Persister Escherichia coli Cells Have a Lower Intracellular pH than Susceptible Cells but Maintain Their pH in Response to Antibiotic Treatment.
Persister Escherichia coli Cells Have a Lower Intracellular pH than Susceptible Cells but Maintain Their pH in Response to Antibiotic Treatment.
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DOI：
10.1128/mbio.00909-21
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发表时间：
2021-08-31
期刊：
影响因子：
6.4
通讯作者：
Pagliara S
中科院分区：
文献类型：
--
作者：
Goode O;Smith A;Zarkan A;Cama J;Invergo BM;Belgami D;Caño-Muñiz S;Metz J;O'Neill P;Jeffries A;Norville IH;David J;Summers D;Pagliara S
持效细胞和活的但不可培养的（VBNC）细胞是两种克隆亚群，它们可以通过大量假定的分子机制在多药暴露中存活。在这里，我们结合联合收割机微流体，延时显微镜，和质粒编码的荧光pH值报告，以测量动态的细胞内pH值的个别persister，VBNC，和敏感的大肠杆菌细胞在氨苄青霉素治疗。我们发现，即使在抗生素暴露之前，持留菌的细胞内pH值也低于VBNC和敏感细胞。然后，我们研究了在persister E中观察到的差异pH调节的分子机制。大肠杆菌细胞，发现这与由tnaA编码的酶-有关。事实上，在ΔtnaA菌株中，我们发现耐药菌、VBNC和敏感性E. coli细胞。全基因组转录组学分析显示，与亲本菌株的表达水平相比，除了下调色氨酸代谢外，ΔtnaA菌株还下调了关键的pH稳态途径，包括对pH的响应、氧化还原和几种羧酸催化过程。我们的研究揭示了pH稳态，证明细胞内pH的调节在克隆群体中是不均匀的，一个亚组的细胞显示出差异的pH调节，以执行专门的功能，包括抗生素治疗后的存活。
Persister and viable but non-culturable (VBNC) cells are two clonal subpopulations that can survive multidrug exposure via a plethora of putative molecular mechanisms. Here, we combine microfluidics, time-lapse microscopy, and a plasmid-encoded fluorescent pH reporter to measure the dynamics of the intracellular pH of individual persister, VBNC, and susceptible Escherichia coli cells in response to ampicillin treatment. We found that even before antibiotic exposure, persisters have a lower intracellular pH than those of VBNC and susceptible cells. We then investigated the molecular mechanisms underlying the observed differential pH regulation in persister E. coli cells and found that this is linked to the activity of the enzyme tryptophanase, which is encoded by tnaA. In fact, in a ΔtnaA strain, we found no difference in intracellular pH between persister, VBNC, and susceptible E. coli cells. Whole-genome transcriptomic analysis revealed that, besides downregulating tryptophan metabolism, the ΔtnaA strain downregulated key pH homeostasis pathways, including the response to pH, oxidation reduction, and several carboxylic acid catabolism processes, compared to levels of expression in the parental strain. Our study sheds light on pH homeostasis, proving that the regulation of intracellular pH is not homogeneous within a clonal population, with a subset of cells displaying a differential pH regulation to perform dedicated functions, including survival after antibiotic treatment.