El sistema toxina-antitoxina,eç, como inhibidor de la proliferación celular e inductor de tolerancia

Resumen

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, methicillin or erythromycin resistant bacteria. In the pSM19035-encoded TA loci, the labile ¿¿ antitoxin binds to free ¿ toxin and neutralizes it phosphotranferase activity. When the ¿ toxin is freed from the ¿ antitoxin, it induces a reversible state of growth arrest in distantly related bacteria as Bacillus subtilis and Escherichia coli. Growth arrest is mainly due to the inhibition of the synthesis of the nucleotide pool, especially GTP and UTP. This inhibition, which is obtained by an unknown mechanism, leads to a drastic reduction on the rate of replication, transcription and translation. However, upon prolonged ¿ toxin action, the cells cannot longer be rescued from their stasis state, and the cells die by lysis. It was found that upon ¿ induction, the repression of several essential genes involved in lipid metabolism could be responsible for cellular lysis. Bacterial populations contain a large fraction of cells susceptible to antibiotics and a small fraction in stasis that are refractory to them, called persisters cells. In this work, we have shown that ¿ toxin induces a small subpopulation that is refractory to the toxin effect. This small subpopulation could be considered as ¿persisters cells¿. In the presence of one antibiotic and the ¿ toxin it was observed an increased multidrug sensitivity, suggesting that ¿ toxin induces a different type of persisters. Furthermore, ¿ action is independent of the culture phase. We have shown that expression of the ¿¿antitoxin only reversed the refractory cells selected by the action of ¿¿toxin. Our results suggested that expression of ¿ toxin should reversibly induce a phenomenon that mimics persistence. However, stasis or dormancy, triggered by the expression of the¿¿ toxin, per se is not sufficient for multidrug tolerance. Indeed, ¿ expression increases multidrug sensitivity. All these results pointed towards ¿¿ system as an ideal candidate for developing new antimicrobials. If we could find a compound that disrupts the ¿¿¿ interaction, it can be considered as an attractive antimicrobial agent. Using molecular dynamics we were able to predict that Asp18 and in minor extent Glu22 residues might be relevant for ¿¿¿ interaction. In order to validate the in silico predictions a mutagenesis analysis on the ¿¿ system was carried out. In addition, a Bioluminescence resonance energy transfer (BRET) assay was developed for high-throughput screening (HTS). For the BRET assay, luc gene was fused to the ¿ gene (Luc-¿) and ¿ to the gfp gene (¿-GFP). Luc-¿ efficiently transfers the excited energy to the fluorescent acceptor molecule (¿-GFP or ¿K46A-GFP) and rendered high bioluminescence BRET signals. The mutagenesis was performed in a ¿ variant with a mutation in the active center (¿K46A-GFP) but that still formed a complex with the Luc-¿ antitoxin. The exchange of Asp18 to Ala from ¿K46A-GFP (D18A) affects Luc-¿·¿D18A K46A-GFP interaction, but exchange of Glu22 to Ala (E22A) does not. With these results, we have validated the hypothesis that it is possible to disrupt a TA module, offering novel and unexploited targets to fight against antibiotic-resistant strains.