A Modular High-Throughput In Vivo Screening Platform Based on Chimeric Bacterial Receptors
A Modular High-Throughput In Vivo Screening Platform Based on Chimeric Bacterial Receptors
复制标题
DOI：
10.1021/acssynbio.6b00288
复制
发表时间：
2017-07-01
影响因子：
4.7
通讯作者：
Draheim, Roger R.
中科院分区：
文献类型：
--
作者：
Lehning, Christina E.;Heidelberger, Jan B.;Draheim, Roger R.
多药耐药（MDR）是一个全球性的问题，需要新的方法。双组分系统是一个有前途的，但尚未开发的新的抗菌药物的目标。它们在细菌中普遍存在，在哺乳动物中不存在，并且它们的活性可以在感知各种刺激时进行调节。筛选预先存在的化合物文库可以揭示抑制毒性调节受体的刺激感知的小分子，减少来自必需受体的信号输出，或鉴定参与毒力的新型SHK的人工刺激配体。这些小分子可能具有理想的治疗特性，以对抗MDR。我们建议，一个模块化的筛选平台，其中的靶向受体的周质结构域融合到一个良好表征的受体，管理荧光报告基因的胞质结构域可以用来快速筛选目前现有的小分子文库。在这里，我们研究了两个以前创建的Tar-EnvZ嵌合体和一个新的NarX-EnvZ嵌合体。我们证明，它是可能的耦合周质刺激感知域的一个不变的胞质结构域，管理动态荧光报告系统的转录。此外，我们表明，芳香调谐，或重新定位的芳香残基在第二个跨膜螺旋（TM 2）的末端，调制基线信号输出从测试的嵌合体，甚至恢复输出从非功能性NarX-EnvZ嵌合体。最后，我们观察到的基线信号输出和同源刺激的反应程度之间的负相关。总之，我们提出，这里描述的平台，荧光大肠杆菌报告菌株与基于质粒的表达的芳香调谐嵌合受体，代表了合成生物学方法，以快速筛选预先存在的化合物库的受体调节活性。
Multidrug resistance (MDR) is a globally relevant problem that requires novel approaches. Two-component systems are a promising, yet untapped target for novel antibacterials. They are prevalent in bacteria and absent in mammals, and their activity can be modulated upon perception of various stimuli. Screening pre-existing compound libraries could reveal small molecules that inhibit stimulus perception by virulence-modulating receptors, reduce signal output from essential receptors or identify artificial stimulatory ligands for novel SHKs that are involved in virulence. Those small molecules could possess desirable therapeutic properties to combat MDR. We propose that a modular screening platform in which the periplasmic domain of the targeted receptors are fused to the cytoplasmic domain of a well characterized receptor that governs fluorescence reporter genes could be employed to rapidly screen currently existing small molecule libraries. Here, we have examined two previously created Tar-EnvZ chimeras and a novel NarX-EnvZ chimera. We demonstrate that it is possible to couple periplasmic stimulus-perceiving domains to an invariable cytoplasmic domain that governs transcription of a dynamic fluorescent reporter system. Furthermore, we show that aromatic tuning, or repositioning the aromatic residues at the end of the second transmembrane helix (TM2), modulates baseline signal output from the tested chimeras and even restores output from a nonfunctional NarX-EnvZ chimera. Finally, we observe an inverse correlation between baseline signal output and the degree of response to cognate stimuli. In summary, we propose that the platform described here, a fluorescent Escherichia coli reporter strain with plasmid-based expression of the aromatically tuned chimeric receptors, represents a synthetic biology approach to rapidly screen pre-existing compound libraries for receptor-modulating activities.