Transcriptional regulation of multi-drug tolerance and antibiotic-induced responses by the histone-like protein Lsr2 in M. tuberculosis.
Transcriptional regulation of multi-drug tolerance and antibiotic-induced responses by the histone-like protein Lsr2 in M. tuberculosis.
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结核分枝杆菌中，组蛋白样LSR2对多药耐受性和抗生素诱导的反应的转录调节。
DOI：
10.1371/journal.ppat.0030087
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发表时间：
2007-06
期刊：
影响因子：
6.7
通讯作者：
Alland, David
中科院分区：
文献类型：
--
作者：
Colangeli, Roberto;Helb, Danica;Vilcheze, Catherine;Hazbon, Manzour Hernando;Lee, Chee-Gun;Safi, Hassan;Sayers, Brendan;Sardone, Irene;Jones, Marcus B;Fleischmann, Robert D;Peterson, Scott N;Jacobs, William R Jr;Alland, David
多重耐药性是一个关键的表型特性，它使感染结核分枝杆菌的哺乳动物的绝育变得复杂。先前的研究已经证实，iniBAC 是一种操纵子，通过相关的泵样活性赋予牛支原体 BCG 多药耐受性，它是由抗生素异烟肼 (INH) 和乙胺丁醇 (EMB) 诱导的。通过研究调节抗生素介导的基因表达的因素，可以更好地了解抗生素诱导基因的功能作用和药物耐受性的调节。对含有与结核分枝杆菌 iniBAC (PiniBAC) 启动子融合的 lacZ 基因的耻垢分枝杆菌菌株进行转座子诱变。具有组成型表达和 EMB 介导的 PiniBAC::lacZ 诱导增加的突变体映射到 lsr2 基因 (MSMEG6065)，这是一种功能未知的小型碱性蛋白，在分枝杆菌中高度保守。这些突变体的菌落形态发生了显着变化，并产生了新的极性脂质。多拷贝结核分枝杆菌 lsr2 (Rv3597c) 的补充使 PiniBAC 表达恢复至基线，逆转了观察到的形态和脂质变化，并将 EMB 对 PiniBAC 的诱导抑制至低于对照耻垢分枝杆菌菌株的水平。 lsr2 敲除的微阵列分析证实了耻垢分枝杆菌 iniA 的上调，并证明了参与细胞壁和代谢功能的基因的上调。在野生型耻垢分枝杆菌 lsr2 敲除中，EMB 处理诱导的 584 个基因中，有 121 个基因被上调（“过度诱导”）至更高水平。上调程度最高的基因和基因簇具有富含腺嘌呤-胸腺嘧啶 (AT) 的 5 素非翻译区域。在结核分枝杆菌中，lsr2 的过度表达抑制了 INH 介导的所有三个 iniBAC 基因以及另一个带注释的泵 efpA 的诱导。 Lsr2 (pI 10.69) 的低分子量和基本特性表明它是一种组蛋白样蛋白，尽管它与此类中的其他蛋白没有表现出序列同源性。与其他组蛋白样蛋白一样，Lsr2 优先结合环状 DNA 来结合 DNA，形成大的寡聚体，抑制 DNase I 活性，并将适度的超螺旋引入松弛的质粒中。 Lsr2 还抑制体外转录和拓扑异构酶 I 活性。 Lsr2 代表一类新型组蛋白样蛋白，可抑制多种 DNA 相互作用酶。 Lsr2 似乎通过优先结合富含 AT 的序列来调节分枝杆菌中的几个重要途径，包括抗生素诱导的基因和与诱导性多药耐受相关的基因。更好地了解 lsr2 的作用可能会为了解抗生素的作用机制以及分枝杆菌适应抗生素治疗等应激的方式提供重要见解。了解用抗生素治疗结核分枝杆菌时刺激的细胞过程可能会提供线索，解释为什么需要数月的治疗和同时使用多种药物才能防止抗生素耐药性。抗生素治疗“开启”或诱导某些结核分枝杆菌基因。这些基因特别令人感兴趣，因为它们似乎可以帮助结核分枝杆菌在抗生素治疗的压力下生存。我们对两种分枝杆菌物种中抗生素诱导基因（包括 iniBAC）调节的研究表明，一种名为 Lsr2 的小蛋白控制 iniBAC 和其他抗生素诱导基因，尤其是与细胞壁相关的基因。 Lsr2 以相对非特异性的方式与 DNA 结合，并且似乎抑制某些必须与 DNA 相互作用作为其功能一部分的酶。这些特性将 Lsr2 与结合特定 DNA 序列的经典基因表达调节因子区分开来，并表明 Lsr2 是一种新型组蛋白样蛋白。这些蛋白质通过改变 DNA 的形状来调节基因，事实上，我们发现 Lsr2 可以通过在 DNA 结构中引入少量卷曲来改变 DNA 的形状。我们的结果表明，Lsr2 是分枝杆菌抗生素诱导反应的主要调节因子。
Multi-drug tolerance is a key phenotypic property that complicates the sterilization of mammals infected with Mycobacterium tuberculosis. Previous studies have established that iniBAC, an operon that confers multi-drug tolerance to M. bovis BCG through an associated pump-like activity, is induced by the antibiotics isoniazid (INH) and ethambutol (EMB). An improved understanding of the functional role of antibiotic-induced genes and the regulation of drug tolerance may be gained by studying the factors that regulate antibiotic-mediated gene expression. An M. smegmatis strain containing a lacZ gene fused to the promoter of M. tuberculosis iniBAC (PiniBAC) was subjected to transposon mutagenesis. Mutants with constitutive expression and increased EMB-mediated induction of PiniBAC::lacZ mapped to the lsr2 gene (MSMEG6065), a small basic protein of unknown function that is highly conserved among mycobacteria. These mutants had a marked change in colony morphology and generated a new polar lipid. Complementation with multi-copy M. tuberculosis lsr2 (Rv3597c) returned PiniBAC expression to baseline, reversed the observed morphological and lipid changes, and repressed PiniBAC induction by EMB to below that of the control M. smegmatis strain. Microarray analysis of an lsr2 knockout confirmed upregulation of M. smegmatis iniA and demonstrated upregulation of genes involved in cell wall and metabolic functions. Fully 121 of 584 genes induced by EMB treatment in wild-type M. smegmatis were upregulated (“hyperinduced”) to even higher levels by EMB in the M. smegmatis lsr2 knockout. The most highly upregulated genes and gene clusters had adenine-thymine (AT)–rich 5-prime untranslated regions. In M. tuberculosis, overexpression of lsr2 repressed INH-mediated induction of all three iniBAC genes, as well as another annotated pump, efpA. The low molecular weight and basic properties of Lsr2 (pI 10.69) suggested that it was a histone-like protein, although it did not exhibit sequence homology with other proteins in this class. Consistent with other histone-like proteins, Lsr2 bound DNA with a preference for circular DNA, forming large oligomers, inhibited DNase I activity, and introduced a modest degree of supercoiling into relaxed plasmids. Lsr2 also inhibited in vitro transcription and topoisomerase I activity. Lsr2 represents a novel class of histone-like proteins that inhibit a wide variety of DNA-interacting enzymes. Lsr2 appears to regulate several important pathways in mycobacteria by preferentially binding to AT-rich sequences, including genes induced by antibiotics and those associated with inducible multi-drug tolerance. An improved understanding of the role of lsr2 may provide important insights into the mechanisms of action of antibiotics and the way that mycobacteria adapt to stresses such as antibiotic treatment. Understanding the cellular processes stimulated when Mycobacterium tuberculosis is treated with antibiotics may provide clues as to why months of therapy and use of several drugs simultaneously are required to prevent antibiotic resistance. Antibiotic treatment “turns on” or induces certain M. tuberculosis genes. These genes are of special interest because they appear to help M. tuberculosis survive the stress of antibiotic treatment. Our study of the regulation of antibiotic-induced genes, including iniBAC, in two mycobacterial species revealed that a small protein called Lsr2 controls iniBAC and other antibiotic-induced genes, especially ones related to the cell wall. Lsr2 binds to DNA in a relatively non-specific manner and appears to inhibit certain enzymes that must interact with DNA as part of their function. These properties differentiate Lsr2 from classical regulators of gene expression that bind to specific DNA sequences, and suggest that Lsr2 is a novel histone-like protein. These proteins regulate genes by changing the way DNA is shaped, and, indeed, we found that Lsr2 can change the shape of DNA by introducing a small number of coils into its structure. Our results suggest that Lsr2 is a major regulator of antibiotic-induced responses in mycobacteria.