Elucidating population-wide mycobacterial replication dynamics at the single-cell level.
Elucidating population-wide mycobacterial replication dynamics at the single-cell level.
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DOI：
10.1099/mic.0.000288
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发表时间：
2016-06
期刊：
影响因子：
--
通讯作者：
Sampson SL
中科院分区：
文献类型：
--
作者：
Mouton JM;Helaine S;Holden DW;Sampson SL
结核分枝杆菌感染导致一系列临床结果，并且通常感染以潜伏的、临床上无症状的状态持续。宿主内的细菌群体可能是异质的，并且认为持久性分枝杆菌来自于存活但非复制（VBNR）细胞的小群体。这些可能是抗生素耐受性，需要长期治疗。对这些持久性分枝杆菌知之甚少，因为它们很难分离。为了解决这个问题，我们已经成功地开发了一个复制报告系统，用于在M。结核这种称为荧光稀释的方法利用了两种荧光报告基因;组成型报告基因允许跟踪细菌，而诱导型报告基因允许测量细菌复制。应用荧光单细胞分析技术对M.结核病鉴定了鼠巨噬细胞中非生长分枝杆菌的独特亚群。感染48 h后，在同一巨噬细胞内观察到VBNR和活跃复制的分枝杆菌的存在。此外，我们的研究结果表明，巨噬细胞摄取导致富集的非或缓慢复制的细菌（所揭示的d-环丝氨酸治疗），这个人口很可能是高度富集的持久性，基于其耐药表型。这些结果证明了新的双荧光报告系统在体外和巨噬细胞感染模型中的成功应用，以提供进入分枝杆菌群体异质性的窗口。
Mycobacterium tuberculosis infections result in a spectrum of clinical outcomes, and frequently the infection persists in a latent, clinically asymptomatic state. The within-host bacterial population is likely to be heterogeneous, and it is thought that persistent mycobacteria arise from a small population of viable, but non-replicating (VBNR) cells. These are likely to be antibiotic tolerant and necessitate prolonged treatment. Little is known about these persistent mycobacteria, since they are very difficult to isolate. To address this, we have successfully developed a replication reporter system for use in M. tuberculosis. This approach, termed fluorescence dilution, exploits two fluorescent reporters; a constitutive reporter allows the tracking of bacteria, while an inducible reporter enables the measurement of bacterial replication. The application of fluorescence single-cell analysis to characterize intracellular M. tuberculosis identified a distinct subpopulation of non-growing mycobacteria in murine macrophages. The presence of VBNR and actively replicating mycobacteria was observed within the same macrophage after 48 h of infection. Furthermore, our results suggest that macrophage uptake resulted in enrichment of non- or slowly replicating bacteria (as revealed by d-cycloserine treatment); this population is likely to be highly enriched for persisters, based on its drug-tolerant phenotype. These results demonstrate the successful application of the novel dual fluorescence reporter system both in vitro and in macrophage infection models to provide a window into mycobacterial population heterogeneity.
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影响因子：
6.4
作者：
Keren I;Minami S;Rubin E;Lewis K
通讯作者：
Lewis K
DOI：
10.1073/pnas.1003219107
发表时间：
2010-07-06
影响因子：
11.1
作者：
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通讯作者：
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影响因子：
30.3
作者：
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影响因子：
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作者：
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通讯作者：
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影响因子：
3.7
作者：
Prakash H;Lüth A;Grinkina N;Holzer D;Wadgaonkar R;Gonzalez AP;Anes E;Kleuser B
通讯作者：
Kleuser B