Mutations in respiratory complex I promote antibiotic persistence through alterations in intracellular acidity and protein synthesis

Abstract

Antibiotic persistence describes the presence of phenotypic variants within an isogenic bacterial population that are transiently tolerant to antibiotic treatment. Perturbations of metabolic homeostasis can promote antibiotic persistence, but the precise mechanisms are not well understood. Here, we use laboratory evolution, population-wide sequencing and biochemical characterizations to identify mutations in respiratory complex I and discover how they promote persistence in Escherichia coli. We show that persistence-inducing perturbations of metabolic homeostasis are associated with cytoplasmic acidification. Such cytoplasmic acidification is further strengthened by compromised proton pumping in the complex I mutants. While RpoS regulon activation induces persistence in the wild type, the aggravated cytoplasmic acidification in the complex I mutants leads to increased persistence via global shutdown of protein synthesis. Thus, we propose that cytoplasmic acidification, amplified by a compromised complex I, can act as a signaling hub for perturbed metabolic homeostasis in antibiotic persisters.

Modulating the evolutionary trajectory of tolerance using antibiotics with different metabolic dependencies

“…Here, we identify a mechanism of SDM tolerance through deletion of the sodium-proton antiporter gene nhaA . Despite the diverse pool of target mutations for evolved tolerance between studies, transcriptomics and proteomics have allowed for the characterization of pathways that are commonly differentially expressed across separate studies 4 , 5 , 13 , 14 , 51 . Consistent with many previously identified antibiotic-tolerant mutants, our transcriptomic analysis of SDM-tolerant mutants revealed upregulation of the SOS response and biofilm formation along with dramatic downregulation of metabolic processes 4 , 14 , 24 , 51 , 52 .…”

“…Despite the diverse pool of target mutations for evolved tolerance between studies, transcriptomics and proteomics have allowed for the characterization of pathways that are commonly differentially expressed across separate studies 4 , 5 , 13 , 14 , 51 . Consistent with many previously identified antibiotic-tolerant mutants, our transcriptomic analysis of SDM-tolerant mutants revealed upregulation of the SOS response and biofilm formation along with dramatic downregulation of metabolic processes 4 , 14 , 24 , 51 , 52 . Metabolic mutations have not only been implicated in tolerance, but also in resistance, as shown by recent studies which found mutations in central carbon metabolism, energy metabolism, and biosynthetic pathway genes associated with antibiotic resistance in in vitro evolved strains and in clinical isolates 28 , 53 .…”

“…Modulation of both intracellular and extracellular pH has been known to alter antibiotic susceptibility 51 , 54 , 55 . For example, mutations in respiratory complex I were found during an in vitro evolution of E. coli , which resulted in cytoplasmic acidification leading to increased antibiotic persistence 51 .…”

“…Modulation of both intracellular and extracellular pH has been known to alter antibiotic susceptibility 51 , 54 , 55 . For example, mutations in respiratory complex I were found during an in vitro evolution of E. coli , which resulted in cytoplasmic acidification leading to increased antibiotic persistence 51 . Antibiotic treatment itself is also thought to trigger changes in intracellular pH, which activates cell stress and contributes to antibiotic-induced cell death, though whether this is achieved through alkalization or acidification may be dependent on the species and antibiotics in question 54 , 56 , 57 .…”

Proteomics in antibiotic resistance and tolerance research: Mapping the resistome and the tolerome of bacterial pathogens

“…Six protein candidates with similar expression profiles across the three evolved populations were obtained, which were GrcA, NuoF, CysP, AhpF, RaiA, and ribosome recycling factor (RRF). Among these, the nuo operon has been identified as a genetic determinant of persistence and was observed to be mutated in multiple tolerant strains [147,[155][156][157]. Besides, RaiA was also found to be synthesized at a high level on E. coli persisters by Semanjski et al [141], and played an important role in forming persisters by Wood and Song [158].…”