Development of a Potent and Functional <i>In Vivo</i> Peptide Competitive Inhibitor for the Toxin MazF

Cell growth regulation granted by toxin-antitoxin systems enables bacteria to fight phage infections, evade host immune defenses, and survive antibiotic treatment. In this work, a potent and specific peptide competitive inhibitor for the <i>Escherichia coli</i> toxin MazF was developed and named Small Antitoxin of MazF (SamF). Employing a set of <i>N</i>-acetylated and <i>C</i>-amidated synthetic peptides, biophysical methods, biochemistry, and molecular biology techniques, we demonstrated that SamF binds tightly and with high specificity to MazF <i>in vitro</i> and <i>in vivo</i>, blocking access to the substrate binding site. Coexpression of SamF with MazF in <i>E. coli</i> efficiently counteracted the metabolic downregulation imposed by the toxin and the formation of antibiotic persisters. Altogether, our data uncovered a new MazF druggable site and an excellent scaffold for the design of antimicrobials. SamF is also a promising tool to study MazF <i>in vitro</i> and its physiological function in bacteria.