Fig 1.
Chemical structure of the bioactive compound assigned as 3β-hydroxyolean-12-en-28-al 3-p-coumarate (oleanolic aldehyde coumarate, OALC).
Fig 2.
Effect of OALC on virulence factors and acylhomoserine lactones production in P. aeruginosa PAO1.
(A) rhamnolipids production, (B) pyocyanin production, (C) Elastase production. The cell density of the bacteria was assessed at 600 nm and elastase production was assessed via an elastolysis assay and calculated as the ratio between A495 and A600. The rhamnolipids production was measured using methylene-blue-based method and expressed in μg mL-1. Pyocyanin was extracted, quantified by absorbance measurements at 380 nm and calculated as the ratio between A380 and A600. (D) Quantification of N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C12-HSL; grey bar) and N-butanoyl-L-homoserine lactone (C4-HSL; clear bar) produced by PAO1 cells. Acylhomoserine lactones were extracted and quantified by mass spectrometry. Error bars represent the standard errors of the means and all experiments were performed in quintuplicate with three independent assays and asterisks indicate samples that are significantly different from the DMSO controls (Student’s t-tests; P ≤ 0.01). Naringenin (Nar) and naringin (Nin) were used as a quorum sensing inhibitor control and negative control, respectively.
Fig 3.
Effect of OALC on QS genes (lasI/R and rhlI/R) and global activator genes (gacA and vfr) expression in P. aeruginosa PAO1.
(A) Effect of OALC on lasR (grey bar) and lasI (clear bar) expression following 18 hours of growth. (B) Effect of OALC on rhlR (grey bar) and rhlI (clear bar) expression following 18 hours of growth. (C) Effect of OALC on gacA (grey bar) and vfr (clear bar) expression following 18 hours of growth. Gene expression was measured as the β-galactosidase activity of the lacZ gene fusions and expressed in Miller units. Error bars represent the standard errors of the means; all experiments were performed in quintuplicate with three independent assays and asterisks indicate samples that are significantly different from the DMSO (Student’s t-tests; P ≤ 0.01).
Fig 4.
Effect of OALC on biofilm formation by P. aeruginosa.
(A) Quantification of biofilm formation by P. aeruginosa PAO1 grown in minimal (grey bar) and complex media (clear bar) after static incubation at 37°C for 24 hours. (B) Quantification of biofilm formation by P. aeruginosa grown in in minimal media (grey bar) and C.F.U. measurement (clear bar) of planktonic bacteria after static incubation at 37°C for 24 hours. Biofilm formation was quantified by crystal violet staining and measured at A590nm. The cell density of the bacteria was assessed at 600 nm. Error bars represent the standard errors of the means; all experiments were performed in quintuplicate with three independent assays and asterisks indicate samples that are significantly different from the DMSO (Student’s t-tests; P ≤ 0.01).
Fig 5.
PAO1 biofilm phenotypes as affected by DMSO, naringin, naringenin or OALC.
(A) Fluorescence microscopy images of PAO1 cells incubated statically at 37°C for 24 hours. Cells were visualized after staining with SYTO-9 (green fluorescence for living bacteria) and propidium iodide (red fluorescence for dead bacteria) furnished in the LIVE/DEAD BacLight kit. (B) Fluorescence microscopic images of biofilm formation by PAO1 cells incubated for 24 hours and then treated for 24 hours with DMSO, naringin, naringenin or OALC. Fluorescence microscopy was achieved by using a Leica DM IRE2 inverted fluorescence microscope using a 40x objective lens and images were false-colored and assembled using Adobe Photoshop.
Fig 6.
Effect of OALC on P. aeruginosa PAO1 motilities.
(A) Swarming motility of P. aeruginosa PAO1 onto LB agar (0.6%) supplemented with glutamate (0.05%), glucose (0.2%) and DMSO (1%) or OALC (200μM final concentration), naringenin (Nar, 4 mM final concentration) or naringin (Nin, 4 mM final concentration). After incubation at 37°C for 24 hours, the zones of migration (down) from the point of inoculation were measured (up) for each condition. (B) Twitching motility of P. aeruginosa PAO1 onto LB agar (1%) supplemented with DMSO (1%), OALC (200μM final concentration), Nar (4 mM final concentration) or Nin (4 mM final concentration). The twitching zones were stained (down) and their diameters (up) measured after incubation at 37°C for 48 h. Error bars represent the standard errors of the means and all experiments were performed in quintuplicate with three independent assays and asterisks indicate samples that are significantly different from the DMSO (Student’s t-tests; P ≤ 0.01).
Fig 7.
Effect of OALC on extracellular polysaccharides production by P. aeruginosa PAO1.
(A) Quantification of total extracellular polysaccharides. The cell density of the bacteria was assessed at 600nm and extracellular polysaccharides production was measured using Phenol-Sulfuric Acid (PSA) method and expressed in μg mL-1 with glucose as standard. (B) Quantification of alginate. The cell density of the bacteria was assessed at 600nm and alginate production was measured using carbazole method and expressed in μg mL-1 with sodium alginate as standard. Error bars represent the standard errors of the means; all experiments were performed in quintuplicate with three independent assays and asterisks indicate samples that are significantly different from the DMSO (Student’s t-tests; P ≤ 0.01). Naringenin (Nar) and naringin (Nin) both at 4 mM final concentration were used as quorum sensing inhibitor positive and negative controls, respectively.
Fig 8.
Synergistic activity of OALC with tobramycin against biofilm-encapsulated P. aeruginosa PAO1.
PAO1 cells were incubated statically for 24 hours in the presence of DMSO, naringin (4 mM), naringenin (4 mM) or OALC (200 μM) and then treated for 24 hours with tobramycin (100 μg mL-1). (A) OALC + tobramycin, (B) tobramycin, (C) naringenin + tobramycin, (D) DMSO + tobramycin, (E) naringin + tobramycin. Assessment of bacterial viability and microscopy were performed as in Fig 6. (F) Quantification of bacterial viability. Error bars represent the standard errors of the means; all experiments were performed in quintuplicate with three independent assays and asterisks indicate samples that are significantly different from the DMSO (Student’s t-tests; P ≤ 0.01).
Fig 9.
Synergistic activity of OALC with tobramycin against biofilm-encapsulated P. aeruginosa PAO1.
PAO1 cells were incubated statically for 24 hours and then treated for 24 hours with tobramycin (100 μg mL-1) and DMSO, naringin (4 mM), naringenin (4 mM) or OALC (200 μM final concentration). (A) OALC + tobramycin, (B) tobramycin, (C) naringenin + tobramycin, (D) DMSO + tobramycin, (E) naringin + tobramycin. Assessment of bacterial viability and microscopy were performed as in Fig 6. (F) Quantification of bacterial viability. Error bars represent the standard errors of the means; all experiments were performed in quintuplicate with three independent assays and asterisks indicate samples that are significantly different from the DMSO (Student’s t-tests; P ≤ 0.01).
Fig 10.
Proposed targets and inhibition cascade occurring in P. aeruginosa in presence of OALC.
In P. aeruginosa, OALC inhibits gacA gene expression (key component of GacS/GacA/Rsm signal transduction pathway) leading to an increased level of free RsmA [95]. The latter negatively regulates las and rhl systems leading to inhibition of rhamnolipids, pyocyanin and elastase production [9] as well as components involved in biofilm formation and maintenance, including motilities (swarming and twitching) and Psl/Pel productions [107] independently of QS systems. Exopolysaccharides production is also reduced directly by RsmA [99].