Development of Ribozyme P in vitro system for Acinetobacter baumannii

DAVIES SALA, C.G.; ABDIAN, P.L.; ZORREGUIETA, A.; TOLMASKY, M.E.

Mar del Plata

Congreso; IX Congreso Argentino de Microbiología General, SAMIGE; 2014

Multidrug resistant (MDR) Acinetobacter baumannii is emerging as the causative agent of numerous nosocomial outbreaks worldwide.  The frail health of hospitalized patients that are usually infected together with its multiresistant nature make A. baumannii a highly problematic pathogen to treat.  Since available antibiotics effective against MDR A. baumannii are running out, newer treatment strategies must be devised. EGS (External Guide Sequence) technology is a type of antisense technology that is utilized to silence essential or resistance genes. It consists of the use of antisense oligonucleotides, known as EGSs, to elicit Ribozyme P (RNase P)-mediated cleavage of a target RNA.  To design EGSs that can silence essential or resistance A. baumannii genes, we first identified and characterized its RNase P enzyme.  Bacterial RNase P is a ribonucleoprotein composed of a catalytic RNA subunit (M1) anda cofactor protein (C5). Its main natural substrates are pre-tRNAs but it can be induced to digest other RNA molecules if an appropriate double stranded three dimensional structure is formed.  Genes with high homology to the E. coli M1 and C5, called M1Ab and C5Ab, were identified within the genome of A. baumannii. Genomic comparisons showed that the M1Ab sequence can be classified as type A, and has high homology with those of other gram-negative opportunistic pathogens such as Escherichia coli and Klebsiella pneumoniae. However, while the M1 sequences of these two enterobacteria were nearly identical, M1Ab presents two 9- and 11-nucleotides gaps located upstream Universally Conserved Region I (CRI) and between CRI and CRII regions. On the other hand, C5Ab is predicted to be a 102 amino acid peptide that includes a 30-amino acids conserved region. The amino acid sequences flanking this conserved region have low homology with the E. coli C5 sequence. However, three-dimensional modeling comparing C5Ab to all known C5 proteins showed that highest structural homology occurs with the E. coli C5 protein. Likewise, C5Ab shows a high pI (10.8) typical of nucleic acid binding proteins, although not as high as that of the E. coli C5, which is 11.8. Following, the M1Ab and C5Ab genes were cloned under the control of the T7 promoter. Using these recombinant clones M1Ab was synthesized in vitro and C5Ab was overexpressed and purified. Both components were used to reconstitute the A. baumannii holoenzyme.  In conclusion, the A. baumannii RNase P was identified, the M1Ab and C5Ab components were characterized in silico, cloned, purified, and used to reconstitute the ribozyme in vitro. Future directions include the utilization of RNase P to test synthetic EGSs that elicit cleavage of mRNA corresponding to essential genes as a first step towards designing new pharmacological tools to control the growing threat of MDR A. baumannii infections.