Differential Carbon Source Utilization Drives Metabolic State and Resuscitation in Antibiotic-Tolerant Persister Cells

Yufei Sun¹Sweta Roy²Qingbo Yang³Yinjie J Tang¹Dacheng Ren^2,4,5*

• ¹Department of Energy, Environmental, and Chemical Engineering, Washington University in St Louis, St Louis, United States
• ²Department of Biomedical and Chemical Engineering, Syracuse University, Syracuse, United States
• ³Cooperative Research, College of Agriculture, Environmental and Human Sciences, Lincoln University, Jefferson City, United States
• ⁴Department of Biology, Syracuse University, Syracuse, United States
• ⁵Department of Civil and Environmental Engineering, Syracuse University, Syracuse, United States

Despite significant advances in medicine, persistent infections remain challenging due to dormant bacterial cells that tolerate conventional antibiotics, even after prolonged high-dose treatments. Specifically, persister cells that are phenotypic variants characterized by high antibiotic tolerance, can resume growth once antibiotic stress is alleviated. Understanding the metabolic shifts of persister cells is important for the development of better therapies. While the general metabolic traits of persister cells have been documented, the precise metabolic shifts during persistence and resuscitation are poorly understood. Here, we applied stable isotope labeling using ¹³C-glucose and ¹³C-acetate to investigate metabolic dynamics in Escherichia coli persisters induced by carbonyl cyanide m-chlorophenyl hydrazone (CCCP). Our results demonstrated major differences in metabolic activities between normal and persister cells. Compared to normal cells, persister cells exhibited significantly reduced metabolism. Peripheral pathways including parts of the central pathway, the pentose phosphate pathway, and the tricarboxylic acid (TCA) cycle, exhibited significantly delayed labeling dynamics in persister cells. Proteinogenic amino acid profiling further demonstrated generalized but reduced labeling in persisters when using glucose as the sole carbon source, indicating a uniform slowdown in protein synthesis. Under acetate conditions, persister cells exhibited a more substantial metabolic shutdown, with markedly reduced labeling across nearly all pathway intermediates and amino acids, this reduction is likely due to the passive diffusion mechanism for acetate entry coupled with ATP demands required to activate acetate for central metabolism. Together, these results revealed metabolic changes in persister cells that are consistent with their dormant nature. The findings may help develop better control strategies.