title: Fluorescence-based reporter for gauging cyclic di-GMP levels in Pseudomonas aeruginosa.
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.
authors.affiliation: Department of International Health, Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Morten T
authors.initials: MT
authors.lastname: Rybtke
authors.forename: Bradley R
authors.initials: BR
authors.lastname: Borlee
authors.forename: Keiji
authors.initials: K
authors.lastname: Murakami
authors.forename: Yasuhiko
authors.initials: Y
authors.lastname: Irie
authors.forename: Morten
authors.initials: M
authors.lastname: Hentzer
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Nielsen
authors.forename: Michael
authors.initials: M
authors.lastname: Givskov
authors.forename: Matthew R
authors.initials: MR
authors.lastname: Parsek
authors.forename: Tim
authors.initials: T
authors.lastname: Tolker-Nielsen
mesh_terms: D018829:Adhesins, Bacterial / Q000235:genetics; D018441:Biofilms; D006152:Cyclic GMP / Q000032:analysis*; D005453:Fluorescence; D017930:Genes, Reporter / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D011550:Pseudomonas aeruginosa / Q000737:chemistry*
chemical_list: D018829:Adhesins, Bacterial; C551088:CdrA protein, Pseudomonas aeruginosa; D049452:Green Fluorescent Proteins; D006152:Cyclic GMP