Transcriptomics of Mycobacterium tuberculosis exposed to zinc limitation, zinc depletion by calprotectin and TPEN, and zinc toxicity

The purpose of this study was to determine the transcriptomes of Mycobacterium tuberculosis m2 6206 (Mtb) exposed to varying concentration of zinc (Zn2+) in vitro that mimic the concentrations of Zn2+ experienced in vivo during infection. Mtb were grown in the chemically defined Sauton's medium without Zn2+ added until expression of Zur-regulated genes were observed, indicating the onset of Zn-limitation. Cells were then treated with with different concentrations of Zn2+ through addition of 500 uM Zn2+, 6uM Zn2+, no added Zn2+, 0.35 mg/mL recombinant calprotectin (CP) or 0.8 µM N,N,N',N'-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine (TPEN). After 24 hours of exposure to the different [Zn2+] conditions, RNA was harvested, prepared for RNA-seq, and sequenced on the Illumina HiSeq platform with a paired-end (2x150bp) configuration. Overall design: Transcriptomes of Mtb exposed to 500 µM Zn2+, 6 µM Zn2+, no added Zn2+, 0.35mg/mL recombinant calprotectin (CP), and 0.8 µM TPEN

本研究旨在解析体外暴露于梯度浓度锌离子（Zn²+）的结核分枝杆菌（Mycobacterium tuberculosis）m2 6206（Mtb）的转录组，所设置的Zn²+浓度模拟了感染过程中体内实际存在的Zn²+水平。结核分枝杆菌先在不含额外Zn²+的化学成分限定Sauton培养基中培养，直至可检测到Zur调控基因（Zur-regulated genes）的表达变化，标志着锌限制培养状态的启动。随后通过添加500 μM Zn²+、6 μM Zn²+、无额外Zn²+、0.35 mg/mL重组钙卫蛋白（calprotectin，CP）或0.8 μM N,N,N',N'-四(2-吡啶甲基)-1,2-乙二胺（TPEN），使菌体接受不同浓度的Zn²+处理。在暴露于不同Zn²+浓度条件24小时后，收集菌体RNA并完成RNA测序建库，采用Illumina HiSeq测序平台以双端（2×150 bp）测序模式进行测序。整体实验设计：本研究的转录组测序样本涵盖暴露于500 μM Zn²+、6 μM Zn²+、无额外Zn²+、0.35 mg/mL重组钙卫蛋白（CP）以及0.8 μM TPEN的结核分枝杆菌转录组。