Dissecting the RecA-(In)dependent Response to Mitomycin C in Mycobacterium tuberculosis Using Transcriptional Profiling and Proteomics Analyses.
Dissecting the RecA-(In)dependent Response to Mitomycin C in Mycobacterium tuberculosis Using Transcriptional Profiling and Proteomics Analyses.
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DOI：
10.3390/cells10051168
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发表时间：
2021-05-11
期刊：
影响因子：
6
通讯作者：
Dziadek J
中科院分区：
文献类型：
--
作者：
Brzostek A;Płociński P;Minias A;Ciszewska A;Gąsior F;Pawełczyk J;Dziadek B;Słomka M;Dziadek J
分枝杆菌利用至少两个独立的全局系统来响应DNA损伤：依赖于LexA/ reca的SOS反应和pafbc调节的途径。细胞内病原体，如结核分枝杆菌，在感染过程中暴露于氧化和亚硝化应激，同时居住在宿主巨噬细胞内。目前对reca对DNA损伤的非依赖性反应的理解是基于耻垢分枝杆菌的腐生模型，耻垢分枝杆菌是一种自由生活的非致病性分枝杆菌。本研究的目的是鉴定致病性细胞内分枝杆菌对DNA损伤的reca不依赖反应的因素。在全球转录分析的帮助下，我们能够解剖reca依赖和reca独立的途径。我们分析了缺乏recA基因的结核分枝杆菌菌株的DNA损伤反应，PafBC调节系统水平不可检测的菌株，以及两个系统同时下调的菌株。RNA-Seq分析与DNA损伤后细胞存活的评估相关，以估计每个系统对遗传毒性药物的总体敏感性的相关性。我们还对丝裂霉素c对结核分枝杆菌菌株的反应进行了全细胞蛋白质组学分析。该方法强调，在依赖RecA的DNA修复过程中，LexA （SOS系统的一个明确定义的关键元件）被蛋白水解失活，我们发现在缺乏RecA的情况下，DNA损伤剂对其的转录抑制。蛋白质组学分析显示，AlkB在ΔrecA pafBCCRISPRi/dCas9菌株中显著过量产生，而Holliday结分解物RuvX是一种DNA损伤反应因子，无论功能RecA和PafBC系统是否存在，该因子都显著上调，因此属于第三类DNA损伤因子：不依赖RecA和PafBC。编码alkA、dnaB和dnaE2的基因在ΔrecA pafBCCRISPRi/dCas9菌株的转录物水平上显著过表达。
Mycobacteria exploit at least two independent global systems in response to DNA damage: the LexA/RecA-dependent SOS response and the PafBC-regulated pathway. Intracellular pathogens, such as Mycobacterium tuberculosis, are exposed to oxidative and nitrosative stress during the course of infection while residing inside host macrophages. The current understanding of RecA-independent responses to DNA damage is based on the saprophytic model of Mycobacterium smegmatis, a free-living and nonpathogenic mycobacterium. The aim of the present study was to identify elements of RecA-independent responses to DNA damage in pathogenic intracellular mycobacteria. With the help of global transcriptional profiling, we were able to dissect RecA-dependent and RecA-independent pathways. We profiled the DNA damage responses of an M. tuberculosis strain lacking the recA gene, a strain with an undetectable level of the PafBC regulatory system, and a strain with both systems tuned down simultaneously. RNA-Seq profiling was correlated with the evaluation of cell survival in response to DNA damage to estimate the relevance of each system to the overall sensitivity to genotoxic agents. We also carried out whole-cell proteomics analysis of the M. tuberculosis strains in response to mitomycin C. This approach highlighted that LexA, a well-defined key element of the SOS system, is proteolytically inactivated during RecA-dependent DNA repair, which we found to be transcriptionally repressed in response to DNA-damaging agents in the absence of RecA. Proteomics profiling revealed that AlkB was significantly overproduced in the ΔrecA pafBCCRISPRi/dCas9 strain and that Holliday junction resolvase RuvX was a DNA damage response factor that was significantly upregulated regardless of the presence of functional RecA and PafBC systems, thus falling into a third category of DNA damage factors: RecA- and PafBC-independent. While invisible to the mass spectrometer, the genes encoding alkA, dnaB, and dnaE2 were significantly overexpressed in the ΔrecA pafBCCRISPRi/dCas9 strain at the transcript level.
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DOI：
10.3109/10409238.2011.576007
发表时间：
2011-06
影响因子：
6.5
作者：
Liu J;Ehmsen KT;Heyer WD;Morrical SW
通讯作者：
Morrical SW
影响因子：
48
作者：
Langmead, Ben;Salzberg, Steven L.
通讯作者：
Salzberg, Steven L.
影响因子：
3.7
作者：
Li X;Jiang X;Xu M;Fang Y;Wang Y;Sun G;Guo J
通讯作者：
Guo J
影响因子：
14.9
作者：
Gupta, Richa;Unciuleac, Mihaela-Carmen;Glickman, Michael S.
通讯作者：
Glickman, Michael S.
影响因子：
2.8
作者：
Dullaghan, EM;Brooks, PC;Davis, EO
通讯作者：
Davis, EO