EAGER: NSF-BSF: Quorum Biosensing Using Magnetic Field-Activated Molecular Machines

细菌细胞之间的通讯，称为群体感应，是控制其种群数量的重要机制。通过用化学信号相互“交谈”，细菌细胞可以决定目前的条件是否有利于它们的繁殖和生物膜的形成。 群体感应的检测和抑制为用治疗诊断剂（诊断和治疗的组合）治疗细菌感染提供了新的可能性。这个EAGER项目解决了群体感应分析及其抑制的基本方面，基于纳米技术方法，使用分子“机器”组装在磁性纳米颗粒上，并在一定距离外激活磁场。成功将开启未来的生物医学应用和环境研究中的其他潜在应用（例如，用于分析和提高水修复效率）和国土安全（例如防止生物恐怖主义）。群体感应是由细菌感应的小分子触发的基因调节机制，以根据可用资源控制细菌种群的密度。使用小分子破坏这种机制是对抗病原菌的新策略。识别这些潜在药物的重要性和优势在于，通过向细菌发出“虚假信息”的信号，细菌不会受到威胁，因此不会产生对药物的耐药性（与传统抗生素不同）。因此，当抗生素不能再使用时，这些药物有望成为抗菌治疗的最前沿。拟议的研究计划将利用一类新的磁场激活分子机器，将在项目框架内开发。这些新机器将被用作独特的生物传感器设备，它们将进入细胞，并将报告（在磁刺激下）不同基因的转录形式（mRNA）的浓度，这些基因负责细菌之间的传感。报告信号将是荧光的，并将被量化。荧光信号将是存在于细胞中的mRNA分子的量的指示。这种方法既可以用于多个基因的定量，也可以用于筛选抑制细菌之间小分子传感的潜在药物（从潜在药物文库中）。由于mRNA的体内定量被认为是在给定时间点细胞状态的现实表示，因此可以预期这种方法在细菌群体感应及其抑制的分析中具有很高的生产率。预期的结果将包括新的群体感应抑制剂，可用作新的抗菌剂，以对抗铜绿假单胞菌相关感染（在未来的其他细菌感染）。具体目标包括：（i）所述分子机器可以进入细菌细胞并检测mRNA的概念证明，（ii）mRNA的特异性检测及其定量，（iii）在抑制剂存在下群体感应下调时低浓度mRNA的生物感应，（iv）以及这些分子机器在高通量筛选群体感应抑制剂中的用途，所述群体感应抑制剂作为新的抗-该奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Communication of bacterial cells to each other, called quorum sensing, is an important mechanism to control their population. By "talking" to each other with chemical signals, bacterial cells can decide if the present conditions are favorable for their multiplication and formation of biofilms. Detection and inhibition of the quorum sensing provides novel possibilities for treating bacterial infections with theranostics (combination of diagnostics and therapy). This EAGER project addresses fundamental aspects of the quorum sensing analysis and its inhibition based on a nano-technological approach using molecular "machines" assembled on magnetic nanoparticles and activated at a distance with an external magnetic field. Success will open future biomedical applications and other potential applications in environmental studies (e.g., for the analysis and enhancement of water remediation efficiency) and homeland security (such as preventing bioterrorism).Quorum sensing is a mechanism of gene regulation triggered by small molecules sensed by bacteria in order to control the density of bacterial population depending on available resources. Disruption of this mechanism using small molecules is a novel strategy to combat pathogenic bacteria. The importance and advantage of identifying such potential drugs is that by signaling bacteria with "disinformation", bacteria are not threatened, hence have no incentive to develop resistance to the drug (unlike with conventional antibiotics). Thus, these drugs are expected to be in the forefront of antibacterial treatments, when antibiotics could not be used anymore. The proposed research program will make use of a novel class of Magnetic Field-activated Molecular Machines that will be developed within the project framework. These new machines will be used as unique biosensor devices that will enter the cells and will report (upon magnetic stimulation) on the concentration of different genes in their transcribed form (mRNA) that are in charge of sensing between bacteria. The reporting signal will be fluorescent and will be quantified. The fluorescent signal will be an indication of the quantities of mRNA molecules that exist in the cells. This approach can be taken both for the quantification of multiple genes, but also for the screening of potential drugs (out of potential drug libraries) that inhibit the sensing of small molecules between bacteria. Since in vivo quantification of mRNA is considered a realistic representation of the state of cells at a given time point, this approach can be expected to be highly productive in the analysis of quorum sensing in bacteria and then its inhibition. The expected results will include novel quorum sensing inhibitors that may be used as new antibacterial reagents to fight Pseudomonas aeruginosa related infections (in the future other bacterial infections). Specific objectives include: (i) a proof of concept that the molecular machines described can enter bacterial cells and detect mRNAs, (ii) specific detection of mRNAs and their quantification, (iii) biosensing of low concentrations of mRNAs upon down regulation of quorum sensing in the presence of inhibitors, (iv) and the use of these molecular machines in high-throughput screens of quorum sensing inhibitors operating as novel anti-bacterial drugs.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.