Department of Chemical and Biomolecular Engineering, University of Houstongrid.266436.3, Houston, Texas, USA.
Microbiol Spectr. 2022 Feb 23;10(1):e0225321. doi: 10.1128/spectrum.02253-21.
Persister cells are a small subpopulation of phenotypic variants that survive high concentrations of bactericidal antibiotics. Their survival mechanisms are not heritable and can be formed stochastically or triggered by environmental stresses such as antibiotic treatment. In this study, high-throughput screening of an Escherichia coli promoter library and subsequent validation experiments identified several genes whose expression was upregulated by antibiotic treatment. Among the identified genes, , and were found to be important in persister cell formation in E. coli as their deletion significantly enhanced the sensitivity of cells to various antibiotics. The GuaA and GuaB enzymes form the upstream reactions of ppGpp (a global persister molecule) biosynthesis, and the deletion of and drastically perturbs the ppGpp regulon in E. coli. WaaG, a lipopolysaccharide glucosyltransferase, plays an important role in shaping the outer membrane structure, and the deletion of dissipates the proton gradient (ΔpH) component of cellular proton motive force (PMF), perturbs cellular ATP production, and reduces type I persister formation in stationary phase. Active respiration in the stationary phase, which drives the PMF, was previously shown to play a critical role in type I persister formation, and our results associated with the deficient strain further corroborate these findings. Persistence is a nonheritable trait by which normal growing cells switch phenotypically to antibiotic tolerant persister cells. This transient state enables persister cells to recover and grow into an antibiotic-sensitive population. Persister cells have been observed in many pathogenic and nonpathogenic bacteria. Previous studies highlight the complexity and diversity of bacterial persister-cell mechanisms, many of which still remain to be elucidated. Here, using promoter and knockout cell libraries in Escherichia coli, we have identified genes that reveal novel persister mechanisms. As persistence is a critical survival strategy that evolved in many bacteria, our study will enhance the current molecular-level understanding of this conserved mechanism.
持久菌细胞是表型变异的一小部分亚群，能够在高浓度杀菌抗生素中存活。它们的生存机制不是可遗传的，可以随机形成，也可以被抗生素治疗等环境压力触发。在这项研究中，通过高通量筛选大肠杆菌启动子文库和随后的验证实验，鉴定了几个基因，这些基因的表达在抗生素处理下被上调。在所鉴定的基因中，GuaA 和 GuaB 酶是 ppGpp（一种全局持久分子）生物合成的上游反应，它们的缺失显著增强了细胞对各种抗生素的敏感性。WaaG 是一种脂多糖葡萄糖基转移酶，在塑造外膜结构方面起着重要作用，其缺失会耗散细胞质子动力势（PMF）的质子梯度（ΔpH）成分，扰乱细胞 ATP 产生，并减少静止期 I 型持久菌的形成。以前的研究表明，静止期的主动呼吸驱动 PMF，在 I 型持久菌的形成中起着关键作用，我们与缺失菌株相关的结果进一步证实了这一发现。持久菌是一种非遗传性特征，使正常生长的细胞在表型上转变为对抗生素有耐受性的持久菌细胞。这种短暂状态使持久菌细胞能够恢复并生长为对抗生素敏感的群体。持久菌已经在许多致病性和非致病性细菌中被观察到。以前的研究强调了细菌持久菌机制的复杂性和多样性，其中许多仍然有待阐明。在这里，我们使用大肠杆菌的启动子和敲除细胞文库，鉴定了揭示新的持久菌机制的基因。由于持久菌是许多细菌进化而来的关键生存策略，我们的研究将增强对这一保守机制的当前分子水平理解。
