellular targets of the novel AMP, EWAMP-R, we generate EWAMP-R-resistant strains of Escherichia coli and Staphylococcus aureus. In E. coli, a nonsynonymous mutation in the clpX gene (1108 C > T) is identified, resulting in an amino acid change in ClpX (R370C). This mutation confers enhanced tolerance to EWAMP-R and induces a “hyperflagellate phenotype” in EWAMP-R-resistant E. coli, as verified by CRISPR/Cas9-generated clpX (1108 C > T) mutants. Further studies reveal that EWAMP-R can bind to the Walker A motif and inhibit the conformational changes of ClpX, suppressing its ATPase activity and the degradation of its substrates, such as LexA 1-84. The accumulation of LexA 1-84 downregulates the SOS response, inducing apoptosis-like death in E. coli. In contrast, EWAMP-R-resistant S. aureus exhibits only a metabolic slowdown. These findings establish ClpX as a novel target for AMPs and highlight EWAMP-R as a promising antibacterial agent. Screening spontaneous mutants reveals AMP EWAMP-R targets ClpX in E. coli, inhibits its ATPase activity, disrupts the SOS response, and induces apoptosis-like cell death. By contrast, in S. aureus, resistance only involves metabolic slowdown.