Characterization of L-arabinose -induced gene expression in Salmonella typhimurium

Objectives Bacterial delivery of therapeutic genes specifically to tumors has been explored recently. A selective induction of the gene expression would be required more appropriate to increase safety of the bacterial gene therapy. We characterized a remote controllable gene expression system with pBAD promoter and L-arabinose in Salmonella typhimurium △ppGpp mutant (SHJ2018).

Methods SHJ2018 was transformed with pBAD-Rluc8 plasmid to create a strain named SHJ2018/pBAD-Rluc8, in which Rluc gene expression would be remote-controlled by L-arabinose. In vitro expression induced by various L-arabinose concentrations was detected by luminometer, cooled-CCD camera or Western blotting. To image bioluminescence signal in living CT26-bearing Balb/c mice, in vivo images were obtained by cooled-CCD camera. In addition, in order to observe the tumor-targeting of SHJ2018, SHJ2018 was transducted with the pBAD-Rluc8 plasmid or pGEM-Rluc8 plasmid.

Results Under the control of pBAD system, the Rluc expression was detected clearly in the group of mice injected with L-arabinose. there was no or very weak expression of Rluc in the control group without L-arabinose. The maximum expression of Rluc in vitro was observed 1.5 hours after induction with 0.2% L-arabinose (p<0.005). Three days after bacterial injection, a strong bioluminescence signal of SHJ2018/pBAD-RLuc8 was detected in the tumor mass. In the group injected with SHJ2018/pBAD-RLuc8, the peak signal was detected 10 hours after induction of 60 mg L-arabinose and the signal increased afterwards in tumor site.

Conclusions We demonstrated that the regulation of pBAD mutant could be tightly controlled by L-arabinose in vitro as well as in vivo.