Regulated antisense RNA eliminates alpha-toxin virulence in Staphylococcusaureus infection

The ability to selectively disrupt gene function remains a critical element in elucidating information regarding gene essentiality for bacterial growt h and/or pathogenesis. In this study,we adapted a tet regulatory expression system for use in Staphylococcus aureus,,vith the goal of downregulating g ene expression via induction of antisense RNA. We demonstrate that this sys tem exhibits a 50- to 100-fold dose-dependent level of induction in bacteri al cells grown in culture (i.e., in vitro) and also functions in mice (i.e. , in vivo) following oral administration of inducer. To determine whether i nduced antisense RNA could interfere with chromosomally derived gene expres sion, we cloned a fragment of the S, aureus alpha-toxin gene (hla) in antis ense orientation downstream of the tet promoter system and introduced the c onstruct into S. aureus. Induced antisense hla RNA downregulated chromosoma lly derived hla gene expression in vitro approximately 14-fold. Similarly, induction of hla antisense RNA in vivo dramatically reduced alpha-toxin exp ression in two different murine models of S. aureus infection. Most importa ntly, this reduction completely eliminated the lethality of the infection. These results indicate that the tet regulatory system functions efficiently in S. aureus and induced antisense RNA can effectively do downregulate chr omosomal gene expression both in vitro and in vivo.