Article Figures & Data
Figures
- Fig 1
Growth kinetics of WT T. forsythia and the S-layer-deficient mutant in a medium containing serum. A total of 105 WT or mutant cells were inoculated into 100 μl of TF medium containing non-heat-inactivated serum on 96-well plates and were incubated for 10 days at 37°C under anaerobic conditions. The cell density (at 600 nm) was measured at 24-h intervals. Circles and squares indicate the growth of the WT and the mutant, respectively. Solid and dashed lines indicate the results with heat-inactivated CS and non-heat-inactivated CS, respectively. Data represent the means ± SD of measurements performed in triplicate.
- Fig 2
Effect of serum on the viability of T. forsythia. (A) WT T. forsythia, S-layer-deficient mutant, and E. coli XL-II cells were exposed to CS or heat-inactivated CS (Hi-CS). Details are given in Materials and Methods. In CLSM analysis, green and orange cells represent viable and dead cells, respectively. (B) The areas occupied by dead and/or live cells in the CLSM images were analyzed, and the ratio of live bacteria to total bacteria was calculated. Data represent means ± SD of measurements performed in triplicate. Asterisks indicate significant differences (***, P < 0.001) as determined by Tukey's honestly significant difference test. The y axis begins with 40%.
- Fig 3
Electron micrographs of T. forsythia exposed to serum. (A and B) WT T. forsythia (A) and the S-layer-deficient mutant (B) were exposed to 100% CS at 37°C for 2 h. Then samples were prepared for observation by TEM. Arrows indicate the disrupted outer membrane. (C) As a control, the mutant treated with PBS was prepared.
- Fig 4
SDS-PAGE analysis of the outer membrane proteins in the WT and the S-layer-deficient mutant. The outer membrane protein fraction of WT T. forsythia and the S-layer-deficient mutant were prepared and separated on a 12.5% SDS-PAGE gel. Details are given in Materials and Methods. Lane 1, WT; lane 2, S-layer-deficient mutant.
- Fig 5
C3b deposition on the surfaces of WT T. forsythia and the S-layer-deficient mutant. (A) WT T. forsythia and the S-layer-deficient mutant were exposed either to 30% human serum (HS) in PBS or to PBS alone at 37°C for 30 min. After washing, the cells were reacted first with an anti-C3 antibody and then with Alexa Fluor 594-conjugated anti-mouse IgG. DAPI staining was performed as well. Blue and red cells represent whole cells and cells with C3b bound to the surface, respectively. (B) The efficiency of C3b deposition on the bacterial cell surface, as shown in the CLSM images, was analyzed. Details are given in Materials and Methods. Data represent means ± SD of measurements performed in triplicate. Significant changes were determined using Bonferroni's t test (***, P < 0.001).
- Fig 6
Binding of factor H to WT T. forsythia and the S-layer-deficient mutant. (A) WT T. forsythia, the S-layer-deficient mutant, an E. coli Omp100-expressing strain (RA11), E. coli RA31, and A. actinomycetemcomitans (Aa) were used. In total, 2 × 109 bacterial cells were used for the factor H binding assay, carried out as described in Materials and Methods. The samples, 8 ng of authentic human factor H (FH), and 0.1 μl of human serum (HS) were first separated by 7.5% SDS-PAGE and then transferred to a nitrocellulose membrane. Immunoblotting was performed as described in Materials and Methods. (B) The intensities of factor H binding were measured with Image Lab and were quantified relative to that for WT T. forsythia. Data represent means ± SD of measurements performed in triplicate. Significant differences between RA31 and RA11, and between WT T. forsythia and the S-layer-deficient mutant, were determined using Student's t test (***, P < 0.001; *, P < 0.05).
- Fig 7
Zymographic analysis of WT T. forsythia and the S-layer-deficient mutant. The protease activities in the supernatants of WT and mutant T. forsythia cells were analyzed using a 7.5% polyacrylamide gel containing gelatin (1 mg/ml). Lane 1, WT; lane 2, S-layer-deficient mutant.
- Fig 8
Coaggregation of T. forsythia with S. sanguinis. Coaggregation of WT T. forsythia and the S-layer-deficient mutant with S. sanguinis was investigated as described in Materials and Methods. A 7-day culture of WT T. forsythia or the mutant was mixed with an overnight culture of S. sanguinis (2:1) in a cuvette, and the mixture was incubated at 37°C. The OD550 of the upper phase in the cuvette was measured using a spectrophotometer at 15-min intervals. A decrease in absorbance was considered to indicate coaggregation. Squares indicate the coaggregation of WT T. forsythia with S. sanguinis. Diamonds indicate the coaggregation of the T. forsythia mutant with S. sanguinis. As controls, the densities of single cells of WT T. forsythia (circles), mutant T. forsythia (triangles), and S. sanguinis (crosses) were measured. Data represent means ± SD of measurements performed in triplicate.
- Fig 9
Coaggregation of T. forsythia with oral bacteria. Coaggregation of WT T. forsythia and the S-layer-deficient mutant with seven species of oral bacteria was investigated as described in Materials and Methods. WT T. forsythia (shaded bars) or the mutant (open bars) was mixed with oral bacteria (2:1) in a cuvette, and the mixture was incubated at 37°C for 60 min. The OD550 of the upper phase in the cuvette was measured using a spectrophotometer. The percentage of coaggregation was calculated as [(initial OD − postincubation OD)/initial OD] × 100. Filled bars indicate the percentage of autoaggregation of partner strains. Aa, A. actinomycetemcomitans. (A) Percentage of coaggregation of stationary-phase cells of T. forsythia (7-day culture) and stationary-phase cells of oral bacteria. (B) Percentage of coaggregation of exponential-phase cells of T. forsythia (3-day culture) and stationary-phase cells of oral bacteria. Data represent means ± SD of measurements performed in triplicate. Significant differences in coaggregation between the WT and mutant T. forsythia strains were determined using Student's t test (*, P < 0.05; **, P < 0.01; ***, P < 0.001).
- Fig 10
Effect of sugar on the coaggregation of T. forsythia with oral bacteria. To observe the effects of sugars on coaggregation activity, 10 mM each sugar was added to the reaction mixture. We used N-acetyl-d-mannosamine, d-glucuronic acid sodium salt, l-(−)-fucose, d-(+)-xylose, and d-(+)-galactose. Coaggregation activity (120 min) was investigated as described in Materials and Methods. The percentage of coaggregation was calculated as [(initial OD − postincubation OD)/initial OD] × 100. Data represent means ± SD of measurements performed in triplicate. Significant differences in coaggregation between bacteria with added sugar and bacteria with no sugar added were determined using Dunnett's test (*, P < 0.05; **, P < 0.01).
Tables
- Table 1
Strains used in this study
Strain Descriptiona Reference or sourceb Tannerella forsythia ATCC 43037 Wild type ATCC ΔtfsAB mutant TfsA and TfsB deletion mutant; Camr 23 Aggregatibacter actinomycetemcomitans HK1651 Wild type ATCC Y4 Wild type ATCC Fusobacterium nucleatum ATCC 25586 Wild type ATCC Porphyromonas gingivalis W83 Wild type ATCC Streptococcus mutans UA159 Wild type ATCC Streptococcus mitis GTC 495 Wild type GTC Streptococcus salivarius GTC 215 Wild type GTC Streptococcus sanguinis GTC 217 Wild type GTC Escherichia coli XL-II endA1 supE44 thi-1 hsdR17 recA1 gyrA96 relA1 lac [F′ proAB lacIqZΔM15 Tn10 (Tetr) Amy Camr] Stratagene RA31 HB101 carrying empty vector (pGEM-T Easy); Ampr 36 RA11 HB101 Omp100-expressing strain; Ampr 36
Supplemental material
Files in this Data Supplement:
- Supplemental file 1 -
Fig. S1. Electron micrographs of T. forsythia exposed to serum (low-power field). Fig. S2. Immunofluorescence CLMS analysis of factor H binding on the bacterial surface of the T. forsythia WT and S-layer-deficient mutant. Fig. S3. Immunofluorescence CLMS analysis of C4BP binding on the bacterial surface of the T. forsythia WT and S-layer-deficient mutant.
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- Supplemental file 2 -
Fig. S1 to S3 legends.
PDF, 66K
- Supplemental file 1 -
