Revealing the Metabolic Activity of Persisters in Mycobacteria by Single-cell D2O Raman Imaging Spectroscopy.
Revealing the Metabolic Activity of Persisters in Mycobacteria by Single-cell D2O Raman Imaging Spectroscopy.
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DOI：
10.1021/acs.analchem.9b03960
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发表时间：
2019-11
影响因子：
7.4
通讯作者：
H. Ueno;Yota Kato;K. Tabata;H. Noji
中科院分区：
文献类型：
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作者：
H. Ueno;Yota Kato;K. Tabata;H. Noji
即使在克隆种群中，细菌细胞的代谢活动也在很大程度上发生了分化。细胞之间的这种代谢差异被认为在表型适应不断变化的环境条件方面发挥了重要作用，例如抗生素的持久性。长期以来，人们一直认为持久者处于一种称为休眠的状态，在这种状态下，细胞代谢不活跃，不会生长。然而，最近的研究表明，某些类型的持久者不一定处于休眠状态，这引发了一场关于持久者机制的辩论。在这里，我们结合单细胞拉曼成像光谱和D2O标记来分析细菌持久细胞的代谢活性。具有代谢活性的细胞通过代谢过程摄取氚，并产生明显的C-D拉曼光谱，这是代谢活动的直接指标。利用这种成像方法，我们表征了污垢分枝杆菌的代谢活性，这是一种快速生长的结核分枝杆菌模型。我们发现，在抗生素利福平的存在下，耻垢分枝杆菌的伴胞细胞表现出一定的代谢活性和活跃的细胞生长。有趣的是，在接触抗生素之前，持久性与生长速度并不相关。这些结果表明，休眠与耻垢分枝杆菌细胞对利福平的持久性无关，这表明持久性的机制在很大程度上取决于抗生素和细菌的类型。我们的结果成功地证明了我们的基于灌注型的单细胞D2O拉曼成像系统在分析细菌宿存细胞的代谢活性和生长方面的潜力。
The metabolic activity of bacterial cells largely differentiates even within a clonal population. Such metabolic divergence among cells is thought to play an important role for phenotypic adaptation to ever-changing environmental conditions, such as antibiotic persistence. It has long been thought that persisters are in a state called dormancy, in which cells are metabolically inactive and do not grow. However, recent studies suggest that some types of persisters are not necessarily dormant, triggering a debate about the mechanisms of persisters. Here, we combined single-cell Raman imaging spectroscopy and D2O labeling to analyze metabolic activities of bacterial persister cells. Metabolically active cells uptake deuterium through metabolic processes and give distinct C-D Raman bands, which are direct indicators of metabolic activity. Using this imaging method, we characterized the metabolic activity of Mycobacterium smegmatis, a fast-growing model for Mycobacterium tuberculosis. We found that persister cells of M. smegmatis show certain metabolic activity and active cell growth in the presence of the antibiotic rifampicin. Interestingly, persistence is not correlated with growth rate prior to antibiotic exposure. These results show that dormancy is not responsible for the persistence of M. smegmatis cells against rifampicin, suggesting that the mechanism of persistence largely varies depending on the type of antibiotics and bacteria. Our results successfully demonstrate the potential of our perfusion based single-cell D2O Raman imaging system for the analysis of the metabolic activity and growth of bacterial persister cells.