Department of Microbiology and Immunology, Weill Cornell Medical College, New York, New York, USA.
Immunology and Microbial Pathogenesis Graduate Program, Weill Cornell Graduate School of Medical Sciences, Cornell University, New York, New York, USA.
Microbiol Spectr. 2024 Jul 2;12(7):e0048724. doi: 10.1128/spectrum.00487-24. Epub 2024 Jun 11.
Iron scavenging is required for full virulence of mycobacterial pathogens. During infection, the host immune response restricts mycobacterial access to iron, which is essential for bacterial respiration and DNA synthesis. The iron-dependent regulator (IdeR) responds to changes in iron accessibility by repressing iron-uptake genes when iron is available. In contrast, iron-uptake gene transcription is induced when iron is depleted. The gene is essential in and is required for bacterial growth. To further study how iron regulates transcription, wee developed an iron responsive reporter system that relies on an IdeR-regulated promoter to drive Cre and mediated recombination in . Recombination leads to the expression of an antibiotic resistance gene so that mutations that activate the IdeR-regulated promoter can be selected. A transposon library in the background of this reporter system was exposed to media containing iron and hemin, and this resulted in the selection of mutants in the antioxidant mycothiol synthesis pathway. We validated that inactivation of the mycothiol synthesis gene results in increased recombination and increased IdeR-regulated promoter activity in the reporter system. Further, we show that vitamin C, which has been shown to oxidize iron through the Fenton reaction, can decrease promoter activity in the mutant. We conclude that the intracellular redox state balanced by mycothiol can alter IdeR activity in the presence of iron.IMPORTANCE is a tractable organism to study mycobacterial gene regulation. We used to construct a novel recombination-based reporter system that allows for the selection of mutations that deregulate a promoter of interest. Transposon mutagenesis and insertion sequencing (TnSeq) in the recombination reporter strain identified genes that impact iron regulated promoter activity in mycobacteria. We found that the mycothiol synthesis gene is required for IdeR mediated transcriptional regulation by maintaining intracellular redox balance. By affecting the oxidative state of the intracellular environment, mycothiol can modulate iron-dependent transcriptional activity. Taken more broadly, this novel reporter system can be used in combination with transposon mutagenesis to identify genes that are required by to overcome temporary or local changes in iron availability during infection.
铁的摄取对于分枝杆菌病原体的完全毒力是必需的。在感染过程中，宿主的免疫反应限制了分枝杆菌获得铁的机会，而铁对细菌的呼吸和 DNA 合成是必不可少的。铁依赖性调节因子（IdeR）通过在铁可用时抑制铁摄取基因来响应铁可及性的变化。相反，当铁耗尽时，铁摄取基因的转录被诱导。该基因在中是必需的，并且是细菌生长所必需的。为了进一步研究铁如何调节转录，我们开发了一种铁反应性报告系统，该系统依赖于 IdeR 调节的启动子来驱动 Cre 和在中的介导重组。重组导致抗生素抗性基因的表达，因此可以选择激活 IdeR 调节启动子的突变。在该报告系统的背景下，转座子文库暴露于含有铁和血红素的培养基中，这导致在抗氧化剂巯基乙胺合成途径中选择了突变体。我们验证了巯基乙胺合成基因的失活导致报告系统中的重组增加和 IdeR 调节启动子活性增加。此外，我们表明，维生素 C 通过芬顿反应已被证明可以氧化铁，可降低突变体中的启动子活性。我们得出结论，由巯基乙胺平衡的细胞内氧化还原状态可以改变铁存在下 IdeR 的活性。重要的是，是研究分枝杆菌基因调控的可处理的生物体。我们使用来构建一种新的基于重组的报告系统，该系统允许选择可调节感兴趣启动子的突变。在重组报告菌株中的转座子诱变和插入测序（TnSeq）鉴定了影响分枝杆菌中铁调节启动子活性的基因。我们发现，巯基乙胺合成基因是必需的，用于 IdeR 介导的转录调节，通过维持细胞内氧化还原平衡。通过影响细胞内环境的氧化状态，巯基乙胺可以调节铁依赖性转录活性。更广泛地说，这种新的报告系统可以与转座子诱变结合使用，以鉴定在感染过程中分枝杆菌克服铁可用性的暂时或局部变化所需的基因。
