A novel inhibitor of P. aeruginosa folate metabolism exploits metabolic differences for narrow-spectrum antibiotic targeting

Pseudomonas aeruginosa is a leading cause of hospital acquired infections for which the development of new antibiotics is urgently needed. Unlike most enteric bacteria, P. aeruginosa lacks thymidine kinase and thymidine phosphorylase activity, and thus cannot scavenge exogenous thymine. An appealing strategy to selectively target P. aeruginosa while leaving the healthy microbiome largely intact would thus be to disrupt thymidine synthesis while providing exogenous thymine. However, this approach was previously intractable because known antibiotics that perturb thymidine synthesis are largely inactive against P. aeruginosa. Here, we characterize a novel dihydrofolate reductase inhibitor, fluorofolin, 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, including critical antibiotic development priorities expressing the beta-lactamases KPC-5 and NDM-1. 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. However, these mutants also decrease pathogenesis, in part due to increased export of quorum sensing precursors leading to decreased virulence factor production. Our findings thus demonstrate how understanding species-specific genetic differences and discovery of an antibiotic with a widely conserved target can enable selective targeting of important pathogens while revealing new tradeoffs between resistance and pathogenesis. To verify the phenotype of two flurofolin resistant mutants in P. Aeruginosa

铜绿假单胞菌（Pseudomonas aeruginosa）是医院获得性感染的首要致病菌之一，亟需开发新型抗生素。与多数肠杆菌科细菌不同，铜绿假单胞菌缺乏胸苷激酶（thymidine kinase）与胸苷磷酸化酶（thymidine phosphorylase）活性，因此无法摄取外源胸腺嘧啶。由此，一种既能选择性靶向铜绿假单胞菌、又能最大程度保留健康微生物组的极具吸引力的策略，便是在提供外源胸腺嘧啶的同时阻断胸苷合成通路。然而，此前该方案难以实施，因为已知的干扰胸苷合成的抗生素对铜绿假单胞菌基本无效。本研究对一种新型二氢叶酸还原酶抑制剂（dihydrofolate reductase inhibitor）氟洛林（fluorofolin）进行了表征，该物质在体外培养体系与小鼠大腿感染模型中均表现出对铜绿假单胞菌的显著抗菌活性。氟洛林对多种临床分离的耐药铜绿假单胞菌菌株均具有抗菌活性，包括表达β-内酰胺酶（beta-lactamases）KPC-5与NDM-1的、属于抗生素开发关键优先级的耐药菌株。值得注意的是，在添加胸腺嘧啶的条件下，氟洛林对铜绿假单胞菌的抗菌活性具有选择性。铜绿假单胞菌对氟洛林的耐药性可通过外排泵（efflux pumps）MexCD-OprJ与MexEF-OprN的过表达产生。然而，此类耐药突变株的致病力也会降低，部分原因是群体感应（quorum sensing）前体物质的外排增加，导致毒力因子生成减少。因此，本研究的发现表明，通过解析物种特异性遗传差异并发现靶向保守靶点的抗生素，不仅可以实现对重要致病菌的选择性靶向，还能揭示耐药性与致病力之间新的权衡关系。为验证两株氟洛林耐药铜绿假单胞菌突变株的表型。