Elucidating bacterial responses to the novel antimicrobial AGXX

摘要 随着抗生素耐药性的迅速传播，迫切需要新的抗菌素战略 损害了目前抗生素的有效性。此外，新药开发没有跟上 抗药性病原体的兴起。铜绿假单胞菌特别难治疗，通常 发现于囊性纤维化患者或烧伤创面患者的肺部。由于新出现的抗生素危机， 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 由于它们具有多种特定的抑制细菌生长的能力，因此具有很好的机会。一个例子是磺胺嘧啶银，一种 常用于治疗或预防急性铜绿假单胞菌伤口感染的外用制剂。 新型含银表面涂料AGXX是近年来发展起来的一种具有广泛应用前景的化合物 抗菌性能。由形成微原电池的两种过渡金属银和Ru组成 细胞，AGXX通过形成活性氧物种，如氢，杀死革兰氏阳性细菌 过氧化氢。然而，它对革兰氏阴性菌如铜绿假单胞菌的影响以及它们对 AGXX暴露还没有被研究过。我们发现AGXX对P. 铜绿假单胞菌即使在亚致死浓度下也是如此，而且这种化合物的效力明显高于银 磺胺嘧啶，烧伤创面治疗的黄金标准。此外，我们发现杀菌剂 在亚致死浓度的情况下，AGXX的活性高达50,000倍 氨基糖苷类药物的浓度，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的分子机制。 AGXX。在目标1中，我们将检验我们的假设，即AGXX诱导的ROS产生扰乱细胞铁硫 因此通过芬顿反应触发羟基自由基的产生。因此，我们预计 观察到膜破裂的增加，潜在地促进了氨基糖苷类物质流入细胞， 这是铜绿假单胞菌同时治疗后细菌杀灭增加的原因 含AGXX和氨基糖苷类药物。此外，我们还将确定联合治疗对P. 铜绿假单胞菌生物膜和周围细胞以及其他临床相关细菌的分离株。在目标2中，我们 将使用独立、公正和有针对性的方法，包括Tnseq和随后的表型 转座子突变体的特性，以鉴定和确定铜绿假单胞菌对和的特异性反应 对AGXX治疗的防御。此外，我们还将评估该化合物在不同细胞中的细胞毒性。 有关联的线条。这些发现将指导制定战略，将AGXX作为一项潜在的 根除铜绿假单胞菌感染的抗菌剂和佐剂。

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.