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.

Fig 1

Schematic drawing of the Copenhagen (A) and Seattle (B) c-di-GMP reporter cassettes. (A) The cassette consists of a transcriptional fusion between the promoter from cdrA (P_cdrA) and a gene encoding either stable GFP or unstable GFP (both designated gfp in this figure) with an optimized ribosomal binding site (RBSII). The transcriptional fusion is followed by two transcriptional terminators (T₀ and T₁). (B) The cassette consists of a transcriptional fusion between the promoter from cdrA, including the native RBS and parts of the coding sequence (P_cdrA-RBS-CDS) and a gene encoding either stable GFP or unstable GFP (gfp). The fusion is linked via an RNase III splice site and is followed by two transcriptional terminators (T₀ and T₁). In addition, both cassettes have flanking NotI restriction sites and a chloramphenicol resistance gene interspersed between the two terminators (omitted for clarity). Individual elements are not drawn to scale.

Fig 2

Colony morphology (top) and fluorescence intensity (bottom) of the plasmid-based reporter strains. (A) P. aeruginosa PAO1 ΔpelΔpsl/pCdrA::gfp^C (normal c-di-GMP level, stable GFP); (B) ΔwspFΔpelΔpsl/pCdrA::gfp^C (increased c-di-GMP level, stable GFP); (C) ΔpelΔpsl/pCdrA::gfp(ASV)^C (unstable GFP). (D) ΔwspFΔpelΔpsl/pCdrA::gfp(ASV)^C (unstable GFP). Shown are 20-h-old colonies.

Fig 3

Fluorescence from P. aeruginosa PAO1/pCdrA::gfp(ASV)^S, ΔfleQ/pCdrA::gfp(ASV)^S, ΔwspFΔpelΔpsl/pCdrA::gfp(ASV)^S, and vector controls (VC). RFU values are arbitrary fluorescence intensity units corrected for cell density. Results are averages of triplicate measurements on test tube cultures in mid-log growth phase.

Fig 4

Test of the plasmid-based reporter strains in shake flasks (A) and microtiter plates (B). (Left) Growth measurements; (right) fluorescence measurements. Results are representative of three independent experiments. ■, P. aeruginosa PAO1 ΔpelΔpsl/pCdrA::gfp^C (normal c-di-GMP level, stable GFP); ▲, ΔwspFΔpelΔpsl/pCdrA::gfp^C (increased c-di-GMP level, stable GFP); □, ΔpelΔpsl/pCdrA::gfp(ASV)^C (unstable GFP); △, ΔwspFΔpelΔpsl/pCdrA::gfp(ASV)^C (unstable GFP).

Fig 5

Treatment of P. aeruginosa PAO1 ΔwspFΔpelΔpsl/pCdrA::gfp^C with SNP in microtiter plates (A) and shake flasks (B). (Left) Growth measurements; (right) fluorescence measurements. Results are based on the Copenhagen group of reporters and are representative of three independent experiments. ⧫, 250 μM SNP; , 125 μM; , 62.5 μM; ♢, 31.25 μM SNP; , 15.63 μM SNP; , 7.81 μM SNP; , 3.91 μM SNP; ○, 1.95 μM SNP; , 0.977 μM SNP; , 0.488 μM; ●, 0.244 μM; , 0.122 μM SNP; ■, 0 μM SNP (untreated).

Fig 6

Fluorescence from P. aeruginosa PAO1/pCdrA::gfp(ASV)^S, ΔwspFΔpelΔpsl/pCdrA::gfp(ASV)^S, ΔpelΔpsl P_BAD::tpbB/pCdrA::gfp(ASV)^S, and vector controls (VC). RFU values are arbitrary fluorescence intensity units corrected for cell density. Results are averages of triplicate measurements on test tube cultures in mid-log growth phase induced with 0.2% l-arabinose.