Antimicrobial resistance is a critical threat to global public health. Beyond antibiotic resistance, persistent infections present another significant challenge in managing bacterial diseases. Ongoing research indicates that, before acquiring resistance, bacteria may first transition into a state of “persistence”, where they survive antibiotic treatments without genetic resistance. These bacterial persisters, phenotypic variants tolerant to high doses of antibiotics, highlight the importance of understanding the mechanisms enabling bacterial survival. In our study, we screened an E. coli reporter library, where promoters of genes were fused to a fast-folding green fluorescent protein (GFP) gene, and discovered that exposure to gentamicin (an aminoglycoside drug) led to reduced expression of aspA. AspA is responsible for breaking down aspartic acid into fumarate and ammonia, which are crucial for carbon and nitrogen metabolism. Notably, deletion of aspA(ΔaspA) considerably enhanced E. coli's tolerance to many aminoglycoside antibiotics. By analyzing the effects of fumarate and ammonia, we found that the increased tolerance stemmed from disturbances in fumarate metabolism, as supplementing cultures with fumarate significantly lowered the survival rate. Aminoglycosides typically inhibit ribosomes, leading to the production of truncated, toxic peptides, and the proton motive force (PMF) is believed to be necessary for their uptake. However, fluorescent assays showed no significant differences in PMF or drug uptake in the ΔaspA strain. Proteomic analysis via LC-MS revealed several pathways that were significantly upregulated or downregulated in the ΔaspA strain compared to wild type. While pathway enrichment analysis did not reveal any critical pathways or protein networks among the downregulated proteins, clusters involved in metabolism and ribosomal processes were upregulated. This observation led to the hypothesis that disturbances in metabolism, particularly due to fumarate, may enhance ribosomal biogenesis and activity. Given this, the activity of promoters associated with ribosomal operons was measured using reporter plasmids, revealing that one of the seven operons (rrnC) exhibited significantly higher activity in the ΔaspA mutant compared to wild-type E. coli. The rrnC operon encodes 16S, 23S, and 5S rRNA, essential for assembling both the small and large ribosomal subunits, and the redundancy of rRNA operons is important for optimal adaptation to changing physiological conditions. In summary, our study uncovers a novel mechanism of aminoglycoside tolerance, wherein the disruption of aspartic acid degradation induces metabolic stress that promotes ribosomal activity and contributes to antibiotic persistence.
- Binder, S., Levitt, A. M., Sacks, J. J. & Hughes, J. M. Emerging Infectious Diseases: Public Health Issues for the 21st Century. Science (1979) 284, 1311–1313 (1999).
- Fisher, R. A., Gollan, B. & Helaine, S. Persistent bacterial infections and persister cells. Nat Rev Microbiol 15, 453–464 (2017).
- Cohen, N. R., Lobritz, M. A. & Collins, J. J. Microbial persistence and the road to drug resistance. Cell Host Microbe 13, 632–642 (2013).
- Taber, H. W., Mueller, J. P., Miller, and, P. F. & Arrow, A. S. Bacterial Uptake of Aminoglycoside Antibiotics. Microbiol Rev 51, 439–457 (1987).
- Ciara, C. et al. rRNA Operon Multiplicity in Escherichia coli and the Physiological Implications of rrn Inactivation. J Bacteriol 177, 4152–4156 (1995).
