Mapping of Mycobacterium tuberculosis katG promoters and their differential expression in infected macrophages
Mapping of Mycobacterium tuberculosis katG promoters and their differential expression in infected macrophages
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DOI：
10.1128/jb.183.13.4033-4039.2001
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发表时间：
2001-07-01
影响因子：
3.2
通讯作者：
Deretic, V
中科院分区：
文献类型：
--
作者：
Master, S;Zahrt, TC;Deretic, V
细胞内致病菌，包括结核分枝杆菌，通常具有多层防御机制，确保它们在宿主吞噬细胞中生存。结核分枝杆菌中的一个这样的防御决定簇是 katG 基因，它编码具有过氧化氢酶、过氧化物酶和过氧化亚硝酸酶活性的酶。 KatG 被认为对于防止吞噬细胞产生的活性氧和氮中间体具有重要作用。然而，KatG 还会激活一线抗结核药物异烟肼，因此使结核分枝杆菌对该化合物极其敏感。在这种情况下，katG 表达是一把双刃剑，因为它是重要的毒力决定因素，但同时其活性水平决定对 INH 的敏感性。因此，描述 katG 的调节和表达非常重要，因为这不仅可以帮助了解结核分枝杆菌如何在宿主中生存和持续存在，而且还可以为更好地管理 INH 治疗提供相关信息。在这里，我们报告了对体外和体内 katG 启动子活性的首次广泛分析。使用 S1 核酸酶保护分析，我们绘制了 katG mRNA 5' 末端图，并证明两个启动子 P(1)furA 和 P(1)katG 控制 katG 的转录，furA 和 katG 基因是从 P(1)furA 共转录的，P(1)furA 和 P(1)katG 启动子在受到过氧化氢和氢过氧化枯烯攻击后均表现出诱导作用，使用 转录融合P(1)furA-gfp、P(1)katG-gfp和P(1)furA-P(1)katG-gfp证实了两个katG启动子的存在。此外，我们还表明，两个启动子在强毒力结核分枝杆菌H37Rv的细胞内生长期间体内表达，P(1)furA在感染早期被诱导，而P(1)katG仅在巨噬细胞中延长生长后才变得活跃。这些研究描绘了furA-katG 区域的转录组织，并表明两个katC 启动子的体内差异调节。这些现象很可能反映了感染周期连续阶段的不同需求，并可能为提高对结核病宿主-病原体相互作用的理解和进一步优化 INH 化疗提供信息。
Intracellular pathogenic bacteria, including Mycobacterium tuberculosis, frequently have multitiered defense mechanisms ensuring their survival in host phagocytic cells. One such defense determinant in M. tuberculosis is the katG gene, which encodes an enzyme with catalase, peroxidase, and peroxynitritase activities. KatG is considered to be important for protection against reactive oxygen and nitrogen intermediates produced by phagocytic cells. However, KatG also activates the front-line antituberculosis drug isoniazid, hence rendering M. tuberculosis exquisitely sensitive to this compound. In this context, katG expression represents a double-edged sword, as it is an important virulence determinant but at the same time its activity levels determine sensitivity to INH, Thus, it is important to delineate the regulation and expression of katG, as this not only can aid understanding of how M. tuberculosis survives and persists in the host but also may provide information of relevance for better management of INH therapy. Here, we report the first extensive analysis of the katG promoter activity examined both in vitro and in vivo. Using S1 nuclease protection analysis, we mapped the katG mRNA 5 ' ends and demonstrated that two promoters, P(1)furA and P(1)katG, control transcription of katG, The furA and katG genes are cotranscribed from P(1)furA, Both P(1)furA and P(1)katG promoters show induction upon challenge with hydrogen peroxide and cumene hydroperoxide, Studies carried out using the transcriptional fusions P(1)furA-gfp, P(1)katG-gfp, and P(1)furA-P(1)katG-gfp, confirmed the existence of two katG promoters. In addition, we showed that both promoters are expressed in vivo during intracellular growth of virulent M. tuberculosis H37Rv, P(1)furA is induced early upon infection, and P(1)katG becomes active only upon extended growth in macrophages. These studies delineate the transcriptional organization of the furA-katG region and indicate differential regulation in vivo of the two katC promoters. These phenomena most likely reflect the differing demands at sequential stages of the infection cycle and may provide information for improved understanding of host-pathogen interactions in tuberculosis and for further optimization of INH chemotherapy.