Data_Sheet_4_Identification and characterization of a small molecule BFstatin inhibiting BrpR, the transcriptional regulator for biofilm formation of Vibrio vulnificus.xlsx

Many pathogenic bacteria form biofilms that are resistant to not only host immune defenses but also antibiotics, posing a need for the development of strategies to control biofilms. In this study, to prevent biofilm formation of the fulminating foodborne pathogen Vibrio vulnificus, chemical libraries were extensively screened to identify a small molecule inhibiting the activity of BrpR, a transcriptional regulator for biofilm genes. Accordingly, the BrpR inhibitor BFstatin [N1-(2-chloro-5-fluorophenyl)-N3-propylmalonamide], with a half-maximal effective concentration of 8.01 μM, was identified. BFstatin did not interfere with bacterial growth or exhibit cytotoxicity to the human epithelial cell line. BFstatin directly bound to BrpR and interrupted its binding to the target promoter DNAs of the downstream genes. Molecular dynamics simulation of the interaction between BFstatin and BrpR proposed that BFstatin modifies the structure of BrpR, especially the DNA-binding domain. Transcriptomic analyses revealed that BFstatin reduces the expression of the BrpR regulon including the cabABC operon and brp locus which contribute to the production of biofilm matrix of V. vulnificus. Accordingly, BFstatin diminished the biofilm levels of V. vulnificus by inhibiting the matrix development in a concentration-dependent manner. Altogether, BFstatin could be an anti-biofilm agent targeting BrpR, thereby rendering V. vulnificus more susceptible to host immune defenses and antibiotics.

诸多致病菌可形成生物膜，此类生物膜不仅能够抵御宿主免疫防御，还可耐受抗生素，因此亟需开发调控生物膜的相关策略。本研究为防控暴发性食源性致病菌创伤弧菌（Vibrio vulnificus）的生物膜形成，通过大规模筛选化学文库，鉴定出一种可抑制生物膜基因转录调控因子BrpR活性的小分子化合物。最终成功鉴定出BrpR抑制剂BFstatin [N1-(2-氯-5-氟苯基)-N3-丙基丙二酰胺]，其半数有效浓度（EC₅₀）为8.01 μM。BFstatin不会干扰细菌生长，也未对人上皮细胞系产生细胞毒性。BFstatin可直接结合BrpR，并阻断其与下游基因的靶启动子DNA的结合。通过BFstatin与BrpR相互作用的分子动力学模拟，推测BFstatin可改变BrpR的结构，尤其是其DNA结合结构域。转录组分析结果显示，BFstatin可下调BrpR调控子的表达，包括参与创伤弧菌生物膜基质合成的cabABC操纵子与brp基因座。因此，BFstatin可通过浓度依赖的方式抑制生物膜基质形成，从而降低创伤弧菌的生物膜生成量。综上，BFstatin可作为靶向BrpR的抗生物膜制剂，使创伤弧菌更易被宿主免疫防御系统清除并对抗生素敏感。