TY - JOUR

T1 - Fluorescence-based reporter for gauging cyclic di-GMP Levels in Pseudomonas aeruginosa.

AU - Rybtke, Morten T

AU - Borlee, Bradley R

AU - Murakami, Keiji

AU - Irie, Yasuhiko

AU - Hentzer, Morten

AU - Nielsen, Thomas E

AU - Givskov, Michael

AU - Parsek, Matthew R

AU - Tolker-Nielsen, Tim

PY - 2012

Y1 - 2012

N2 - The increased tolerance towards 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 biofilm formation is believed to be a key aspect in the development of novel anti-pathogenic 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 anti-pathogenic 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 c-di-GMP level reporters by transcriptionally fusing the cyclic di-GMP responsive cdrA promoter to genes encoding Gfp. We show that the reporter constructs give a fluorescent read-out 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 biofilm formation 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 anti-pathogenic compounds targeting cyclic di-GMP-signaling, as well as for use in research aiming at understanding the biofilm biology of P. aeruginosa.

AB - The increased tolerance towards 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 biofilm formation is believed to be a key aspect in the development of novel anti-pathogenic 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 anti-pathogenic 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 c-di-GMP level reporters by transcriptionally fusing the cyclic di-GMP responsive cdrA promoter to genes encoding Gfp. We show that the reporter constructs give a fluorescent read-out 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 biofilm formation 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 anti-pathogenic compounds targeting cyclic di-GMP-signaling, as well as for use in research aiming at understanding the biofilm biology of P. aeruginosa.

U2 - 10.1128/AEM.00414-12

DO - 10.1128/AEM.00414-12

M3 - Journal article

C2 - 22582064

VL - 78

SP - 5060

EP - 5069

JO - Applied and Environmental Microbiology

JF - Applied and Environmental Microbiology

SN - 0099-2240

IS - 15

ER -