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A Modular High-Throughput In Vivo Screening Platform Based on Chimeric Bacterial Receptors
Christina E Lehning1, Jan B Heidelberger2, John Reinhard3
1Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark , Building 220, Kemitorvet, DK-2800, Kgs. Lyngby, Denmark.
ACS Synthetic Biology
|April 5, 2017
Summary
This study presents a novel screening platform using chimeric receptors in E. coli to find new antibacterials targeting multidrug resistance (MDR). The system rapidly screens compound libraries for molecules that modulate bacterial two-component systems.
Area of Science:
- Synthetic Biology
- Microbiology
- Drug Discovery
Background:
- Multidrug resistance (MDR) is a major global health threat necessitating novel antibacterial strategies.
- Bacterial two-component systems (TCS) are essential for bacterial survival and virulence, making them promising targets for new drugs.
- TCS are absent in mammals, offering a selective therapeutic window.
Purpose of the Study:
- To develop and validate a modular screening platform for identifying small molecules that modulate bacterial TCS activity.
- To assess the utility of chimeric receptors fused to a fluorescence reporter system for high-throughput screening.
- To investigate the impact of aromatic tuning in transmembrane helices on receptor signaling and responsiveness.
Main Methods:
- Construction of chimeric receptors by fusing periplasmic stimulus-sensing domains to the cytoplasmic domain of EnvZ, a well-characterized TCS component.
- Utilized a fluorescent Escherichia coli reporter strain for real-time monitoring of transcriptional output.
- Examined Tar-EnvZ and NarX-EnvZ chimeras, including aromatic residue repositioning (aromatic tuning) within the second transmembrane helix (TM2).
Main Results:
- Successfully coupled bacterial periplasmic stimulus-perception domains to a reporter-governing cytoplasmic domain.
- Demonstrated that aromatic tuning modulates baseline signaling and can restore function to a previously non-functional chimera.
- Observed an inverse correlation between baseline signal output and the response magnitude to specific stimuli.
Conclusions:
- The developed fluorescent reporter system with plasmid-based chimeric receptors is a viable synthetic biology approach for rapid screening of compound libraries.
- This platform can identify novel modulators of bacterial TCS, offering potential therapeutic agents against MDR.
- Aromatic tuning is a key strategy for optimizing chimeric receptor function and signal output.