Das Kishore, Garnica Omar, Dhandayuthapani Subramanian
Center of Emphasis in Infectious Diseases and Department of Biomedical Sciences, Paul L. Foster School of Medicine, Texas Tech University Health Sciences Center El Paso, El Paso, TX, 79905, USA.
Center of Emphasis in Infectious Diseases and Department of Biomedical Sciences, Paul L. Foster School of Medicine, Texas Tech University Health Sciences Center El Paso, El Paso, TX, 79905, USA; Graduate School of Biomedical Sciences, Texas Tech University Health Sciences Center El Paso, El Paso, TX 79905, USA.
Tuberculosis (Edinb). 2019 May;116S:S19-S27. doi: 10.1016/j.tube.2019.04.007. Epub 2019 Apr 27.
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.
我们最近报道，体外和细胞内有机过氧化物应激会氧化耻垢分枝杆菌的OhrR，氧化后的OhrR会解除对Ohr表达的抑制。在此我们证明，OhrR-Ohr系统对于重组分枝杆菌蛋白的表达以及将结核分枝杆菌（Mtb）抗原递送至吞噬体区室非常有用。携带表达Ohr-T85BCFP和Ohr-MtrA的质粒构建体的重组耻垢分枝杆菌菌株，在用叔丁基过氧化氢（t-BHP）诱导后，以剂量依赖方式显示出融合蛋白的表达。耻垢分枝杆菌表达的Ohr-T85BCFP融合蛋白可通过在融合蛋白的C末端添加9x组氨酸标签进行亲和纯化。此外，用带有ohr-T85BCFP构建体的重组耻垢分枝杆菌或卡介苗（BCG）菌株感染的小鼠骨髓来源巨噬细胞（BMDM），在没有任何外源添加诱导剂的情况下显示出T85BCFP蛋白的表达。此外，用带有ohr-T85BCFP构建体的重组卡介苗或结核分枝杆菌感染的BMDM，与仅携带对照质粒的菌株相比，能够以更高水平有效地将抗原递送至T细胞。总之，这些结果表明，OhrR-Ohr系统是研究分枝杆菌生物学和发病机制的一种新型诱导系统。
