Utility of OhrR-Ohr system for the expression of recombinant proteins in mycobacteria and for the delivery of M. tuberculosis antigens to the phagosomal compartment.

We have recently reported that in vitro and intracellular organic peroxide stress oxidizes OhrR of Mycobacterium smegmatis and that the oxidized OhrR consequently derepresses the expression of Ohr. Here we demonstrate that the OhrR-Ohr system is highly useful for the expression of recombinant mycobacterial proteins and also for the delivery of Mycobacterium tuberculosis (Mtb) antigens to the phagosomal compartments. Recombinant M. smegmatis strains, which bear plasmid constructs to express Ohr-T85BCFP and Ohr-MtrA, showed expression of fusion proteins upon induction with t-butyl hydroperoxide (t-BHP) in a dose dependent manner. The M. smegmatis expressed Ohr-T85BCFP fusion could be affinity purified by adding a 9x histidine tag to the C-terminal end of the fusion protein. Further, mouse bone marrow derived macrophages (BMDMs) infected with either recombinant M. smegmatis or BCG strains with ohr-T85BCFP construct showed expression of T85BCFP protein without any exogenously added inducer. In addition, BMDMs infected with either recombinant BCG or Mtb with ohr-T85BCFP construct could effectively deliver the antigens to T-cells at higher levels than strains bearing the control plasmid alone. Altogether, these results suggest that the OhrR-Ohr system is a novel inducible system to study the biology and pathogenesis of mycobacteria.