氮杂环卡宾银配合物 SBC3靶向大肠杆菌 TDRS杀菌的作用机制

The N-heterocyclic carbine-silver complex SBC3 inhibits Escherichia coli by targeting TDRS

摘要:

新型氮杂环卡宾银配合物(N-heterocyclic carbene silver, Ag-NHC)兼具优异的稳定性、水溶性和杀菌活性，是具有巨大应用前景的抗菌候选物。 目的 探讨合成的1,3-二苄基Ag-NHC [1,3-dibenzyl-4,5-diphenylimidazol-2-ylidene silver (I) acetate, SBC3]对大肠杆菌的抗菌活性及作用机制。 方法 采用可见分光光度法检测SBC3的抗菌活性；使用透射电子显微镜观察SBC3处理后大肠杆菌DHB4菌株的形态学变化；通过5,5′-二硫代双(2-硝基苯甲酸) [5,5′-dithiobis-(2-nitrobenzoic acid), DTNB]实验检测SBC3处理后DHB4胞内硫氧还蛋白(thioredoxin, Trx)及硫氧还蛋白还原酶(thioredoxin reductase, TrxR)的活性和谷胱甘肽(glutathione, GSH)的含量；利用流式细胞术检测SBC3对DHB4胞内活性氧(reactive oxygen species, ROS)含量的影响，并通过还原剂二硫苏糖醇(dithiothreitol, DTT)进行挽救实验。通过在实验室连续传代，获得SBC3耐受性大肠杆菌菌株SBC3-resistant strains (SRSs)。其中SRS3、SRS4和SRS7菌株对SBC3的最小抑菌浓度(minimal inhibitory concentration, MIC)依次为24、32和56 μg/mL，分别为野生菌株MIC的3倍、4倍和7倍。将对应的菌株分别命名为SRS3、SRS4和SRS7，并使用这3种菌株复测上述指标；采用Western blotting检测SBC3对Trx1及 S-谷胱甘肽化蛋白( S-glutathionylated proteins, S-PSSG)表达量的影响。 结果 SBC3对实验菌株的MIC为8.0-30.0 μg/mL；SBC3处理后的DHB4菌株发生肿胀，并伴有内容物流出；SBC3可显著抑制DHB4胞内Trx和TrxR的活性，明显降低GSH的含量，同时升高ROS水平。此外，SBC3处理后的SRS3、SRS4和SRS7菌株胞内Trx和TrxR的蛋白活性降低，GSH含量和 S-PSSG的蛋白表达水平降低，但与DHB4菌株相比有不同程度的升高。 结论 SBC3通过靶向大肠杆菌的硫醇依赖的氧化还原系统(thiol-dependent redox system, TDRS)系统发挥其抗菌作用，为SBC3作为新型抗菌剂的开发设计提供了新思路。

Abstract:

N-Heterocyclic carbine-silver (Ag-NHC) complexes possessing excellent stability, water solubility, and bactericidal activity are antimicrobial candidates with great potential. Objective To study the inhibitory activity and mechanism of a novel Ag-NHC complex 1,3-dibenzyl-4,5-diphenylimidazol-2-ylidene silver (I) acetate (SBC3), synthesized by Matthias Tacke’s team against Escherichia coli. Methods Visible spectrophotometry was employed to examine the antimicrobial activity of SBC3. Transmission microscopy was employed to observe the morphological changes of DHB4 cells post SBC3 treatment. Flow cytometry was performed to detect the effect of SBC3 on the intracellular reactive oxygen species (ROS) content. The 5,5′- dithiobis-(2-nitrobenzoic acid) (DTNB) assay was used to determine the intracellular thioredoxin (Trx) and thioredoxin reductase (TrxR) activities and the content of glutathione (GSH) post SBC3 treatment. Dithiothreitol (DTT), the ROS scavenger, was added to rescue DHB4 from ROS. SBC3-resistant strains (SRSs) were obtained by successive passaging in the laboratory. The obtained strains showed the minimal inhibitory concentrations (MICs) against SBC3 being 24, 32, and 56 μg/mL, respectively, which were 3, 4, and 7 folds of the MIC of WT. The three strains were named SRS3, SRS4, and SRS7 and then used to retest the above indicators. Western blotting was performed to determine the expression levels of Trx1 and S-glutathionylated proteins ( S-PSSG) post SBC3 treatment. Results The MIC values of SBC3 against tested pathogens were 8.0–30.0 μg/mL. The DHB4 cells treated with SBC3 underwent swelling, which was accompanied by contents leakage. SBC3 significantly inhibited the Trx and TrxR activities, reduced the GSH content, and elevated the ROS level in DHB4. SBC3 treatment decreased the Trx and TrxR activities, reduced the GSH content, and down-regulated the expression of S-PSSG in SRS3, SRS4, and SRS7. However, all the above indicators were increased to different extent compared with those in DHB4. Conclusion SBC3 can target the thiol-dependent redox system (TDRS) of E. coli to exert antibacterial effects. This study provides a new idea for the design of SBC3 as a novel antimicrobial agent.