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A folate inhibitor exploits metabolic differences in Pseudomonas aeruginosa for narrow-spectrum targeting
Connor Chain1, Joseph P Sheehan1, Xincheng Xu2,3
1Department of Molecular Biology, Princeton University, Princeton, NJ, USA.
Abstract:
Pseudomonas aeruginosa is a leading cause of hospital-acquired infections for which the development of antibiotics is urgently needed. Unlike most enteric bacteria, P. aeruginosa lacks enzymes required to scavenge exogenous thymine. An appealing strategy to selectively target P. aeruginosa is to disrupt thymidine synthesis while providing exogenous thymine. However, known antibiotics that perturb thymidine synthesis are largely inactive against P. aeruginosa.Here we characterize fluorofolin, a dihydrofolate reductase (DHFR) inhibitor derived from Irresistin-16, that exhibits significant activity against P. aeruginosa in culture and in a mouse thigh infection model. Fluorofolin is active against a wide range of clinical P. aeruginosa isolates resistant to known antibiotics. Metabolomics and in vitro assays using purified folA confirm that fluorofolin inhibits P. aeruginosa DHFR. Importantly, in the presence of thymine supplementation, fluorofolin activity is selective for P. aeruginosa. Resistance to fluorofolin can emerge through overexpression of the efflux pumps MexCD-OprJ and MexEF-OprN, but these mutants also decrease pathogenesis. Our findings demonstrate how understanding species-specific genetic differences can enable selective targeting of important pathogens while revealing trade-offs between resistance and pathogenesis.
Insights
A new antibiotic, fluorofolin, effectively targets Pseudomonas aeruginosa by inhibiting dihydrofolate reductase (DHFR). Supplementing with thymine enhances its selectivity, offering a promising strategy against hospital-acquired infections.
Area of Science:
- Microbiology
- Infectious Diseases
- Drug Discovery
Background:
- Pseudomonas aeruginosa is a major cause of hospital-acquired infections.
- There is an urgent need for new antibiotics against P. aeruginosa.
- P. aeruginosa's unique metabolic pathway for thymine scavenging presents a potential therapeutic target.
Purpose of the Study:
- To characterize fluorofolin, a novel dihydrofolate reductase (DHFR) inhibitor, for its activity against P. aeruginosa.
- To investigate the selectivity of fluorofolin, particularly in the presence of thymine.
- To understand the mechanisms of resistance and their impact on P. aeruginosa pathogenesis.
Main Methods:
- In vitro assays using purified P. aeruginosa folA (encoding DHFR).
- Metabolomic analysis to confirm enzyme inhibition.
- Testing activity against clinical P. aeruginosa isolates resistant to existing antibiotics.
- In vivo efficacy studies in a mouse thigh infection model.
- Analysis of resistance mechanisms, including efflux pump overexpression.
Main Results:
- Fluorofolin demonstrates significant activity against P. aeruginosa in vitro and in vivo.
- The compound inhibits P. aeruginosa DHFR, confirmed by metabolomics and enzyme assays.
- Fluorofolin exhibits selectivity for P. aeruginosa when thymine is supplemented.
- Resistance can arise from efflux pump overexpression, but these mutants show reduced virulence.
Conclusions:
- Fluorofolin is a potent inhibitor of P. aeruginosa DHFR with broad activity against resistant strains.
- Thymine supplementation enables selective targeting of P. aeruginosa, minimizing impact on other bacteria.
- Understanding species-specific genetics can lead to targeted therapies with reduced resistance-virulence trade-offs.
