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. 2021 Mar 18;65(4):e02431-20.
doi: 10.1128/AAC.02431-20. Print 2021 Mar 18.

Induction of Native c-di-GMP Phosphodiesterases Leads to Dispersal of Pseudomonas aeruginosa Biofilms

Jens Bo Andersen  1 , Kasper Nørskov Kragh  1 , Louise Dahl Hultqvist  1 , Morten Rybtke  1 , Martin Nilsson  1 , Tim Holm Jakobsen  1 , Michael Givskov  1   2 , Tim Tolker-Nielsen  3
Affiliations

Induction of Native c-di-GMP Phosphodiesterases Leads to Dispersal of Pseudomonas aeruginosa Biofilms

Jens Bo Andersen et al. Antimicrob Agents Chemother. .

Abstract

A decade of research has shown that the molecule c-di-GMP functions as a central second messenger in many bacteria. A high level of c-di-GMP is associated with biofilm formation, whereas a low level of c-di-GMP is associated with a planktonic single-cell bacterial lifestyle. c-di-GMP is formed by diguanylate cyclases and is degraded by specific phosphodiesterases. We previously presented evidence that the ectopic expression of the Escherichia coli phosphodiesterase YhjH in Pseudomonas aeruginosa results in biofilm dispersal. More recently, however, evidence has been presented that the induction of native c-di-GMP phosphodiesterases does not lead to a dispersal of P. aeruginosa biofilms. The latter result may discourage attempts to use c-di-GMP signaling as a target for the development of antibiofilm drugs. However, here, we demonstrate that the induction of the P. aeruginosa c-di-GMP phosphodiesterases PA2133 and BifA indeed results in the dispersal of P. aeruginosa biofilms in both a microtiter tray biofilm assay and a flow cell biofilm system.

Keywords: Pseudomonas aeruginosa; biofilm dispersal; c-di-GMP; phosphodiesterase.

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Figures

FIG 1
FIG 1
Amount of biofilm formed in microtiter tray wells by P. aeruginosa, P. aeruginosa PBAD-bifA, and P. aeruginosa PBAD-PA2133 (A) and by P. aeruginosa wspF, P. aeruginosa wspF PBAD-bifA, and P. aeruginosa wspF PBAD-PA2133 (B) after 10 h of growth in the presence (+) or absence (−) of arabinose. Biofilm biomass was determined by the use of a crystal violet staining assay. Average amounts of biofilm biomass from three replicate cultures are shown. The bars indicate standard deviations, and the asterisks indicate significant differences (***, P < 0.001; ****, P < 0.0001). wt, wild type.
FIG 2
FIG 2
c-di-GMP levels gauged by the use of a cdrA-gfp fluorescent reporter in cultures formed in microtiter tray wells by P. aeruginosa/pCdrA-gfp, P. aeruginosa PBAD-bifA/pCdrA-gfp, and P. aeruginosa PBAD-PA2133/pCdrA-gfp (A) and by P. aeruginosa wspF pel psl/pCdrA-gfp, P. aeruginosa wspF pel psl PBAD-bifA/pCdrA-gfp, and P. aeruginosa wspF pel psl PBAD-PA2133/pCdrA-gfp (B) after 10 h of growth in the presence (+) or absence (−) of arabinose. The average amount of GFP fluorescence/OD600 was calculated from 2 biological replicates, each with three technical replicates. The bars indicate standard deviations, and the asterisks indicate significant differences (*, P < 0.05; ****, P < 0.0001).
FIG 3
FIG 3
Biofilms of P. aeruginosa (A and D), P. aeruginosa PBAD-bifA (B and E), and P. aeruginosa PBAD-PA2133 (C and F) were grown in microtiter tray wells for 8 h, after which an arabinose inducer (+) or a water control (−) was added, and incubation was continued for 1 h (A to C) or 2 h (D to F). The biofilms were initiated with various dilutions of the overnight cultures in order to establish biofilms with different degrees of maturity. The numbers at the x axis refer to the dilution of the inoculum. Biofilm biomass was determined by the use of a crystal violet staining assay. Average amounts of biofilm biomass from three replicate cultures are shown. The bars indicate standard deviations, and the asterisks indicate significant differences (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).
FIG 4
FIG 4
Biofilms of P. aeruginosa wspF (A and D), P. aeruginosa wspF PBAD-bifA (B and E), and P. aeruginosa wspF PBAD-PA2133 (C and F) were grown in microtiter tray wells for 8 h, after which an arabinose inducer (+) or a water control (−) was added, and incubation was continued for 1 h (A to C) or 2 h (D to F). The biofilms were initiated with various dilutions of the overnight cultures in order to establish biofilms with different degrees of maturity. The numbers at the x axis refer to the dilution of the inoculum. Biofilm biomass was determined by the use of a crystal violet staining assay. Average amounts of biofilm biomass from four replicate cultures are shown. The bars indicate standard deviations, and the asterisks indicate significant differences (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).
FIG 5
FIG 5
Biofilms of P. aeruginosa wspF, P. aeruginosa wspF PBAD-bifA, and P. aeruginosa wspF PBAD-PA2133 were grown in flow cells for 68 h. Automated CLSM was used to capture images every 60 min at three different locations in three biofilms of each of the three strains. The amount of biofilm biomass was subsequently quantified using the Measurement Pro feature of Imaris. The average biomass/CLSM image of the three P. aeruginosa wspF, P. aeruginosa wspF PBAD-bifA, and P. aeruginosa wspF PBAD-PA2133 biofilms as a function of time is shown. Bars indicate standard deviations. The final biomass (after 68 h of cultivation) was used to assess statistical significance. The biomass of the wspF biofilm differed significantly from that of the wspF PBAD-bifA biofilm (P = 0.048), the biomass of the wspF biofilm differed significantly from that of the wspF PBAD-PA2133 biofilm (P = 0.0001), and the biomass of the wspF PBAD-bifA biofilm differed significantly from that of the wspF PBAD-PA2133 biofilm (P = 0.040).
FIG 6
FIG 6
Biofilms of P. aeruginosa wspF, P. aeruginosa wspF PBAD-bifA, and P. aeruginosa wspF PBAD-PA2133 were grown in flow cells for 68 h, after which the medium was shifted to arabinose-containing medium. Subsequently, automated CLSM was used to capture images every 14 min at three different locations in three biofilms of each of the three strains. The amount of biofilm biomass/CLSM image was quantified using the Measurement Pro feature of Imaris. The relative biomasses of the three P. aeruginosa wspF, P. aeruginosa wspF PBAD-bifA, and P. aeruginosa wspF PBAD-PA2133 biofilms after the shift to arabinose-containing medium are shown. Bars indicate standard deviations. The biomass after 5.5 h of PDE induction was used to assess statistical significance. The biomass of the wspF biofilm differed significantly from that of the wspF PBAD-bifA biofilm (P = 0.033), the biomass of the wspF biofilm differed significantly from that of the wspF PBAD-PA2133 biofilm (P = 0.0001), and the biomass of the wspF PBAD-bifA biofilm differed significantly from that of the wspF PBAD-PA2133 biofilm (P = 0.046).
FIG 7
FIG 7
Biofilms of P. aeruginosa wspF, P. aeruginosa wspF PBAD-bifA, and P. aeruginosa wspF PBAD-PA2133 were grown in flow cells for 68 h, after which the medium was shifted to arabinose-containing medium. Subsequently, automated CLSM was used to capture images every 14 min at three different locations in three biofilms of each of the three strains. Representative images of the biofilms immediately before the shift to arabinose-containing medium and 2.5 and 5.5 h after the shift to arabinose-containing medium are shown.
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