Fluorescence-based reporter for gauging cyclic di-GMP levels in Pseudomonas aeruginosa

Morten T Rybtke, Bradley R Borlee, Keiji Murakami, Yasuhiko Irie, Morten Hentzer, Thomas E Nielsen, Michael Givskov, Matthew R Parsek, Tim Tolker-Nielsen

Research output: Contribution to journalArticle

90 Citations (Scopus)

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.
Original languageEnglish
Pages (from-to)5060-5069
Number of pages10
JournalApplied and Environmental Microbiology
Volume78
Issue number15
DOIs
Publication statusPublished - Aug 2012

Fingerprint

Pseudomonas aeruginosa
biofilm
fluorescence
Fluorescence
Biofilms
antibiotics
targeting
tolerance
bronchopneumonia
cystic fibrosis
second messengers
pathogenicity
immune system
nitric oxide
gauges
green fluorescent protein
infection
gauge
turnover
drug

Keywords

  • Adhesins, Bacterial
  • Biofilms
  • Cyclic GMP
  • Fluorescence
  • Genes, Reporter
  • Green Fluorescent Proteins
  • Plasmids
  • Promoter Regions, Genetic
  • Pseudomonas aeruginosa

Cite this

Rybtke, M. T., Borlee, B. R., Murakami, K., Irie, Y., Hentzer, M., Nielsen, T. E., ... Tolker-Nielsen, T. (2012). Fluorescence-based reporter for gauging cyclic di-GMP levels in Pseudomonas aeruginosa. Applied and Environmental Microbiology, 78(15), 5060-5069. https://doi.org/10.1128/AEM.00414-12

Fluorescence-based reporter for gauging cyclic di-GMP levels in Pseudomonas aeruginosa. / Rybtke, Morten T; Borlee, Bradley R; Murakami, Keiji; Irie, Yasuhiko; Hentzer, Morten; Nielsen, Thomas E; Givskov, Michael; Parsek, Matthew R; Tolker-Nielsen, Tim.

In: Applied and Environmental Microbiology, Vol. 78, No. 15, 08.2012, p. 5060-5069.

Research output: Contribution to journalArticle

Rybtke, MT, Borlee, BR, Murakami, K, Irie, Y, Hentzer, M, Nielsen, TE, Givskov, M, Parsek, MR & Tolker-Nielsen, T 2012, 'Fluorescence-based reporter for gauging cyclic di-GMP levels in Pseudomonas aeruginosa', Applied and Environmental Microbiology, vol. 78, no. 15, pp. 5060-5069. https://doi.org/10.1128/AEM.00414-12
Rybtke, Morten T ; Borlee, Bradley R ; Murakami, Keiji ; Irie, Yasuhiko ; Hentzer, Morten ; Nielsen, Thomas E ; Givskov, Michael ; Parsek, Matthew R ; Tolker-Nielsen, Tim. / Fluorescence-based reporter for gauging cyclic di-GMP levels in Pseudomonas aeruginosa. In: Applied and Environmental Microbiology. 2012 ; Vol. 78, No. 15. pp. 5060-5069.
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