Fig 1.
Structural features of flavan-3-ols, proanthocyanidins and quercetin-3-O-glucuronid tested for antiadhesive and ant-gingipain activity against P. gingivalis; compounds not isolated from Rumex acetosa are marked by asterisk and have been used to obtain complete structure-activity relations; G: gallic acid.
Fig 2.
7 epicatechin-(4β→8)-epicatechin, 14 epicatechin-(4β-→8)-epicatechin(4β→8,2β→O→7)-epicatechin, 5 epicatechin-3-O-gallate, 15 quercetin-3-O-glucuronide, 8 epicatechin-3-O-gallate-(4β→8)-epicatechin-3-O-gallate, IS internal standard epigallocatechin-3-O-gallate.
Fig 3.
Influence of RA1 on the relative adhesion of FITC-labeled P. gingivalis to KB cells in the coincubation assay (90 min), determined by flow cytometric analysis.
Results are related to the untreated control (UC). The protease inhibitor Nα-tosyl-L-lysine chloromethyl ketone hydrochloride (TLCK, 1 mM) served as positive control (PC). Values represent the mean ± SD from three independent experiments; * p < 0.05; **: p < 0.01.
Fig 4.
Representative confocal laser scanning microscopy images of FITC labeled P. gingivalis during different incubation protocols with KB cells.
(A) Coincubation (90 min) of KB cells with FITC-labeled P. gingivalis (BCR 100:1) and RA1 (10 μg/mL); 1: untreated control 2: RA1 (10 μg/mL). (B) Preincubation of KB cells with RA1 (10 μg/mL) for 12 h and subsequent infection with P. gingivalis (BCR 100:1) for 90 min; 3: untreated control; 4: RA1 (10 μg/mL). Bacteria are stained with FITC (green), nuclei of KB cells with DAPI (blue) and endosomes with Dextran Texas Red (red).
Fig 5.
Representative fluorescence microscopy images of FITC labeled P. gingivalis adherent to murine oral mucosa sections from four independent experiments; data sets 4 and 5 originate as technical replicates from the same experiment to indicate intraassay reproducibility A 1–5: untreated control B 1–5: positive control, pretreated with 5 mM TLCK for 90 minutes C 1–5: RA1 100 μg/mL (preincubation of bacteria for 90 minutes).
Magnification: 100 ×. Images are equalized in brightness, contrast and fluorescence intensity.
Table 1.
Influence of flavan-3-ols and proanthocyanidins from RA1 on the adhesion of P. gingivalis to KB cells in the coincubation adhesion assay.
Fig 6.
Influence of RA1 (5, 10 and 50 μg/mL) and compounds 1 to 14 (5, 10, 50 μM) on Arg-gingipain (A) and Lys-gingipain (B) protease activity in relation to untreated P.gingivalis.
Leupeptin served as positive control (PC), untreated bacteria as negative control (UC). Data are mean ± SD from 3 independent experiments with n = 5 replicates. * p < 0.05; **: p < 0.01; ***: p < 0.001.
Table 2.
Influence of RA1 on P. gingivalis-mediated hemagglutination.
Fig 7.
Protein-ligand docking of epicatechin-3-O-gallate-(4β→8)-epicatechin-3-O-gallate 10 into the binding cavity of Arg-gingipain.
(A) 3D model; protein: green: hydrophobic, purple: polar, red: exposed; ligand: yellow: carbon, light grey: hydrogen, red: oxygen, blue: nitrogen; (B) 2D model.
Table 3.
Docking scores for compounds 1 to 14 from RA1 for the proteolytic domain of Rgp and for the hemaglutinin (HA) domain and correlation of the respective docking scores for Rgp with Arg-gingipain activity as determined within the protease assay (Rgp activity at 50 μM in %, related to the untreated control groups).