Purine and pyrimidine analogues differentially regulate cell wall precursor biosynthesis to control β-lactam susceptibility in methicillin resistant Staphylococcus aureus

Maintaining the efficacy of {beta}-lactam antibiotics against Staphylococcus aureus is a clinical priority given the prevalence of methicillin-resistant S. aureus (MRSA). We previously showed that the pyrimidine analogues 5-fluorouracil (5-FU) and 5-fluorouridine (5-FUrd) synergize with {beta}-lactams. Here, we extended this by evaluating additional nucleotide metabolism-targeting agents. Gemcitabine (Gem) and mitomycin C (Mito), like 5-FU and 5-FUrd, exhibited intrinsic anti-MRSA activity and potentiated {beta}-lactams, whereas the purine analogue 6-thioguanine (6-TG) showed distinct, often antagonistic effects. Transcriptomic analysis revealed that pyrimidine-targeting agents repress lysine and glutamate biosynthesis, while 6-TG induced these pathways, implicating amino acid metabolism in {beta}-lactam potentiation. Consistent with this, pyrimidine analogues also suppressed GlmS expression, potentially limiting UDP-GlcNAc production required for cell wall synthesis, and synergized with fosfomycin. Fluorescence microscopy confirmed that the potentiation of oxacillin activity by pyrimidine-targeting agents, but not 6-TG, was accompanied by impaired peptidoglycan synthesis. Additionally, glutathione-mediated attenuation of killing implicated reactive oxygen species in the bactericidal activity of cloxacillin combinations. Finally, these agents displayed strong anti-biofilm activity, further enhanced in combination with daptomycin and rifampicin. Together, these findings highlight the potential of pyrimidine analogues to potentiate cell wall-targeting antibiotics and identify an important role for modulation of cell wall precursor pathways in this anti-MRSA activity. ImportanceDrug interactions can complicate the treatment of antimicrobial resistant infections in patients undergoing treatment for cancer highlighting the importance of understanding the effects of anti-cancer drugs on pathogens like MRSA. Here, we investigated several drugs that target nucleotide metabolism and are used to treat cancer, fungal, and viral infections, both alone and in combination with commonly used penicillin-type antibiotics. We found that pyrimidine analogue drugs enhanced the activity of these antibiotics against MRSA, whereas the purine analogue 6-thioguanine reduced antibiotic effectiveness. These drugs altered the bacterial cell wall and other metabolic pathways linked to antibiotic susceptibility. Our findings reveal the potential to repurpose certain anticancer drugs to improve treatment of MRSA infections, while also cautioning that some drug combinations may interfere with antibiotic therapy.