Article Figures & Data
Figures
- FIG. 1.
Construction of recombinant L. casei expressing OmpC. (a) Specific antibody for the detection of OmpC. Purified recombinant OmpC and anti-OmpC antibodies were analyzed by immunoblotting. Approximately 108 CFU of SE whole-cell extract (cell), the corresponding volume of culture supernatant (Sup), and 25 ng of purified OmpC (rOmpC) were applied. The sizes of the molecular mass markers are shown in the left margin. (b) Detection and quantification of OmpC expressed by recombinant L. casei. Whole-cell extracts of OmpC-expressing L. casei and a nonexpressing strain (corresponding to 2 × 108 CFU/lane) were applied to immunoblotting. The blot was conjugated with anti-OmpC antibody and Alexa Fluor 488-labeled IgG. An OmpC-specific band was visualized using Molecular Imager FX and analyzed with Quantity One (Bio-Rad). The sizes of the molecular mass markers are shown in the left margin. LCO, OmpC-expressing L. casei; LCN, nonexpressing L. casei. The values 50, 25, 12.5, and 6.25 refer to 50, 25, 12.5, and 6.25 ng of recombinant OmpC/lane, respectively. (c) Flow cytometric analysis of recombinant L. casei. Bacterial cells labeled with anti-OmpC antibody and Alexa Fluor 488-conjugated IgG. Ten thousand events were analyzed and are shown in histogram form. The gray-shaded area represents LCN cells, and the nonshaded solid line represents LCO cells. (d) Evaluation of cell viability by flow cytometry. Bacterial cells (LCO or LCN) at exponential phase (8 h) and stationary phase (24 h) were stained with SYTO 9 and propidium iodide. The percentages of damaged and/or intact cells were calculated by using CellQuest software. The result shown is representative of two independent experiments.
- FIG. 2.
TNF-α induction and LDH release caused by recombinant lactobacilli. (a) TNF-α released by RAW264.7 cells; (b) LDH release induced by recombinant L. casei; (c) TNF-α released by murine peritoneal macrophages. All three assays were performed using cell cultures incubated for 24 h. The concentrations of TNF-α in the culture supernatant or the OD450 are described in the left margin. The concentrations of heat-killed bacteria added to the cell cultures are shown in the bottom margin (in μg/ml). Solid bars represent LCO cells, and open bars represent LCN cells. The data are presented as the means plus the standard deviations (SD) (n = 3). The results shown are representative of three independent experiments. *, P < 0.05; **, P < 0.01.
- FIG. 3.
TNF-α induction by LCN with or without rOmpC. A mixture of LCN (10 μg/ml) and rOmpC (or LPS) was added to a RAW264.7 cell culture. The concentrations of released TNF-α in the culture supernatant are described in the left margin. The data are presented as the means plus the SD (n = 3). The results shown are representative of three independent experiments. No significant difference was shown. 1n, 1 ng/ml; 1μ, μg/ml; 100p, 100 pg/ml.
- FIG. 4.
Importance of phagocytosed bacteria in TNF-α induction and the frequency of phagocytosis. (a) TNF-α release elicited by recombinant lactobacilli with or without cytochalasin D (5 μg/ml) supplementation. Cytochalasin D was added to the RAW264.7 cell culture (described as cyD+) 30 min before the addition of 10 μg of LCO (solid bar) or 10 μg of LCN (open bar)/ml. The concentrations of released TNF-α (ng/ml) are indicated in the left margin. The data are presented as the mean plus the SD (n = 3). (b) FACS analysis of phagocytosis of lactobacilli. RAW264.7 cells were cultured with FITC-labeled bacteria (10 μg/ml) for 0, 0.5, 2, 5, or 24 h and then collected. The bold gray line and thin black line represent LCN and LCO, respectively. The results shown are representative of two independent experiments. *, P < 0.05; **, P < 0.01.
- FIG. 5.
Evaluation of N-acetylmuramidase sensitivity. The reduction of the OD600 caused by cell lysis was measured at different time points. This result is representative of three independent experiments. Symbols: •, LCO cells; ○, LCN cells.
- FIG. 6.
Duration of TNF-α release induced by recombinant lactobacilli. LCO cells (•) or LCN cells (○) were added to a RAW264.7 cell culture, followed by incubation for 1 h (indicated by an arrow in the bottom margin) to allow phagocytosis. Excess bacteria that were not internalized were then removed by replacing the medium. Each culture supernatant was collected at 2, 4, 6, or 8 h. This result is representative of three independent experiments.
- FIG. 7.
TNF-α production induced by the ICW or CWRF of recombinant lactobacilli. Cell components were prepared from each concentration of heat-killed bacteria and added to RAW264.7 cell cultures. The concentrations of the added cell components described in the bottom margin are not the actual number but correspond to the concentration of heat-killed bacteria. The concentrations of released TNF-α are indicated in the left margin. The data are presented as the means + the SD (n = 3). The results are representative of two independent experiments. Bars: ▪, ICW from LCO cells; □, ICW from LCN cells; ▥, CWRF from LCO cells; ▦, CWRF from LCN cells. *, P < 0.05; **, P < 0.01.
