Table_5_One-Year Old Dormant, “Non-culturable” Mycobacterium tuberculosis Preserves Significantly Diverse Protein Profile.XLSX

For adaptation to stressful conditions, Mycobacterium tuberculosis (Mtb) is prone to transit to a dormant, non-replicative state, which is believed to be the basis of the latent form of tuberculosis infection. Dormant bacteria persist in the host for a long period without multiplication, cannot be detected from biological samples by microbiological methods, however, their “non-culturable” state is reversible. Mechanisms supporting very long capacity of mycobacteria for resuscitation and further multiplication after prolonged survival in a dormant phase remain unclear. Using methods of 2D electrophoresis and MALDI-TOF analysis, in this study we characterized changes in the proteomic profile of Mtb stored for more than a year as dormant, non-replicating cells with a negligible metabolic activity, full resistance to antibiotics, and altered morphology (ovoid forms). Despite some protein degradation, the proteome of 1-year-old dormant mycobacteria retained numerous intact proteins. Their protein profile differed profoundly from that of metabolically active cells, but was similar to the proteome of the 4-month-old dormant bacteria. Such protein stability is likely to be due to the presence of a significant number of enzymes involved in the protection from oxidative stress (katG/Rv1908, sodA/Rv3846, sodC/Rv0432, bpoC/Rv0554), as well as chaperones (dnaJ1/Rv0352, htpG/Rv2299, groEL2/Rv0440, dnaK/Rv0350, groES/Rv3418, groEL1/Rv3417, HtpG/Rv2299c, hspX/Rv2031), and DNA-stabilizing proteins. In addition, dormant cells proteome contains enzymes involved in specific metabolic pathways (glycolytic reactions, shortened TCA cycle, degradative processes) potentially providing a low-level metabolism, or these proteins could be “frozen” for usage in the reactivation process before biosynthetic processes start. The observed stability of proteins in a dormant state could be a basis for the long-term preservation of Mtb cell vitality and hence for latent tuberculosis.

为适应压力环境，结核分枝杆菌（Mycobacterium tuberculosis，简称Mtb）倾向于过渡至一种休眠的非复制状态，此状态被认为是潜伏性结核感染的基础。这些休眠细菌在宿主体内长期存活而不进行繁殖，无法通过微生物学方法从生物样本中检测到，然而，其“不可培养”的状态是可逆的。本研究中，我们采用二维电泳和基质辅助激光解吸电离飞行时间质谱分析（MALDI-TOF分析）技术，对储存超过一年的Mtb休眠、非复制细胞进行了蛋白质组学特征的表征。这些细胞具有可忽略的代谢活性、对抗生素的全抗性以及形态的改变（卵圆形）。尽管存在某些蛋白质降解，但一岁大的休眠分枝杆菌的蛋白质组仍然保留了众多完整的蛋白质。它们的蛋白质谱与代谢活跃细胞的蛋白质谱存在显著差异，但与四个月大的休眠细菌的蛋白质组相似。这种蛋白质的稳定性很可能归因于大量参与抵抗氧化应激（katG/Rv1908、sodA/Rv3846、sodC/Rv0432、bpoC/Rv0554）的酶以及伴侣蛋白（dnaJ1/Rv0352、htpG/Rv2299、groEL2/Rv0440、dnaK/Rv0350、groES/Rv3418、groEL1/Rv3417、HtpG/Rv2299c、hspX/Rv2031）以及DNA稳定蛋白的存在。此外，休眠细胞的蛋白质组还包含参与特定代谢途径（糖酵解反应、缩短的TCA循环、降解过程）的酶，这些酶可能提供低水平的代谢活动，或者这些蛋白质可能在生物合成过程开始之前“冻结”以用于再激活过程。观察到的休眠状态下蛋白质的稳定性可能是Mtb细胞长期保存活力的基础，从而是潜伏性结核病的基础。