EGS Technology: Characterization of Acinetobacter baumannii RNase P

ANGELES ZORREGUIETA; CAROL DAVIES SALA; MARCELO E TOLMASKY; SAUMYA JANI; PATRICIA L ABDIAN

New Orleans

Congreso; ASM microbe 2017; 2017

American Society for Microbiology

Background: EGS technology consists of inhibiting gene expression by utilizing an antisense oligonucleotide analog, known as EGS, to induce cleavage of a target RNA molecule by the bacterial RNase P. Since different bacterial species can harbor the same resistance gene, e.g., the aminoglycoside resistance aac(6ʹ)-Ib, an EGS that elicits degradation of the resistance mRNA could be combined with the antibiotic to overcome resistance in several bacteria. However, the success of this approach requires that the RNase P versions of the target bacteria are capable of utilizing the EGS as substrate. One important pathogen that naturally harbors aac(6ʹ)-Ib is Acinetobacter baumannii. Therefore, we identified and characterized this bacterium RNase P.Methods: aac(6ʹ)-Ib and the M1 RNA subunit of the A. baumannii RNase P were transcribed using a commercial kit, the gene coding for the C5 protein subunit was cloned fused to a histidine tag, and the protein was overexpressed and purified by affinity chromatography. mRNA cleavage assays were carried by mixing the C5 and M1 subunits and labeled substrate. The products were identified using denaturing gel electrophoresis.Results: A. baumannii genes potentially coding for an RNA species with conserved regions characteristic of the M1 catalytic subunit and a 120 amino acids peptide that includes a conserved region nearly identical to the E. coli C5 protein were identified and cloned. The M1 and C5 components were obtained and tested to determine functionality in vitro. The reconstituted A. baumannii RNase P digested a tRNA substrate as well as the aac(6?)-Ib mRNA in the presence of an EGS that was proved functional when tested in the presence of the E. coli RNase P. Furthermore, heterologous combination were also active in vitro, and an M1 thermosensitive E. coli mutant transformed with a clone coding for the A. baumannii M1 grew at the nonpermissive temperature.Conclusions: We identified and isolated the A. baumannii RNase P an showed that it can cleave a target mRNA in the presence of an EGS that was functional when tested with the E. coli enzyme. These results indicate that a unique EGS could be used to induce cleavage of aac(6?)-Ib in different bacterial backgrounds.