Table_5_Iron deprivation enhances transcriptional responses to in vitro growth arrest of Mycobacterium tuberculosis.xlsx

The establishment of Mycobacterium tuberculosis (Mtb) long-term infection in vivo depends on several factors, one of which is the availability of key nutrients such as iron (Fe). The relation between Fe deprivation inside and outside the granuloma, and the capacity of Mtb to accumulate lipids and persist in the absence of growth is not well understood. In this context, current knowledge of how Mtb modifies its lipid composition in response to growth arrest, depending on iron availability, is scarce. To shed light on these matters, in this work we compare genome-wide transcriptomic and lipidomic profiles of Mtb at exponential and stationary growth phases using cultures with glycerol as a carbon source, in the presence or absence of iron. As a result, we found that transcriptomic responses to growth arrest, considered as the transition from exponential to stationary phase, are iron dependent for as many as 714 genes (iron-growth interaction contrast, FDR <0.05), and that, in a majority of these genes, iron deprivation enhances the magnitude of the transcriptional responses to growth arrest in either direction. On the one hand, genes whose upregulation upon growth arrest is enhanced by iron deprivation were enriched in functional terms related to homeostasis of ion metals, and responses to several stressful cues considered cardinal features of the intracellular environment. On the other hand, genes showing negative responses to growth arrest that are stronger in iron-poor medium were enriched in energy production processes (TCA cycle, NADH dehydrogenation and cellular respiration), and key controllers of ribosomal activity shut-down, such as the T/A system mazE6/F6. Despite of these findings, a main component of the cell envelope, lipid phthiocerol dimycocerosate (PDIM), was not detected in the stationary phase regardless of iron availability, suggesting that lipid changes during Mtb adaptation to non-dividing phenotypes appear to be iron-independent. Taken together, our results indicate that environmental iron levels act as a key modulator of the intensity of the transcriptional adaptations that take place in the bacterium upon its transition between dividing and dormant-like phenotypes in vitro.

结核分枝杆菌（Mycobacterium tuberculosis, Mtb）在宿主体内建立长期感染的过程受多种因素调控，其中关键营养物质（如铁元素，Fe）的可获得性是重要一环。目前学界对于肉芽肿内外的铁匮乏环境，与Mtb在生长停滞状态下积累脂质并持续存活的能力之间的关联仍缺乏充分认知；同时，关于Mtb如何根据铁元素可获得性调整自身脂质组成以应对生长停滞的现有研究也十分有限。为阐明上述科学问题，本研究以甘油作为碳源构建培养体系，分别在含铁与缺铁的培养条件下，对处于指数生长期与稳定生长期的Mtb开展全基因组转录组与脂质组谱分析，并进行对比研究。结果显示，从指数生长期向稳定生长期转变的生长停滞过程中，细菌的转录组应答反应与铁元素密切相关：共计714个基因呈现铁-生长交互效应（错误发现率False Discovery Rate, FDR < 0.05），且其中大多数基因的转录应答幅度在铁匮乏条件下，无论上调或下调方向均得到增强。一方面，在生长停滞阶段被铁匮乏进一步增强上调表达的基因，其功能富集于金属离子稳态维持、以及多种胞内环境应激响应相关的生物学过程——后者被认为是胞内病原菌感染的核心特征之一。另一方面，在缺铁培养基中对生长停滞呈现更强抑制性应答的基因，则富集于能量产生相关通路（如三羧酸循环、NADH脱氢反应与细胞呼吸），以及核糖体活性关闭的关键调控因子，例如毒素-抗毒素（Toxin-Antitoxin, T/A）系统mazE6/F6。尽管获得上述发现，本研究未在稳定生长期的菌体中检测到细胞包膜的主要组分——菌醇二分枝杆菌酸酯（phthiocerol dimycocerosate, PDIM），无论培养体系是否含铁。该结果提示，Mtb适应非分裂表型过程中的脂质变化，似乎不受铁元素水平的调控。综上，本研究结果表明，环境中铁元素水平是调控该细菌在体外从分裂表型向类休眠表型转变过程中，转录适应反应强度的关键调节因子。