Pandeya Ankit, Yang Ling, Alegun Olaniyi, Karunasena Chamikara, Risko Chad, Li Zhenyu, Wei Yinan
Department of Chemistry, College of Arts and Sciences, University of Kentucky, Lexington, KY, United States of America.
Centre for Applied Energy and Research, University of Kentucky, Lexington, KY, United States of America.
PLoS One. 2021 Nov 12;16(11):e0260023. doi: 10.1371/journal.pone.0260023. eCollection 2021.
Antibiotic resistance is a major public health concern. The shrinking selection of effective antibiotics and lack of new development is making the situation worse. Gram-negative bacteria more specifically pose serious threat because of their double layered cell envelope and effective efflux systems, which is a challenge for drugs to penetrate. One promising approach to breach this barrier is the "Trojan horse strategy". In this technique, an antibiotic molecule is conjugated with a nutrient molecule that helps the antibiotic to enter the cell through dedicated transporters for the nutrient. Here, we explored the approach using biotin conjugation with a florescent molecule Atto565 to determine if biotinylation enhances accumulation. Biotin is an essential vitamin for bacteria and is obtained through either synthesis or uptake from the environment. We found that biotinylation enhanced accumulation of Atto565 in E. coli. However, the enhancement did not seem to be due to uptake through biotin transporters since the presence of free biotin had no observable impact on accumulation. Accumulated compound was mostly in the periplasm, as determined by cell fractionation studies. This was further confirmed through the observation that expression of streptavidin in the periplasm specifically enhanced the accumulation of biotinylated Atto565. This enhancement was not observed when streptavidin was expressed in the cytoplasm indicating no significant distribution of the compound inside the cytoplasm. Using gene knockout strains, plasmid complementation and mutagenesis studies we demonstrated that biotinylation made the compound a better passenger through OmpC, an outer membrane porin. Density functional theory (DFT)-based evaluation of the three-dimensional geometries showed that biotinylation did not directly stabilize the conformation of the compound to make it favorable for the entry through a pore. Further studies including molecular dynamics simulations are necessary to determine the possible mechanisms of enhanced accumulation of the biotinylated Atto565.
抗生素耐药性是一个重大的公共卫生问题。有效抗生素的选择范围不断缩小，且缺乏新的研发成果，这使得情况愈发严峻。革兰氏阴性菌因其双层细胞膜和有效的外排系统，尤其构成了严重威胁，这对药物的渗透来说是一项挑战。一种有前景的突破这一障碍的方法是“特洛伊木马策略”。在这项技术中，抗生素分子与一种营养分子结合，该营养分子帮助抗生素通过专门的营养转运蛋白进入细胞。在此，我们探索了将生物素与荧光分子Atto565结合的方法，以确定生物素化是否能增强积累。生物素是细菌必需的维生素，可通过合成或从环境中摄取获得。我们发现生物素化增强了Atto565在大肠杆菌中的积累。然而，这种增强似乎并非由于通过生物素转运蛋白摄取，因为游离生物素的存在对积累没有可观察到的影响。通过细胞分级分离研究确定，积累的化合物大多存在于周质中。通过观察周质中链霉亲和素的表达特异性增强了生物素化Atto565的积累，这一点得到了进一步证实。当链霉亲和素在细胞质中表达时未观察到这种增强，这表明该化合物在细胞质内没有显著分布。通过基因敲除菌株、质粒互补和诱变研究，我们证明生物素化使该化合物成为通过外膜孔蛋白OmpC的更好的载体。基于密度泛函理论（DFT）对三维几何结构的评估表明，生物素化并没有直接稳定化合物的构象，使其有利于通过孔进入。需要进一步的研究，包括分子动力学模拟，以确定生物素化Atto565积累增强的可能机制。
