Locked nucleic acid-containing external guide sequences induce amikacin susceptibility on Escherichia coli harboring aac(6?)-Ib.

SOLER BISTUE, ALFONSO; MARTIN FA; HA H; JOAQUIN J

Philadelphia

Congreso; 109thASM General Meeting; 2009

American Society for Microbiology

Background: Antisense strategies targeting antibioticresistance genes may be useful to extend the life of antibiotics. ExternalGuide Sequences (EGSs) are small RNAs that induce RNase P-mediated cleavage of acomplementary mRNA by forming a precursor tRNA-like complex.  Anypharmacological application requires the development of nuclease-resistantRNA analogs that are active asEGSs.  We have developed oligomersincluding locked nucleic acid (LNA) acid residues that inhibit expression of aac(6')-Ib.Methods: In vitro activity of EGSs was assessed by incubatingthem in the presence of 5? end radiolabeledaac(6´)-Ib mRNA,  M1 RNAand C5 protein followed by 6% denaturing polyacrylamidegel electrophoresis.  In vivo activity ofEGS was determined by following growth of the hyperpermeable E.coli strain AS19 harboring aac(6')-Ib when cultured in the presenceof EGSs and amikacin. Internalization of Alexa Fluor 488 Green-conjugated EGSs was visualized by fluorescence microscopy.Results: Several LNA/DNA cooligomers with different number and configurations of LNA substitutions were able to elicit RNase P-mediated digestion of aac(6')-Ib mRNA with varying efficiencies. Conversely phosphorotioates, 2-O'-methyl RNA and phosphorodiamidate morpholino oligomerswere not recognized as substrates by RNAse P. Although replacements of two Cs among the 5' ACCA consensus end were sufficient to induce mRNA cleavage,further replacements in the antisense region of the EGS improved this activity.   The number and position of LNA substitutions were important for high efficiency of mRNA cleavage. The most active LNA/DNA EGSs could penetrate E.coliAS19 and interfere with resistance to amikacin.   Conclusion: LNA/DNA EGSs were able to elicit aac(6')-Ib mRNA degradation by RNase P and cause anincrease in susceptibility to amikacin in the hyperpermeable E. coli AS19. Our results suggest thatLNA/DNA EGSs might be an effective tool to preserve the efficacy ofaminoglycosides and overcome the problems caused by the spread of aac(6')-Ib among pathogenic gram negatives.