Modern antibiotics have been developed with the aim of destroying cellular function; however, the risk of antibiotic-resistance is increasing continuously. As a result, antimicrobial peptide (AMP) is considered a novel strategy to substitute traditional drugs. This study focused on revealing the antibacterial mechanism(s) of periplanetasn-4, an AMP identified from Cockroach. To elucidate whether periplanetasin-4 generates reactive oxygen species (ROS), a crucial stress factor for cell death, intracellular ROS was measured in Escherichia coli. The degree of membrane and DNA damage was determined using the properties that ROS causes oxidative stress to cell components. Unlike normal cell death, membrane depolarization was observed but DNA fragmentation did not occur. In addition, accumulation of nitric oxide (NO), a free radical with high toxicity, was measured and the byproduct of NO also induced severe intracellular damage. Periplanetasin-4-induced NO also impacted on cytosol calcium levels and triggered lipid peroxidation and DNA oxidation. These features were weakened when NO synthesis was interrupted, and this data suggested that perplanetasin-4-induced NO participates in E. coli cell damage. Moreover, this AMP-induced NO stimulates expression of SOS repair proteins and activation of RecA, a bacterial caspase-like protein. Features of nitrosative damage did not occur especially without dinF gene which is associated with oxidative stress. Therefore, it was indicated that when there is a NO signal, dinF promotes cell death. In conclusion, the combined investigations demonstrated that the antibacterial mechanism(s) of periplanetasin-4 was a NO-induced cell death, and dinF gene is closely related to cell death pathway.

Graphic Abstract

中文翻译：

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dinF 在大肠杆菌中引发由美国蟑螂的 Periplanetasin-4 诱导的一氧化氮信号传导

现代抗生素的开发旨在破坏细胞功能。然而，抗生素耐药性的风险正在不断增加。因此，抗菌肽（AMP）被认为是替代传统药物的新策略。这项研究的重点是揭示 periplanetasn-4（一种从蟑螂中鉴定出的 AMP）的抗菌机制。为了阐明 periplanetasin-4 是否产生活性氧 (ROS)，这是细胞死亡的关键应激因素，在大肠杆菌中测量了细胞内 ROS. 使用 ROS 对细胞成分造成氧化应激的特性来确定膜和 DNA 损伤的程度。与正常细胞死亡不同，观察到膜去极化但未发生 DNA 断裂。此外，测量到一氧化氮 (NO) 的积累，这是一种具有高毒性的自由基，并且 NO 的副产物也引起了严重的细胞内损伤。Periplanetasin-4 诱导的 NO 也影响细胞溶质钙水平并引发脂质过氧化和 DNA 氧化。当 NO 合成中断时，这些特征会减弱，该数据表明 perplanetasin-4 诱导的 NO 参与了大肠杆菌细胞损伤。此外，这种 AMP 诱导的 NO 刺激 SOS 修复蛋白的表达和 RecA（一种细菌半胱天冬酶样蛋白）的激活。特别是在没有与氧化应激相关的dinF基因的情况下，不会发生亚硝化损伤的特征。因此，表明当有NO信号时，dinF促进细胞死亡。总之，联合研究表明，periplanetasin-4 的抗菌机制是 NO 诱导的细胞死亡，并且dinF基因与细胞死亡途径密切相关。

图形摘要