CONICET | Buscador de Institutos y Recursos Humanos

Background: The dissemination of AAC(6')-I-type acetyltransferases has rendered amikacin and other aminoglycosides all but useless in some parts of the world. Developing compounds that interfere with expression of the resistance may extend the useful life of these and other antibiotics. External guide sequences (EGSs) are short antisense oligoribonucleotides that induce RNase P-mediated cleavage of a target RNA by forming a precursor tRNA-like complex. Methods: Several 13-nucleotide EGSs were designed to complement single stranded regions of aac(6)-Ib mRNA. Their capacity to bind to the messenger in vitro was determined by electrophoretic mobility shift assays. In vitro EGS-mediated cleavage of 5 end radiolabeled mRNA was assesed in the presence of M1 RNA and C5 protein, the two subunits of RNAse P, and analyzed on 6% denaturing polyacrylamide gel electrophoresis. In vivo activities of EGSs were determined following the growth of amikacin-containing cultures of E.coli BL21(DE3) harboring recombinant plasmids coding for AAC(6)-Ib and the EGS to be tested. The levels of aac(6)-Ib mRNA were inquired in vivo by northern blot. Results: EGSs complementary to locations within the five single stranded regions in the mRNA that encodes AAC(6)-Ib were analyzed. While small variations in the location targeted by different EGSs resulted in big changes in efficiency of binding to native aac(6)-Ib mRNA, most of them induced high levels of RNase P-mediated cleavage in vitro. In vivo assays indicated that the EGSs that exhibited the strongest binding efficiency to the mRNA in vitro interfered with expression of the resistance phenotype in vivo. A significant reduction on the aac(6)-Ib mRNA was observed in strain expressing EGS C3, the EGS with the strongest effect in vivo, when compared to sense or missense controls. Conclusion: There was a correlation of in vitro binding of EGSs to the mRNA and efficient interference with expression of resistance to amikacin. These results indicate that RNase P-mediated degradation of mRNA induced by the use of EGSs could be a viable strategy topreserve the efficacy of amikacin.