Fluorescence-Based Reporter for Gauging Cyclic Di-GMP Levels in Pseudomonas aeruginosa

Publication: Research - peer-reviewJournal article – Annual report year: 2012

  • Author: Rybtke, Morten T., Denmark

    Department of International Health, Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark

  • Author: Borlee, Bradley R., United States

    Department of Microbiology, University of Washington, United States

  • Author: Murakami, Keiji, United States

    Department of Microbiology, University of Washington, United States

  • Author: Irie, Yasuhiko

    Department of Microbiology, University of Washington, Seattle, Washington, USA

  • Author: Hentzer, Morten, Denmark

    Medicinal Chemistry Research, Lundbeck Research Denmark, Denmark

  • Author: Nielsen, Thomas Eiland

    Organic Chemistry, Department of Chemistry, Technical University of Denmark, Denmark

  • Author: Givskov, Michael, Denmark

    Department of International Health, Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark

  • Author: Parsek, Matthew R., United States

    Department of Microbiology, University of Washington, United States

  • Author: Tolker-Nielsen, Tim, United States

    Department of International Health, Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, United States

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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.
Original languageEnglish
JournalApplied and Environmental Microbiology
Publication date2012
Volume78
Journal number15
Pages5060-5069
ISSN0099-2240
DOIs
StatePublished
CitationsWeb of Science® Times Cited: 7
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