Bis-(3'-5')-cyclic dimeric GMP regulates antimicrobial peptide resistance in Pseudomonas aeruginosa

Abstract

Bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) is an intracellular second messenger that controls the lifestyles of many bacteria. A high intracellular level of c-di-GMP induces a biofilm lifestyle, whereas a low intracellular level of c-di-GMP stimulates dispersal of biofilms and promotes a planktonic lifestyle. Here, we used the expression of different reporters to show that planktonic cells, biofilm cells, and cells dispersed from biofilms (DCells) had distinct intracellular c-di-GMP levels. Proteomics analysis showed that the low intracellular c-di-GMP level of DCells induced the expression of proteins required for the virulence and development of antimicrobial peptide resistance in Pseudomonas aeruginosa. In accordance with this, P. aeruginosa cells with low c-di-GMP levels were found to be more resistant to colistin than P. aeruginosa cells with high c-di-GMP levels. This finding contradicts the current dogma stating that dispersed cells are inevitably more susceptible to antibiotics than their sessile counterparts.

Fig 1

(A) Expression of p_cdrA-gfp fusion in P. aeruginosa PAO1 (PCells), PAO1ΔwspF (BCells), PAO1ΔwspF/p_lac-yhjH, and PAO1/p_lac-yhjH (DCells) strains. Means and standard deviations (SD) in relative fluorescence units (RFU) from triplicate experiments are shown. *, P < 0.01. (B) Biofilm formation of P. aeruginosa PAO1 (PCells), PAO1ΔwspF (BCells), and PAO1/p_lac-yhjH (DCells) strains in microplates. Means and SD from triplicate experiments are shown. *, P < 0.01.

Fig 2

β-Galactosidase activity of P. aeruginosa strains grown as planktonic cells or biofilm cells containing the pel-lacZ biosensors. SNP was added to both PAO1 and the PAO1ΔwspF (BCells) at final concentration of 5 μM, whereas 0.25, 0.5, and 1% arabinose was added to the PAO1/p_BAD-yhjH strain. For the biofilm cells, the β-galactosidase activity was measured in the dispersed cells. Means and SD in β-galactosidase activity from triplicate experiments are shown. *, P < 0.01.

Fig 3

Pyoverdine production by P. aeruginosa PAO1 (PCells), PAO1ΔwspF (BCells), and PAO1/p_lac-yhjH (DCells). The pyoverdine fluorescence levels (excitation wavelength, 400 nm; emission wavelength, 450 nm) of supernatants of P. aeruginosa overnight cultures were recorded using the Tecan Infinite Pro2000 microplate reader.

Fig 4

P_pmr-gfp expression in P. aeruginosa PAO1 (PCells), PAO1ΔwspF (BCells), and PAO1/p_lac-yhjH (DCells) strains. Overnight cultures were diluted 10-fold into fresh ABTGC medium with or without 1 μg of colistin ml⁻¹. Portions (3 μl) of cultures representing each condition were spotted onto cover slides after 7 h of growth for imaging by fluorescence microscopy. The level of fluorescence of 30 individual p_pmr-gfp tagged bacterial cells was measured for each sample by using ImageJ. Means and SD in relative fluorescence intensity units (RFU) from 30 individual cells are shown. *, P < 0.01.

Fig 5

Colistin resistance assay. P. aeruginosa PAO1 (PCells) (A), PAO1ΔwspF (BCells) (B), and PAO1/p_lac-yhjH (DCells) (C) were cultivated at 37°C in ABTGC medium with 0, 0.25, or 2 μg of colistin ml⁻¹. The OD₆₀₀ was monitored for 10 h. Means and SD from triplicate experiments are shown. (D) Fast-kill assay of P. aeruginosa PAO1 (PCells), PAO1ΔwspF (BCells), and PAO1/p_lac-yhjH (DCells) by 4 μg of colistin ml⁻¹. The proportion of dead bacterial cells was monitored by using the Live/Dead BacLight bacterial viability kits (Invitrogen) after 10 min of treatment. *, P < 0.01.

Fig 6

Colistin resistance of planktonic cells (PCells), biofilm cells (BCells), and dispersed cells (DCells). Planktonic cells (PCells) of PAO1 (A), biofilm-dispersed cells (BCells) from PAO1 biofilm by 5 μM SNP (B), planktonic cells of (PCells) PAO1/p_BAD-yhjH (C), and biofilm-dispersed cells (DCells) from PAO1/p_BAD-yhjH biofilms by 1% arabinose (D) were cultivated at 37°C in ABTGC medium with 0 or 4 μg of colistin/ml. The OD₆₀₀ was monitored for 300 min. Means of three replicates are shown. (E and F) Biofilms formed by PAO1 strain on glass slides were submerged into ABTGC medium with 0 (E) and 4 (F) μg of colistin ml⁻¹ for 2 h. Live and dead cells in treated biofilms were stained by using Live/Dead BacLight bacterial viability kits, followed by confocal laser scanning microscopy imaging.