Down-regulation of gene expression in Gram-positive bacteria using RNase P

"Shoot the messenger"-strategies is a new innovative tool in functional genomic research of both bacteria and mammalian cells. This approach also holds promise for the development of a new class of therapeutics using RNA-molecules to silence unwanted gene expression in vivo. We will explore and use the capabilities of the endogenous, essential ribonuclease P (RNase P) to target, cleave and inactivate specific messenger RNAs (mRNAs) in efforts to understand the evolution, spread and persistence of pathogen ic lineages of antimicrobial resistant enterococci. A technique to down-regulate gene expression in Gram-positive bacteria (Enterococcus faecium) using external guide sequences (EGSs) will be developed through the identification of enterococcal RNase P su bstrate recognition and activity. EGSs are anti-sense oligos that bind sequence-specifically to mRNA of selected target genes. This binding mimics natural RNase P targets in the cell and induces RNase P-mediated mRNA cleavage. The technique will be used i n ongoing projects to validate mechanisms and factors involved in the persistence of antimicrobial resistance, chromosomal gene transfer events as well as essential factors for infection and colonisation using enterococci as a clinical relevant model syst em. A tool for efficient knockdown of gene expression is needed in E. faecium as a traditional knockout technique involving allelic replacement is difficult to apply in E. faecium.