5 ± 3.5 2.0 ± 0.9 6.9 ± 1.4 KDP150 (ΔfimA) 52.5 ± 3.5* 1.7 ± 0.7* 23.7 ± 5.6** MPG67 (Δmfa1) 35.8 ± 3.6** 2.7 ± 1.6** 20.9 ± 4.4** MPG4167 (ΔfimAΔmfa1) 32.3 ± 3.8** 3.0 ± 1.6** 20.5 ± 4.3** KDP129 (Δkgp) 39.8 ± 3.2 2.2 ± 1.2 19.6 ± 5.4** KDP133 (ΔrgpAΔrgpB) 41.0 ± STI571 nmr 5.7 2.2 ± 1.0 45.9 ± 4.5** KDP136 (ΔrgpAΔrgpBΔkgp) 43.0 ± 1.4 2.1 ± 0.8 22.2 ± 2.4** a)Number of peaks was evaluated in an area sized 90 (x axis) × 2 (y axis) μm. The mean ± SE of 10 areas was shown. *p < 0.05 and **p < 0.01 in comparison with the

wild type using a Scheffe test. Figure 3 Homotypic biofilm formation by P. gingivalis wild-type strain and mutants in dTSB. P. gingivalis strains were stained with CFSE (green) and incubated in dTSB for 24 hours. After washing, the biofilms that developed on the coverglasses were observed with a CLSM equipped with a 40× objective. Optical sections were obtained along the z axis at 0.7-μm intervals, and images of the x-y and x-z planes were reconstructed with selleck chemical imaging software, as described in the text. Upper panels indicate z stacks of the x-y sections. Lower panels

show x-z sections. The experiment was repeated independently three times with each strain in triplicate. Representative images are shown. Quantitative analysis of biofilms in dTSB In the early maturation phase, the biovolumes of the biofilms were significantly increased find more in all of tested mutants as compared to the wild type (Figure 4). Deletion of long fimbriae resulted in the opposite tendency from the initial attachment phase, suggesting that this molecule has distinct roles under the different

phases. Figure 4 Quantification of homotypic biofilms formed by P. gingivalis wild-type strain and mutants in dTSB. Biofilms were formed as described in the legend to Figure 3, and 10 fields per a sample were randomly recorded and quantified, similar to the method described in the legend to Figure 2. Statistical analysis was performed with a Scheffe test. *p < 0.05 and **p < 0.01 cAMP in comparison to the wild-type strain. Exopolysaccharide production under proliferation conditions As extracellular polysaccharide is important for the development of biofilm communities, we examined the influences of fimbriae and gingipains on the accumulation of exopolysaccharide in P. gingivalis biofilms. To visualize and quantify exopolysaccharide accumulation in biofilms under the proliferation condition, 4′,6-diamino-2-phenylindole (DAPI)-labeled P. gingivalis cells and fluorescein isothiocyanate (FITC)-labeled exopolysaccharide were examined by confocal microscopy with digitally reconstructed image analysis.