Division of Molecular Therapeutics and Formulation, School of Pharmacy, University of Nottingham, Nottingham NG7 2RD, U.K.
Nottingham University Biodiscovery Institute, National Biofilms Innovation Centre, School of Life Sciences, University of Nottingham, Nottingham NG7 2RD, U.K.
ACS Appl Mater Interfaces. 2021 Apr 28;13(16):19230-19243. doi: 10.1021/acsami.1c00907. Epub 2021 Apr 14.
The selective isolation of bacteria from mixed populations has been investigated in varied applications ranging from differential pathogen identification in medical diagnostics and food safety to the monitoring of microbial stress dynamics in industrial bioreactors. Selective isolation techniques are generally limited to the confinement of small populations in defined locations, may be unable to target specific bacteria, or rely on immunomagnetic separation, which is not universally applicable. In this proof-of-concept work, we describe a novel strategy combining inducible bacterial lectin expression with magnetic glyconanoparticles (MGNPs) as a platform technology to enable selective bacterial isolation from cocultures. An inducible mutant of the type 1 fimbriae, displaying the mannose-specific lectin FimH, was constructed in allowing for "on-demand" glycan-binding protein presentation following external chemical stimulation. Binding to glycopolymers was only observed upon fimbrial induction and was specific for mannosylated materials. A library of MGNPs was produced via the grafting of well-defined catechol-terminal glycopolymers prepared by reversible addition-fragmentation chain transfer (RAFT) polymerization to magnetic nanoparticles. Thermal analysis revealed high functionalization (≥85% polymer by weight). Delivery of MGNPs to cocultures of fluorescently labeled bacteria followed by magnetic extraction resulted in efficient depletion of type 1 fimbriated target cells from wild-type or afimbriate . Extraction efficiency was found to be dependent on the molecular weight of the glycopolymers utilized to engineer the nanoparticles, with MGNPs decorated with shorter Dopa-(ManAA) mannosylated glycopolymers found to perform better than those assembled from a longer Dopa-(ManAA) analogue. The extraction efficiency of fimbriated was also improved when the counterpart strain did not harbor the genetic apparatus for the expression of the type 1 fimbriae. Overall, this work suggests that the modulation of the genetic apparatus encoding bacterial surface-associated lectins coupled with capture through MGNPs could be a versatile tool for the extraction of bacteria from mixed populations.
从混合群体中选择性分离细菌已在各种应用中进行了研究，包括医学诊断和食品安全中的差异病原体识别、工业生物反应器中微生物应激动力学的监测等。选择性分离技术通常仅限于将小种群限制在特定位置，可能无法针对特定细菌，或者依赖于不是普遍适用的免疫磁分离。在这项概念验证工作中，我们描述了一种新的策略，该策略结合了诱导型细菌凝集素表达和磁性糖纳米粒子（MGNP）作为平台技术，使从共培养物中选择性分离细菌成为可能。构建了一种 1 型菌毛的诱导型突变体，该突变体表达了甘露糖特异性凝集素 FimH，可在外部化学刺激后“按需”呈现糖结合蛋白。只有在诱导菌毛后才观察到与糖聚合物的结合，并且该结合是甘露糖基化材料特有的。通过将通过可逆加成-断裂链转移（RAFT）聚合制备的具有明确邻苯二酚末端糖聚合物接枝到磁性纳米粒子上来制备 MGNP 文库。热分析表明，功能化程度很高（重量至少 85%的聚合物）。将 MGNP 递送到荧光标记细菌的共培养物中，然后进行磁性提取，可有效地从野生型或无菌毛的细菌中耗尽 1 型菌毛靶细胞。发现提取效率取决于用于工程纳米颗粒的糖聚合物的分子量，用较短的 Dopa-（ManAA）甘露糖化糖聚合物修饰的 MGNP 比用较长的 Dopa-（ManAA）类似物组装的 MGNP 表现更好。当对照菌株不具有表达 1 型菌毛的遗传装置时，菌毛的提取效率也得到了提高。总体而言，这项工作表明，与细菌表面相关的凝集素的遗传装置的调节与通过 MGNP 进行捕获相结合可能是从混合群体中提取细菌的通用工具。
