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

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 - 2012/8

Y1 - 2012/8

N2 - 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 inthe 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 regulatorof biofilm formation,and cyclic di-GMP signaling is now regarded as a potential target for thedevelopment of antipathogenic compounds. Here we describe the development of fluorescent monitors thatcan gauge the cellular level of cyclic di-GMP inP. aeruginosa. We have created cyclic di-GMPlevelreporters 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 cyclicdi-GMP mediated by treatment of P. aeruginosa with the phosphodiesterase inducer nitric oxide.Consideringthat 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 atunderstanding the biofilm biology of P. aeruginosa.

AB - 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 inthe 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 regulatorof biofilm formation,and cyclic di-GMP signaling is now regarded as a potential target for thedevelopment of antipathogenic compounds. Here we describe the development of fluorescent monitors thatcan gauge the cellular level of cyclic di-GMP inP. aeruginosa. We have created cyclic di-GMPlevelreporters 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 cyclicdi-GMP mediated by treatment of P. aeruginosa with the phosphodiesterase inducer nitric oxide.Consideringthat 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 atunderstanding the biofilm biology of P. aeruginosa.

U2 - 10.1128/AEM.00414-12

DO - 10.1128/AEM.00414-12

M3 - Journal article

C2 - 22582064

SN - 0099-2240

VL - 78

SP - 5060

EP - 5069

JO - Applied and Environmental Microbiology

JF - Applied and Environmental Microbiology

IS - 15

ER -