靶向α-酮戊二酸脱氢酶增强纳米银的抗菌作用

Targeting alpha-ketoglutarate dehydrogenase enhances antibacterial activity of silver nanoparticles

摘要:

【目的】纳米银(silver nanoparticles, AgNPs)的生物安全性一直受业界诟病，扩大纳米银的治疗窗将为治疗人和动物多耐药性细菌感染提供有效的备选药物。本研究拟用三羧酸循环的重要成员α-酮戊二酸(alpha-ketoglutaric acid, AKG)对纳米银进行表面修饰以提高其抗菌的生物安全性。【方法】芦丁在常温下合成纳米银，用全波长分光光度计、粒度仪及透射电镜进行表征。加1 mmol/L聚乙烯吡咯烷酮(polyvinylpyrrolidone, PVP)作为稳定剂(PVP-AgNPs)，另加10 mmol/L AKG作为封端剂(PVP-AgNPs@AKG)，比较2种纳米银的抗菌性及对人正常宫颈上皮细胞(human cervical epithelial cells, HCerEpic)的毒性作用，再分析2种纳米银对大肠杆菌(Escherichia coli) BW25113能量代谢、抗氧化应激和无氧呼吸相关基因表达等的影响。【结果】PVP-AgNPs@AKG对多株革兰阳性细菌和革兰阴性细菌的最小抑菌浓度(minimal inhibit concentration, MIC)和最低杀菌浓度(minimum bactericidal concentration, MBC)均比PVP-AgNPs低50%或50%以上，而对HCerEpic细胞的毒性无显著差异。与PVP-AgNPs相比，PVP-AgNPs@AKG在MIC浓度下对E. coli α-酮戊二酸脱氢酶活性的抑制作用增强，AKG蓄积，ATP水平显著降低，同时活性氧(reactive oxygen species, ROS)的水平显著升高，soxS表达上调，但是，厌氧呼吸相关的arcA、fnr、fdnH基因表达上调的程度显著降低。【结论】AKG修饰纳米银能通过靶向α-酮戊二酸脱氢酶抑制细菌的能量代谢，使其对氧化损伤更敏感，从而获得更强的抗菌能力，是一种扩大纳米银治疗窗的有效手段。

Abstract:

[Objective] The biosafety of silver nanoparticles (AgNPs) has been a subject of concern due to the narrow therapeutic window. Expanding the therapeutic window could facilitate the application of AgNPs in the treatment of multi-drug resistant bacterial infections in humans and animals. This study aimed to enhance the biosafety of AgNPs by modifying their surface with alpha-ketoglutaric acid (AKG), a crucial component of the tricarboxylic acid cycle. [Methods] Silver ion was reduced to AgNPs by rutin at room temperature, and then AgNPs were stabilized with 1 mmol/L polyvinylpyrrolidone (PVP) solution to generate PVP-AgNPs. AKG (10 mmol/L) was added to generate PVP-AgNPs@AKG. The prepared AgNPs were characterized by a full-wavelength spectrophotometer, a particle size analyzer, and a transmission electron microscope. The antibacterial activities of PVP-AgNPs and PVP-AgNPs@AKG were evaluated based on minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), time-kill curve, and post-antibiotic effect. The cytotoxicity of the prepared AgNPs to human cervical epithelial cells (HCerEpic) was examined by the MTT assay and flow cytometry. Furthermore, the effects of the prepared AgNPs on the energy metabolism, oxidative stress, and expression of genes involved in anaerobic respiration of Escherichia coli BW25113 were studied. [Results] The MIC and MBC of PVP-AgNPs@AKG against Gram-positive and Gram-negative bacteria were 50% or above 50% lower than those of PVP-AgNPs. PVP-AgNPs@AKG and PVP-AgNPs showed no significant difference in the cytotoxicity to HCerEpic cells. Compared with PVP-AgNPs, PVP-AgNPs@AKG at the MIC showed significantly enhanced inhibitory effect on the α-ketoglutarate dehydrogenase in Escherichia coli, increased accumulation of AKG, lowered ATP level, and elevated reactive oxygen species level. Moreover, PVP-AgNPs@AKG significantly up-regulated the expression of soxS and down-regulated the expression of genes involved in anaerobic respiration, such as arcA, fnr, and fdnH. [Conclusion] The findings suggested that PVP-AgNPs@AKG disrupted the energy metabolism by targeting α-ketoglutarate dehydrogenase, rending bacteria more vulnerable to oxidative damage. Modifying with AKG would be a potential method to expand the therapeutic window of AgNPs.