Marine-inspired antimicrobial peptides disrupt gene expression on DNA level

Genome mining of Streptomyces sp. H-KF8 combined with sequence engineering yielded two serum-stable, non-cytotoxic, non-lytic antimicrobial peptides, L3 and L3-K. Initial studies in uropathogenic Escherichia coli suggested membrane effects and nucleoid relaxation, prompting a comprehensive investigation of their mode of action. Tandem mass tag (TMT)-based quantitative proteomics revealed extensive proteome remodelling, with 175 and 120 differentially expressed proteins (DEPs) after treatment with L3 and L3-K, respectively. L3 induced predominantly upregulated responses linked to metabolism, RNA processing, transport, and homeostasis, whereas L3-K mainly caused downregulation of proteins involved in metabolism, transport, and cell structure. Both peptides disrupted ABC transporter–mediated nutrient uptake and elicited stress responses, while L3 specifically perturbed the mal regulon, indicative of broader transcriptional dysregulation. Complementary fluorescent dye displacement and in vitro transcription/translation assays demonstrated non-specific DNA binding, stronger for L3 than L3-K, and potent inhibition of transcriptional and translational processes. Strikingly, inhibitory concentrations paralleled their minimum inhibitory concentrations, directly linking DNA binding and interference with central information processing to antimicrobial activity. Taken together, these findings reveal that L3 and L3-K primarily act by targeting DNA and interfering with the transcription–translation machinery. Beyond offering mechanistic insights, this study also underscores their promise as scaffolds for developing next-generation antimicrobial peptides with DNA-binding, non-membrane-lytic activity.

对链霉菌属（Streptomyces sp.）菌株H-KF8开展基因组挖掘并结合序列工程改造，成功获得两种血清稳定、无细胞毒性、非溶菌性的抗菌肽L3与L3-K。针对尿路致病性大肠杆菌（uropathogenic Escherichia coli）的初步研究显示，二者可引发膜损伤与类核松弛效应，这推动了对其作用模式的全面探究。基于串联质量标签（Tandem mass tag, TMT）的定量蛋白质组学分析揭示了广泛的蛋白质组重塑：经L3与L3-K处理后，分别有175种和120种差异表达蛋白（differentially expressed proteins, DEPs）发生表达变化。L3主要诱导与代谢、RNA加工、物质转运及稳态维持相关的上调应答，而L3-K则主要使参与代谢、物质转运与细胞结构构建的蛋白表达下调。两种抗菌肽均会破坏ATP结合盒转运体（ATP-binding cassette transporter, ABC transporter）介导的营养摄取过程并引发应激应答；其中L3可特异性干扰mal操纵子（mal regulon），提示存在更广泛的转录失调。配套的荧光染料置换实验与体外转录/翻译实验（in vitro transcription/translation assays）证实，两种肽均能非特异性结合DNA，且L3的结合能力强于L3-K，同时可强效抑制转录与翻译过程。值得注意的是，其抑菌浓度与最低抑菌浓度（minimum inhibitory concentrations, MIC）相符，直接将DNA结合与中枢信息加工过程的干扰与抗菌活性关联起来。综上，本研究表明L3与L3-K主要通过靶向DNA并干扰转录-翻译机器发挥抗菌作用。本研究不仅为抗菌肽的作用机制提供了新见解，同时也凸显了二者作为开发新一代具备DNA结合活性、非膜溶菌型抗菌肽支架的潜力。