A toxin-antitoxin module as a target for antimicrobial development.

Abstract The emergence and spread of pathogenic bacteria that have become resistant to multiple antibiotics through lateral gene transfer have created the need of novel antimicrobials. Toxin–antitoxin (TA) modules, which have been implicated in plasmid maintenance and stress management, are ubiquitous among plasmids from vancomycin or methicillin resistant bacteria. In the Streptococcus pyogenes pSM19035-encoded TA loci, the labile e antitoxin binds to free ζ toxin and neutralizes it. When the ζ toxin is freed from the e antitoxin, it induces a reversible state of growth arrest with a drastic reduction on the rate of replication, transcription and translation. However, upon prolonged ζ toxin action, the cells can no longer be rescued from their stasis state. A compound that disrupts the e·ζ interaction can be considered as an attractive antimicrobial agent. Gene e was fused to luc (Luc-e antitoxin) and ζ to the gfp gene (ζ-GFP). Luc-e or e antitoxin neutralizes the toxic effect of the ζ or ζ-GFP toxin. In the absence of the antitoxin, free ζ or ζ-GFP triggers a reversible loss of cell proliferation, but the ζK46A-GFP variant fails to block growth. Bioluminescence resonance energy transfer (BRET) assay was developed for high-throughput screening (HTS). To develop the proper controls, molecular dynamics studies were used to predict that the Asp18 and/or Glu22 residues might be relevant for e·ζ interaction. Luc-e efficiently transfers the excited energy to the fluorescent acceptor molecule (ζ-GFP or ζK46A-GFP) and rendered high bioluminescence BRET signals. The exchange of Asp18 to Ala from ζ (D18A) affects Luc-e·ζD18A K46A-GFP interaction. In this study, we validate the hypothesis that it is possible to disrupt a TA module and offer a novel and unexploited targets to fight against antibiotic-resistant strains.