Lu Wen-Jung, Lin Hsuan-Ju, Hsu Pang-Hung, Lin Hong-Ting Victor
Department of Food Science, National Taiwan Ocean University, No. 2, Pei-Ning Road, Keelung 202, Taiwan.
Department of Bioscience and Biotechnology, National Taiwan Ocean University, No. 2, Pei-Ning Road, Keelung 202, Taiwan.
Antibiotics (Basel). 2020 Sep 24;9(10):639. doi: 10.3390/antibiotics9100639.
Multidrug efflux pumps play an essential role in antibiotic resistance. The conventional methods, including minimum inhibitory concentration and fluorescent assays, to monitor transporter efflux activity might have some drawbacks, such as indirect evidence or interference from color molecules. In this study, MALDI-TOF MS use was explored for monitoring drug efflux by a multidrug transporter, and the results were compared for validation with the data from conventional methods. Minimum inhibitory concentration was used first to evaluate the activity of drug transporter AcrB, and this analysis showed that the overexpressing AcrB exhibited elevated resistance to various antibiotics and dyes. Fluorescence-based studies indicated that AcrB in could decrease the accumulation of intracellular dyes and display various efflux rate constants for different dyes, suggesting AcrB's efflux activity. The MALDI-TOF MS analysis parameters were optimized to maintain a detection accuracy for AcrB's substrates; furthermore, the MS data showed that overexpressing AcrB led to increased ions abundancy of various dyes and drugs in the extracellular space at different rates over time, illustrating continuous substrate efflux by AcrB. This study concluded that MALDI-TOF MS is a reliable method that can rapidly determine the drug pump efflux activity for various substrates.
多药外排泵在抗生素耐药性中起着至关重要的作用。监测转运蛋白外排活性的传统方法，包括最低抑菌浓度法和荧光测定法，可能存在一些缺点，如间接证据或颜色分子的干扰。在本研究中，探索了使用基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）来监测多药转运蛋白的药物外排，并将结果与传统方法的数据进行比较以进行验证。首先使用最低抑菌浓度来评估药物转运蛋白AcrB的活性，该分析表明过表达的AcrB对各种抗生素和染料表现出更高的抗性。基于荧光的研究表明，AcrB可以减少细胞内染料的积累，并对不同染料显示出不同的外排速率常数，这表明AcrB具有外排活性。对MALDI-TOF MS分析参数进行了优化，以保持对AcrB底物的检测准确性；此外，质谱数据表明，过表达的AcrB导致细胞外空间中各种染料和药物的离子丰度随时间以不同速率增加，这说明AcrB持续进行底物外排。本研究得出结论，MALDI-TOF MS是一种可靠的方法，可以快速确定各种底物的药物泵外排活性。
