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 [Soutschek et al. 2004]. 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 mRNAs in our research efforts to understand the evolution, spread and persi stence of pathogenic lineages of antimicrobial resistant enterococci. A functional genetic knockdown technique to down-regulate gene expression in Gram-positive bacteria (Enterococcus faecium) using external guide sequences (EGSs) will be developed throu gh the identification of RNase P substrate 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 cle avage. One major advantage of this approach is to overcome the technological problems encountered in E. faecium when using traditional knockout techniques. Moreover, the problem of disrupting genes by resistance cassettes in multi-resistant clinical isola tes of any species will be bypassed. Finally, anti-sense technology offers an attractive alternative for microbial genomics exploring essential genes and clustered regions as it allows subtle control of gene expression rather than knockout. The technique will be used in 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 relev ant model system. A successful technique may also have a therapeutic potential by targeting expression of antimicrobial resistance, virulence factors and essential elements for bacterial growth.