Discovery of a LuxR-type regulator involved in isoniazid-dependent gene regulation in Mycobacterium smegmatis
Discovery of a LuxR-type regulator involved in isoniazid-dependent gene regulation in Mycobacterium smegmatis
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DOI：
10.1016/j.jiac.2022.12.011
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发表时间：
2023-01-24
影响因子：
2.2
通讯作者：
Kaneko, Yukihiro
中科院分区：
文献类型：
--
作者：
Sakiyama, Arata;Oinuma, Ken-Ichi;Kaneko, Yukihiro
目的：大多数非结核分枝杆菌对抗结核药物异烟肼（INH）表现出内在耐药性。我们之前发现耻垢分枝杆菌的吡嗪酰胺酶/烟酰胺酶（PzaA）具有水解INH的酶活性，这可能有助于产生内在耐药性。此外，在缺氮条件下，INH 会强烈诱导 PzaA 表达，但这种现象的确切机制仍不清楚。在这里，我们的目的是使用转录组学方法揭示 PzaA 的 INH 依赖性诱导机制。方法：进行 RNA 测序以鉴定除 pzaA 之外的 INH 诱导基因。采用 cDNA 末端分析的 5' 快速扩增来鉴定 INH 诱导的转录单位的转录起始位点。通过INH水解实验和定量逆转录PCR等基因缺失和互补实验，证实了包含pzaA的基因簇中发现的LuxR样调节基因（MSMEI_1050）的功能。结果：RNA测序显示INH在缺氮条件下强烈诱导的23个基因，其中17个位于包含pzaA的基因簇中。该簇包含至少三个转录单位，包括含有 MSMEI_1050 的非 INH 诱导型单顺反子单位。该基因的缺失使耻垢分枝杆菌失去了对INH反应的能力，而互补则恢复了这种能力。结论：MSMEI_1050在INH依赖性基因调控中发挥着关键作用。确切的作用机制有待未来的研究确定。
Objective: Most non-tuberculous mycobacteria exhibit intrinsic resistance against the anti-tuberculosis drug isoniazid (INH). We previously found that a pyrazinamidase/nicotinamidase of Mycobacterium smegmatis , named PzaA, has an enzymatic activity to hydrolyze INH, which may contribute to intrinsic resistance. Furthermore, PzaA expression is strongly induced by INH under nitrogen-depleted conditions, although the precise mechanism of this phenomenon remains unclear. Here, we aimed to reveal the mechanism underlying the INH-dependent induction of PzaA using a transcriptomic approach.Methods: RNA sequencing was performed to identify INH-inducible genes other than pzaA. 5 ' rapid amplification of cDNA ends analysis was employed to identify the transcription start sites of INH-induced transcription units. The function of a LuxR-like regulator gene (MSMEI_1050) found within the gene cluster containing pzaA was confirmed by gene deletion and complementation experiments involving INH hydrolysis assay and quantitative reverse transcription PCR.Results: RNA sequencing revealed 23 genes that INH strongly induced under conditions of nitrogen depletion, 17 of which were in a gene cluster containing pzaA. This cluster comprised at least three transcription units, including a non-INH-inducible monocistronic unit containing MSMEI_1050. Deletion of this gene deprived M. smegmatis of the ability to respond to INH, and complementation restored this ability.Conclusions: MSMEI_1050 plays a key role in INH-dependent gene regulation. The precise mechanism of action is to be determined in future studies.