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Abstract：
Nitrate respiration is a widespread mode of anaerobic energy generation used by many bacterial pathogens, and the respiratory nitrate reductase, Nar, has long been known to reduce chlorate to the toxic oxidizing agent chlorite. Here, we demonstrate the antibacterial activity of chlorate against Pseudomonas aeruginosa, a representative pathogen that can inhabit hypoxic or anoxic host microenvironments during infection. Aerobically grown P. aeruginosa cells are tobramycin sensitive but chlorate tolerant. In the absence of oxygen or an alternative electron acceptor, cells are tobramycin tolerant but chlorate sensitive via Nar-dependent reduction. The fact that chlorite, the product of chlorate reduction, is not detected in culture supernatants suggests that it may react rapidly and be retained intracellularly. Tobramycin and chlorate target distinct populations within metabolically stratified aggregate biofilms; tobramycin kills cells on the oxic periphery, whereas chlorate kills hypoxic and anoxic cells in the interior. In a matrix populated by multiple aggregates, tobramycin-mediated death of surface aggregates enables deeper oxygen penetration into the matrix, benefiting select aggregate populations by increasing survival and removing chlorate sensitivity. Finally, lasR mutants, which commonly arise in P. aeruginosa infections and are known to withstand conventional antibiotic treatment, are hypersensitive to chlorate. A lasR mutant shows a propensity to respire nitrate and reduce chlorate more rapidly than the wild type does, consistent with its heightened chlorate sensitivity. These findings illustrate chlorate\'s potential to selectively target oxidant-starved pathogens, including physiological states and genotypes of P. aeruginosa that represent antibiotic-tolerant populations during infections.IMPORTANCE The anaerobic growth and survival of bacteria are often correlated with physiological tolerance to conventional antibiotics, motivating the development of novel strategies targeting pathogens in anoxic environments. A key challenge is to identify drug targets that are specific to this metabolic state. Chlorate is a nontoxic compound that can be reduced to toxic chlorite by a widespread enzyme of anaerobic metabolism. We tested the antibacterial properties of chlorate against Pseudomonas aeruginosa, a pathogen that can inhabit hypoxic or anoxic microenvironments, including those that arise in human infection. Chlorate and the antibiotic tobramycin kill distinct metabolic populations in P. aeruginosa biofilms, where chlorate targets anaerobic cells that tolerate tobramycin. Chlorate is particularly effective against P. aeruginosalasR mutants, which are frequently isolated from human infections and more resistant to some antibiotics. This work suggests that chlorate may hold potential as an anaerobic prodrug.
摘要：
硝酸盐呼吸是许多细菌病原体使用的厌氧能量产生的广泛模式，和呼吸硝酸还原酶，不，长期以来已知将氯酸盐还原为有毒氧化剂亚氯酸盐。这里,我们证明了氯酸盐对铜绿假单胞菌的抗菌活性，一种代表性的病原体，可以在感染期间生活在缺氧或缺氧的宿主微环境中。需氧生长的铜绿假单胞菌细胞对妥布霉素敏感但对氯酸盐耐受。在没有氧气或替代电子受体的情况下，细胞耐受妥布霉素，但通过Nar依赖性还原对氯酸盐敏感。绿泥石的事实，氯酸盐还原的产物，在培养上清液中未检测到，表明它可能会快速反应并保留在细胞内。妥布霉素和氯酸盐靶向代谢分层聚集生物膜内的不同群体；妥布霉素杀死有氧外周细胞，而氯酸盐杀死内部的缺氧和缺氧细胞。在由多个聚合填充的矩阵中，妥布霉素介导的表面聚集体死亡能够使氧气更深地渗透到基质中，通过增加存活率和消除氯酸盐敏感性，使选定的聚集种群受益。最后,lasR突变体,通常出现在铜绿假单胞菌感染中，已知可以承受常规抗生素治疗，对氯酸盐过敏。lasR突变体显示出比野生型更快地呼吸硝酸盐和减少氯酸盐的倾向，与提高的氯酸盐敏感性一致。这些发现说明了氯酸盐选择性靶向氧化剂饥饿的病原体的潜力，包括代表感染期间抗生素耐受人群的铜绿假单胞菌的生理状态和基因型。细菌的厌氧生长和存活通常与对常规抗生素的生理耐受性相关。激发在缺氧环境中针对病原体的新策略的开发。一个关键的挑战是确定特定于这种代谢状态的药物靶标。氯酸盐是一种无毒化合物，可以通过广泛的厌氧代谢酶还原为有毒的亚氯酸盐。我们测试了氯酸盐对铜绿假单胞菌的抗菌性能，一种可以生活在缺氧或缺氧微环境中的病原体，包括那些在人类感染中出现的。氯酸盐和抗生素妥布霉素杀死铜绿假单胞菌生物膜中不同的代谢群体，氯酸盐靶向耐受妥布霉素的厌氧细胞。氯酸盐对铜绿假单胞菌突变体特别有效，它们经常从人类感染中分离出来，对某些抗生素更具耐药性。这项工作表明，氯酸盐可能具有作为厌氧前药的潜力。
