Velayati Ali Akbar, Abeel Thomas, Shea Terrance, Konstantinovich Zhavnerko Gennady, Birren Bruce, Cassell Gail H, Earl Ashlee M, Hoffner Sven, Farnia Parissa
Mycobacteriology Research Centre, National Research Institute of Tuberculosis and Lung Disease (NRITLD), Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Genome Sequencing & Analysis Program, The Broad Institute of MIT & Harvard, Cambridge, MA, USA; Delft Bioinformatics Lab, Delft University of Technology, Delft, The Netherlands.
Int J Mycobacteriol. 2016 Mar;5(1):66-73. doi: 10.1016/j.ijmyco.2015.12.001. Epub 2015 Dec 28.
OBJECTIVE/BACKGROUND: Mycobacterium tuberculosis (MTB) causes active tuberculosis (TB) in only a small percentage of infected people. In most cases, the infection is clinically latent, where bacilli can persist in human hosts for years without causing disease. Surprisingly, the biology of such persister cells is largely unknown. This study describes the isolation, identification, and whole-genome sequencing (WGS) of latent TB bacilli after 782days (26months) of latency (the ability of MTB bacilli to lie persistent).
The in vitro double-stress model of latency (oxygen and nutrition) was designed for MTB culture. After 26months of latency, MTB cells that persisted were isolated and investigated under light and atomic force microscopy. Spoligotyping and WGS were performed to verify the identity of the strain.
We established a culture medium in which MTB bacilli arrest their growth, reduce their size (0.3-0.1μm), lose their acid fastness (85-90%) and change their shape. Spoligopatterns of latent cells were identical to original H37Rv, with differences observed at spacers two and 14. WGS revealed only a few genetic changes relative to the already published H37Rv reference genome. Among these was a large 2064-bp insertion (RvD6), which was originally detected in both H37Ra and CDC1551, but not H37Rv.
Here, we show cell-wall free cells of MTB bacilli in their latent state, and the biological adaptation of these cells was more phenotypic in nature than genomic. These cell-wall free cells represent a good model for understanding the nature of TB latency.
目的/背景：结核分枝杆菌（MTB）仅在一小部分感染者中引发活动性结核病（TB）。在大多数情况下，感染处于临床潜伏状态，此时杆菌可在人类宿主中持续存在数年而不引发疾病。令人惊讶的是，此类持留菌的生物学特性在很大程度上尚不清楚。本研究描述了潜伏782天（26个月）后潜伏性结核杆菌的分离、鉴定及全基因组测序（WGS）（MTB杆菌的持续存在能力）。
设计MTB培养的体外双应激潜伏模型（氧气和营养）。潜伏26个月后，分离出持续存在的MTB细胞，并在光学显微镜和原子力显微镜下进行研究。进行寡核苷酸分型和WGS以验证菌株的身份。
我们建立了一种培养基，其中MTB杆菌停止生长，体积减小（0.3 - 0.1μm），失去抗酸性（85 - 90%）并改变形状。潜伏细胞的寡核苷酸分型模式与原始H37Rv相同，在间隔区2和14处观察到差异。WGS显示相对于已发表的H37Rv参考基因组仅有少数基因变化。其中有一个2064 bp的大插入片段（RvD6），最初在H37Ra和CDC1551中均有检测到，但在H37Rv中未检测到。
在此，我们展示了处于潜伏状态的MTB杆菌的无细胞壁细胞，并且这些细胞的生物学适应性在本质上更多是表型而非基因组方面的。这些无细胞壁细胞代表了一个理解结核病潜伏本质的良好模型。
