Reengineering redox sensitive GFP to measure mycothiol redox potential of Mycobacterium tuberculosis during infection.
Reengineering redox sensitive GFP to measure mycothiol redox potential of Mycobacterium tuberculosis during infection.
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DOI：
10.1371/journal.ppat.1003902
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发表时间：
2014-01
期刊：
影响因子：
6.7
通讯作者：
Singh A
中科院分区：
文献类型：
--
作者：
Bhaskar A;Chawla M;Mehta M;Parikh P;Chandra P;Bhave D;Kumar D;Carroll KS;Singh A
结核分枝杆菌(Mtb)在宿主吞噬细胞内遇到的氧化性有害环境中生存。为了保护自己免受氧化应激，Mtb产生毫摩尔浓度的霉菌硫醇(MSH)，它作为主要的细胞质氧化还原缓冲区发挥作用。在这里，我们介绍了一种新的系统，用于实时成像感染期间结核分枝杆菌细胞内的霉菌硫醇氧化还原电位(EMSH)。我们证明了Mtb MSH依赖的氧化还原酶(mycoredosin-1；mrx1)与氧化还原敏感的绿色荧光蛋白(roGFP2；mrx1-roGFP2)的偶联能够以前所未有的灵敏度和特异性测量分枝杆菌内EMSH的动态变化。利用mrx1-roGFP2，我们首次报道了在不同分枝杆菌物种、基因突变和耐药患者分离株中EMSH的定量测量。这些细胞研究首次揭示，巨噬细胞和亚空泡腔内的环境导致结核分枝杆菌人群EMSH的异质性。这种新型生物传感器的进一步应用表明，用抗结核药物处理感染结核分枝杆菌的巨噬细胞可以诱导EMSH的氧化位移，这表明巨噬细胞内环境和抗生素协同破坏了MSH的动态平衡，从而发挥了有效的Mtb杀伤作用。最后，我们分析了不同EMSH的结核分枝杆菌细胞在感染过程中的膜完整性，发现EMSH高的亚群对临床相关的抗生素敏感，而低EMSH的亚群促进了抗生素的耐受。综上所述，这些数据表明MSH氧化还原信号在调节抗结核药物治疗后分枝杆菌存活方面的重要性。我们预计，mrx1-roGFP2将对我们理解MTB的氧化还原生物学做出主要贡献，并将导致针对氧化还原代谢的新策略，以控制MTB的持久性。全球约30%的人口感染结核分枝杆菌(Mtb)。结核分枝杆菌在宿主吞噬细胞中的持久性取决于其抵抗氧化剂介导的抗菌反应的能力。霉硫醇(MSH)是主要的抗氧化剂，提供丰富的还原当量来源，保护结核分枝杆菌免受感染过程中遇到的氧化应激。大多数关于结核分枝杆菌氧化还原信号的研究依赖于对全细胞提取液中MSH的化学分析，这会产生氧化伪影，并阻止对感染过程中MSH氧化还原状态的动态成像。我们成功地开发了一种基于基因编码的氧化还原敏感荧光探针的新型非侵入性工具，用于实时测量结核分枝杆菌感染期间的霉菌硫醇氧化还原电位(EMSH)。我们首次揭示了强毒和无毒分枝杆菌菌株的EMSH，包括耐药的临床分离株。我们使用了这项技术，并得出了令人惊讶的结论：在单个感染的巨噬细胞内，单个结核分枝杆菌的氧化还原特征存在异质性。重要的是，我们发现抗结核药物加速了感染巨噬细胞内结核分枝杆菌的氧化应激，氧化还原异质性有助于药物耐受人群的出现。这些发现对抗结核药物治疗后分枝杆菌的持久性有一定的影响。
Mycobacterium tuberculosis (Mtb) survives under oxidatively hostile environments encountered inside host phagocytes. To protect itself from oxidative stress, Mtb produces millimolar concentrations of mycothiol (MSH), which functions as a major cytoplasmic redox buffer. Here, we introduce a novel system for real-time imaging of mycothiol redox potential (EMSH) within Mtb cells during infection. We demonstrate that coupling of Mtb MSH-dependent oxidoreductase (mycoredoxin-1; Mrx1) to redox-sensitive GFP (roGFP2; Mrx1-roGFP2) allowed measurement of dynamic changes in intramycobacterial EMSH with unprecedented sensitivity and specificity. Using Mrx1-roGFP2, we report the first quantitative measurements of EMSH in diverse mycobacterial species, genetic mutants, and drug-resistant patient isolates. These cellular studies reveal, for the first time, that the environment inside macrophages and sub-vacuolar compartments induces heterogeneity in EMSH of the Mtb population. Further application of this new biosensor demonstrates that treatment of Mtb infected macrophage with anti-tuberculosis (TB) drugs induces oxidative shift in EMSH, suggesting that the intramacrophage milieu and antibiotics cooperatively disrupt the MSH homeostasis to exert efficient Mtb killing. Lastly, we analyze the membrane integrity of Mtb cells with varied EMSH during infection and show that subpopulation with higher EMSH are susceptible to clinically relevant antibiotics, whereas lower EMSH promotes antibiotic tolerance. Together, these data suggest the importance of MSH redox signaling in modulating mycobacterial survival following treatment with anti-TB drugs. We anticipate that Mrx1-roGFP2 will be a major contributor to our understanding of redox biology of Mtb and will lead to novel strategies to target redox metabolism for controlling Mtb persistence. Approximately 30% of the global population is infected with Mycobacterium tuberculosis (Mtb). Persistence of Mtb in host phagocytes depends on its ability to resist oxidant-mediated antibacterial responses. Mycothiol (MSH) is the main antioxidant that provides an abundant source of reducing equivalent, which protects Mtb from oxidative stress encountered during infection. The majority of research into redox signaling in Mtb has relied on chemical analysis of MSH in whole cell extract, which creates oxidation artifacts and prohibits dynamic imaging of MSH redox state during infection. We have successfully developed a novel and noninvasive tool based on genetically encoded redox sensitive fluorescent probes to perform real-time measurement of mycothiol redox potential (EMSH) in Mtb during infection. For the first time we reveal the EMSH of virulent and avirulent mycobacterial strains, including drug-resistant clinical isolates. We used this technology and came to the surprising conclusion that within a single infected macrophage there is heterogeneity in the redox signature of individual Mtb bacilli. Importantly, we show that anti-TB drugs accelerate oxidative stress in Mtb within infected macrophages and redox heterogeneity can contribute to emergence of drug tolerant population. These findings have implications for mycobacterial persistence following treatment with anti-TB drugs.
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影响因子：
3.6
作者：
Chawla M;Parikh P;Saxena A;Munshi M;Mehta M;Mai D;Srivastava AK;Narasimhulu KV;Redding KE;Vashi N;Kumar D;Steyn AJ;Singh A
通讯作者：
Singh A
为什么要治愈结核病需要长期治疗？
影响因子：
15.8
作者：
Connolly, Lynn E.;Edelstein, Paul H.;Ramakrishnan, Lalita
通讯作者：
Ramakrishnan, Lalita
DOI：
10.1084/jem.175.4.1111
发表时间：
1992-04-01
期刊：
The Journal of experimental medicine
影响因子：
--
作者：
Chan J;Xing Y;Magliozzo RS;Bloom BR
通讯作者：
Bloom BR
影响因子：
7.4
作者：
Haramaki, N;Han, D;Packer, L
通讯作者：
Packer, L
影响因子：
4.8
作者：
Boshoff, Helena I. M.;Xu, Xia;Barry, Clifton E., III
通讯作者：
Barry, Clifton E., III