Related Experiment Video
Updated: May 22, 2026
Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa
Published on: June 30, 2016
Fluorescence-based reporter for gauging cyclic di-GMP levels in Pseudomonas aeruginosa
Morten T Rybtke1, Bradley R Borlee, Keiji Murakami
1Department of International Health, Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
Abstract:
The increased tolerance toward the host immune system and antibiotics displayed by biofilm-forming Pseudomonas aeruginosa and other bacteria in chronic infections such as cystic fibrosis bronchopneumonia is of major concern. Targeting of biofilm formation is believed to be a key aspect in the development of novel antipathogenic drugs that can augment the effect of classic antibiotics by decreasing antimicrobial tolerance. The second messenger cyclic di-GMP is a positive regulator of biofilm formation, and cyclic di-GMP signaling is now regarded as a potential target for the development of antipathogenic compounds. Here we describe the development of fluorescent monitors that can gauge the cellular level of cyclic di-GMP in P. aeruginosa. We have created cyclic di-GMP level reporters by transcriptionally fusing the cyclic di-GMP-responsive cdrA promoter to genes encoding green fluorescent protein. We show that the reporter constructs give a fluorescent readout of the intracellular level of cyclic di-GMP in P. aeruginosa strains with different levels of cyclic di-GMP. Furthermore, we show that the reporters are able to detect increased turnover of cyclic di-GMP mediated by treatment of P. aeruginosa with the phosphodiesterase inducer nitric oxide. Considering that biofilm formation is a necessity for the subsequent development of a chronic infection and therefore a pathogenicity trait, the reporters display a significant potential for use in the identification of novel antipathogenic compounds targeting cyclic di-GMP signaling, as well as for use in research aiming at understanding the biofilm biology of P. aeruginosa.
Insights
Researchers developed fluorescent reporters to measure cyclic di-GMP levels in Pseudomonas aeruginosa. This tool aids in developing new drugs to combat antibiotic tolerance in chronic infections like cystic fibrosis.
Area of Science:
- Microbiology
- Bacterial Pathogenesis
- Drug Discovery
Background:
- Biofilm formation in Pseudomonas aeruginosa contributes to antibiotic tolerance and chronic infections, such as cystic fibrosis.
- Targeting biofilm formation is crucial for developing novel antipathogenic strategies to overcome antimicrobial resistance.
Purpose of the Study:
- To develop fluorescent biosensors for quantifying cyclic di-GMP levels in P. aeruginosa.
- To utilize these biosensors for identifying new antipathogenic compounds targeting cyclic di-GMP signaling.
Main Methods:
- Constructed reporter strains by fusing the cyclic di-GMP-responsive cdrA promoter to green fluorescent protein genes.
- Validated reporter sensitivity across P. aeruginosa strains with varying cyclic di-GMP levels.
- Assessed reporter function in response to nitric oxide-induced changes in cyclic di-GMP turnover.
Main Results:
- Successfully created fluorescent reporters that accurately reflect intracellular cyclic di-GMP concentrations in P. aeruginosa.
- Demonstrated the reporters' ability to detect alterations in cyclic di-GMP metabolism.
- Confirmed the utility of reporters in identifying modulators of cyclic di-GMP signaling.
Conclusions:
- Developed novel fluorescent tools for real-time monitoring of cyclic di-GMP in P. aeruginosa.
- These reporters hold significant potential for drug discovery targeting bacterial biofilm formation and chronic infections.
- The tools will advance research into the fundamental biology of P. aeruginosa biofilms.
