Elucidating bacterial responses to the novel antimicrobial AGXX

总结 随着抗生素耐药性的迅速蔓延，迫切需要新的抗菌策略。 从而影响了现有抗生素的有效性。此外，新药开发也没有跟上 抗药性病原体的增加。铜绿假单胞菌特别难以治疗， 在囊性纤维化患者或烧伤患者的肺部发现。由于抗生素危机的出现， 目前的工作重点是寻找替代治疗策略。含银化合物代表这样的化合物。 由于其多特异性抑制细菌生长的能力，一个例子是磺胺嘧啶银， 局部制剂，其通常被施用以治疗或预防急性铜绿假单胞菌伤口感染。 新型含银表面涂层AGXX是最近开发的一种有前途的化合物， 抗菌性能。由两种过渡金属银和钌组成，形成微电流 细胞，AGXX杀死革兰氏阳性菌通过形成活性氧，如氢 过氧化物然而，它对革兰氏阴性菌如铜绿假单胞菌的作用以及它们对 AGXX暴露尚未研究。我们发现AGXX在P. 即使在亚致死浓度下，该化合物也能抑制铜绿假单胞菌，并且该化合物比银更有效 磺胺嘧啶，烧伤治疗的黄金标准。此外，我们发现， 亚致死浓度的AGXX的活性在亚致死浓度的存在下高达50，000倍。 氨基糖苷类药物浓度，表明AGXX作为佐剂的潜在应用。 我们现在将研究氨基糖苷类抗生素协同作用背后的分子机制， AGXX.在目标1中，我们将测试我们的假设，即AGXX诱导的ROS产生破坏了细胞铁硫代谢。 团簇池并因此通过芬顿反应引发羟基自由基产生。因此，我们希望 观察到膜破坏增加，可能促进氨基糖苷类药物流入细胞， 其负责在同时处理铜绿假单胞菌后观察到的增加的细菌杀灭 AGXX和氨基糖苷类药物。此外，我们将确定联合治疗对P。 铜绿假单胞菌生物膜和持留细胞以及其他临床相关细菌物种的分离物。在目标2中， 将使用独立的无偏见和有针对性的方法，包括Tnseq和随后的表型 转座子突变体的表征，以鉴定和确定铜绿假单胞菌特异性反应， 抗AGXX治疗。此外，我们将评估该化合物在不同细胞中的细胞毒性， 相关的线。这些研究结果将指导制定战略的努力，将AGXX作为一种潜在的 在根除铜绿假单胞菌感染中的抗微生物剂和佐剂。

SUMMARY New antimicrobial strategies are badly needed, as the spread of antibiotic resistance is rapidly compromising the effectiveness of current antibiotics. Moreover, new drug development has not kept pace with the rise of drug-resistant pathogens. Pseudomonas aeruginosa is particularly difficult to treat and commonly found in the lungs of cystic fibrosis patients or in patients with burn wounds. Due to the emerging antibiotic crisis, efforts are now focused on finding alternative treatment strategies. Silver-containing compounds represent such opportunity due to their multi-specific ability to inhibit bacterial growth. One example is silver sulfadiazine, a topical formulation that is often administered to treat or prevent acute P. aeruginosa wound infections. The novel silver containing surface coating AGXX was recently developed as a promising compound with antimicrobial properties. Composed of the two transition metals silver and ruthenium which form a micro-galvanic cell, AGXX kills gram-positive bacteria through the formation of reactive oxygen species, such as hydrogen peroxide. However, its effect on gram-negative bacteria such as P. aeruginosa as well as their responses to AGXX exposure have not yet been studied. We found that AGXX elicits strong proteotoxic effects in P. aeruginosa even at sublethal concentrations, and that the compound is significantly more potent than silver sulfadiazine, the gold standard for the treatment of burn wounds. Moreover, we discovered that the bactericidal activity of sublethal concentrations of AGXX is up to 50,000-fold higher in the presence of sublethal aminoglycosides concentrations, indicating a potential application for AGXX as an adjuvant. We will now investigate the molecular mechanism behind the synergy of aminoglycoside antibiotics and AGXX. In Aim 1, we will test our hypothesis that AGXX-induced ROS production disrupts the cellular iron-sulfur cluster pool and therefore triggers hydroxyl radical production via the Fenton reaction. As a result, we expect to observe increased membrane disruption, potentially facilitating an elevated aminoglycoside influx into the cell, which is responsible for increased bacterial killing that is observed after simultaneous treatment of P. aeruginosa with AGXX and aminoglycosides. Furthermore, we will determine the effect a combinational treatment has on P. aeruginosa biofilms and persister cells and on isolates of other clinically relevant bacterial species. In Aim 2, we will use independent unbiased and targeted approaches, including Tnseq and subsequent phenotypic characterization of transposon mutants, to identify and determine P. aeruginosa-specific responses to and defenses against AGXX treatment. Moreover, we will assess the cytotoxicity of this compound in different cell lines of relevance. These findings will guide efforts to devise strategies to implement AGXX as a potential antimicrobial and adjuvant in eradicating P. aeruginosa infections.