Li Yanan, Wood Thomas K, Zhang Weiwei, Li Chenghua
State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products, Ningbo University, Ningbo, China.
Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, Qingdao, China.
Front Microbiol. 2023 Mar 16;14:1127018. doi: 10.3389/fmicb.2023.1127018. eCollection 2023.
A small subpopulation of AJ01 that was exposed to tetracycline at 10 times the minimal inhibitory concentration (MIC) still survived, named tetracycline-induced persister cells in our previous work. However, the formation mechanisms of persister is largely unknown. Here, we investigated tetracycline-induced AJ01 persister cells by transcriptome analysis and found that the purine metabolism pathway was significantly downregulated, which was consistent with lower levels of ATP, purine, and purine derivatives in our metabolome analysis. Inhibition of the purine metabolism pathway by 6-mercaptopurine (6-MP, inhibits ATP production), increased persister cell formation and accompanied with the decreasing intracellular ATP levels and increasing cells with protein aggresome. On the other hand, the persister cells had reduced intracellular tetracycline concentrations and higher membrane potential after 6-MP treatment. Inhibition of the membrane potential by carbonyl cyanide m-chlorophenyl hydrazone reversed 6-MP-induced persistence and resulted in higher levels of intracellular tetracycline accumulation. Meanwhile, cells with 6-MP treatment increased the membrane potential by dissipating the transmembrane proton pH gradient, which activated efflux to decrease the intracellular tetracycline concentration. Together, our findings show that reduction of purine metabolism regulates AJ01 persistence and is associated with protein aggresome formation and intracellular tetracycline efflux.
一小部分暴露于10倍最小抑菌浓度（MIC）四环素的AJ01亚群仍存活，在我们之前的工作中被命名为四环素诱导的持留菌细胞。然而，持留菌的形成机制在很大程度上尚不清楚。在这里，我们通过转录组分析研究了四环素诱导的AJ01持留菌细胞，发现嘌呤代谢途径显著下调，这与我们代谢组分析中较低水平的ATP、嘌呤和嘌呤衍生物一致。用6-巯基嘌呤（6-MP，抑制ATP产生）抑制嘌呤代谢途径，增加了持留菌细胞的形成，并伴随着细胞内ATP水平的降低和含有蛋白质聚集体的细胞增加。另一方面，6-MP处理后，持留菌细胞的细胞内四环素浓度降低，膜电位升高。用羰基氰化物间氯苯腙抑制膜电位可逆转6-MP诱导的持留现象，并导致细胞内四环素积累水平升高。同时，经6-MP处理的细胞通过消散跨膜质子pH梯度增加膜电位，从而激活外排以降低细胞内四环素浓度。总之，我们的研究结果表明，嘌呤代谢的减少调节了AJ01的持留现象，并与蛋白质聚集体的形成和细胞内四环素外排有关。
