A toxin-antitoxin module as a target for antimicrobial development.
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 epsilon antitoxin binds to free zeta toxin and neutralizes it. When the zeta toxin is freed from the epsilon antitoxin, it induces a reversible state of growth arrest with a drastic reduction on the rate of replication, transcription and translation. However, upon prolonged zeta toxin action, the cells can no longer be rescued from their stasis state. A compound that disrupts the epsilon.zeta interaction can be considered as an attractive antimicrobial agent. Gene epsilon was fused to luc (Luc-epsilon antitoxin) and zeta to the gfp gene (zeta-GFP). Luc-epsilon or epsilon antitoxin neutralizes the toxic effect of the zeta or zeta-GFP toxin. In the absence of the antitoxin, free zeta or zeta-GFP triggers a reversible loss of cell proliferation, but the zetaK46A-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 epsilon.zeta interaction. Luc-epsilon efficiently transfers the excited energy to the fluorescent acceptor molecule (zeta-GFP or zetaK46A-GFP) and rendered high bioluminescence BRET signals. The exchange of Asp18 to Ala from zeta (D18A) affects Luc-epsilon.zetaD18A 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.
Department of Microbial Biotechnology, Centro Nacional de Biotecnología, CSIC, E-28049 Madrid, Spain.
Virginia S
VS
Lioy
Oscar
O
Rey
Dolors
D
Balsa
Teresa
T
Pellicer
Juan C
JC
Alonso
D000890:Anti-Infective Agents / Q000494:pharmacology*; D000992:Antitoxins / Q000378:metabolism*; D001412:Bacillus subtilis / Q000166:cytology / Q000187:drug effects; D001681:Biological Assay; D015195:Drug Design; D004926:Escherichia coli / Q000187:drug effects / Q000378:metabolism; D008163:Luminescent Measurements; D008826:Microbial Sensitivity Tests; D050296:Microbial Viability / Q000187:drug effects; D056004:Molecular Dynamics Simulation; D009154:Mutation / Q000235:genetics; D010455:Peptides / Q000494:pharmacology; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013297:Streptococcus pyogenes / Q000187:drug effects / Q000378:metabolism; D014118:Toxins, Biological / Q000378:metabolism* / Q000494:pharmacology
D000890:Anti-Infective Agents; D000992:Antitoxins; D010455:Peptides; D011993:Recombinant Fusion Proteins; D014118:Toxins, Biological