Inactivation of the Organic Hydroperoxide Stress Resistance Regulator OhrR Enhances Resistance to Oxidative Stress and Isoniazid in Mycobacterium smegmatis
Inactivation of the Organic Hydroperoxide Stress Resistance Regulator OhrR Enhances Resistance to Oxidative Stress and Isoniazid in Mycobacterium smegmatis
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DOI：
10.1128/jb.02252-14
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发表时间：
2015-01-01
影响因子：
3.2
通讯作者：
Dhandayuthapani, Subramanian
中科院分区：
文献类型：
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作者：
Saikolappan, Sankaralingam;Das, Kishore;Dhandayuthapani, Subramanian
有机过氧化氢胁迫抗性调节剂（organic hydroperoxide stress resistance regulator, OhrR）是一种MarR类型的转录调节剂，主要调控细菌中有机过氧化氢还原酶（organic hydroperoxide reducase, Ohr）的表达。在分枝杆菌中，编码这些蛋白质的基因只存在于少数物种中，其中包括快速生长的耻垢分枝杆菌。为了描述Ohr和OhrR在氧化应激防御中的作用，我们通过同源重组分别删除OhrR和Ohr基因，构建了缺乏这些蛋白表达的菌株。OhrR突变株（MS Delta OhrR）的OhrR表达上调数倍，这在转录物和蛋白水平上都可以观察到。在异丙烯过氧化氢（CHP）和过氧化叔丁基过氧化氢（t-BHP）诱导的褐皮藻野生型菌株（MSWt）中也发现了类似的Ohr表达上调。MS Delta ohrR中Ohr表达的升高与CHP和t-BHP对氧化应激的抗性增强以及抗结核药物异烟肼（INH）的抑制作用相关。此外，该突变株在巨噬细胞的细胞内区室中表现出显著提高的存活率。相比之下，缺乏Ohr的菌株（MS Delta Ohr）或缺乏Ohr和OhrR的菌株（MS Delta Ohr - OhrR）对氢过氧化物和INH的抗性有限或没有。此外，这些菌株在细胞内存活与野生型没有显著差异。电泳迁移迁移试验（emsa）显示，过表达和纯化的OhrR与ohr-ohrR基因间区具有更大的亲和力，这种相互作用取决于OhrR的氧化还原状态。这些结果表明，Ohr-OhrR是耻垢分枝杆菌一个重要的过氧化应激反应系统。
The organic hydroperoxide stress resistance regulator (OhrR) is a MarR type of transcriptional regulator that primarily regulates the expression of organic hydroperoxide reductase (Ohr) in bacteria. In mycobacteria, the genes encoding these proteins exist in only a few species, which include the fast-growing organism Mycobacterium smegmatis. To delineate the roles of Ohr and OhrR in defense against oxidative stress in M. smegmatis, strains lacking the expression of these proteins were constructed by deleting the ohrR and ohr genes, independently and together, through homologous recombination. The OhrR mutant strain (MS Delta ohrR) showed severalfold upregulation of Ohr expression, which could be observed at both the transcript and protein levels. Similar upregulation of Ohr expression was also noticed in an M. smegmatis wild-type strain (MSWt) induced with cumene hydroperoxide (CHP) and t-butyl hydroperoxide (t-BHP). The elevated Ohr expression in MS Delta ohrR correlated with heightened resistance to oxidative stress due to CHP and t-BHP and to inhibitory effects due to the antituberculosis drug isoniazid (INH). Further, this mutant strain exhibited significantly enhanced survival in the intracellular compartments of macrophages. In contrast, the strains lacking either Ohr alone (MS Delta ohr) or both Ohr and OhrR (MS Delta ohr-ohrR) displayed limited or no resistance to hydroperoxides and INH. Additionally, these strains showed no significant differences in intracellular survival from the wild type. Electrophoretic mobility shift assays (EMSAs) revealed that the overexpressed and purified OhrR interacts with the ohr-ohrR intergenic region with a greater affinity and this interaction is contingent upon the redox state of the OhrR. These findings suggest that Ohr-OhrR is an important peroxide stress response system in M. smegmatis.