Figure 1.
The iacP mutant strain has two different types of flagellar filaments.
(A) The bacteria cell surface was immunogold-labeled and negatively stained for transmission electron microscopy. The bacteria were deposited on the grid and incubated with an anti-FljB primary antibody; this was followed by incubation with protein G–gold and negative staining with uranyl acetate. The images at the bottom panel show higher magnification of electron micrographs. The 10-nm gold particles (arrows) decorate the flagella structures. The images are representative of three independent experiments. Scale bars, 0.5 µm. (B) The flagellar filaments were stained with FITC-conjugated anti-FljB antibody and rhodamine-conjugated anti-FliC antibody. The nucleus and bacteria were stained with Hoechst 33342 dye (original magnification, × 630) (Right panel). For each strain, the number of flagella per cell was quantified from at least 50 cells. Error bars indicate the mean ± SD (standard deviation) of three independent experiments. Asterisks indicate statistically significant difference from the control group, determined by a Student’s t test (P < 0.05). (C) To visualize the flagellar filaments on a single bacterium, the images in B panel show magnified views of at least 100 cells.
Figure 2.
Flagella associated with fljB were observed in the iacP fliC double mutant.
(A) The levels of FljB and FliC in the pellet and culture supernatants from all strains were determined by Western blotting with anti-FljB and anti-FliC antibodies. An anti-DnaK antibody was used as a loading control for cytoplasmic proteins. (B) A transmission electron micrograph of double mutant iacP and each fljB, fliC and triple mutant iacP fljB fliC was negatively stained with uranyl acetate. A higher magnification images is shown in the bottom panels (Right panel). The average number of flagella was determined from electron micrographs of at least 50 cells per strain. Representative images of three independent experiments are shown. The error bars represent ± SD of the mean. Scale bars, 2µm. (C) The flagellar filaments were visualized in different strains after immunostaining (as in Figure 1B and 1C). Bacterial DNA was stained with the DAPI (Right panel). Quantification of the flagella numbers of all strains in single bacterial cells. The error bars correspond to means ± SD. (D) Complementation of the fljB or the fliC genes in double mutant strains was examined by Western blotting and immunostaining. An anti-DnaK antibody was used as a control for cytoplasmic proteins (Right panel). To visualize flagellar filaments at the bacterial cells, the iacP fljB, the iacP fliC double mutant strain carrying the fljB or fliC gene on a plasmid were stained as described in Figures 1 and 2. The bacteria were stained with Hoechst 33342 dye.
Figure 3.
NF-κB activation is increased in Raw 264.7 cells infected with the iacP mutant.
(A) The expression of NF-κB p65 was determined using immunoblot analysis with an anti-NF-κB p65 antibody in the cytoplasmic and nuclear fractions of Salmonella-infected Raw 264.7 cells. The anti-GAPDH and anti-Lamin B antibodies were used as cytoplasmic and nuclear protein markers, respectively (Right panel). Quantification of the amount of nuclear and cytoplasmic p65 expression levels in Raw 264.7 macrophages. Bars correspond to means ± SD. (B) The nuclear localization of NF-κB p65 as monitored by indirect immunofluorescence was used as readout for NF-κB activation. The cell nuclei were stained with DAPI, and p65 was visualized with FITC-conjugated anti-p65 antibody (Right panel). Quantification of nuclear p65 activation in Raw 264.7 cells was performed by densitometry analysis. Images from five different confocal planes (at least 30 cells) per sample were analyzed to determine the quantification of nuclear NF-κB p65. The error bars represent the means ± SD for three individual experiments. Analysis by Student’s t test indicated that the differences were statistically significant (*, P < 0.05).
Figure 4.
The iacP mutant induced the nuclear translocation of NF-κB in BMDM.
(A) The expression levels of NF-κB p65 in the cytoplasmic and nuclear fractions were determined using immunoblot analysis with an anti-NF-κB p65 antibody. The data were normalized relative to Lamin B (nuclear protein) or GAPDH (cytoplasmic protein) as a protein loading control (Right panel). The expression levels of p65 in the nuclear and cytoplasmic fractions were quantified by densitometry analysis. Each band in blots was densitometrically normalized to GAPDH and Lamin B, and the levels of p65 in cytosolic and nuclear fractions are expressed as the mean ± SD from three separate experiments. (B) Nuclear localization of NF-κB p65 was detected by immunofluorescence staining in primary cultures of BMDMs. DNA in the cell nucleus was visualized by DAPI staining, and p65 was stained with a FITC-conjugated anti-p65 antibody (Right panel). NF-κB p65 nuclear activation was quantified by densitometry analysis. The data shown are representative of at least three independent experiments. The error bars correspond to the means ± SD. *, P < 0.05 (statistically significant difference from the control group).
Figure 5.
The iacP mutant enhanced cytokine-induced nuclear translocation of NF-κB in BMDM.
The mRNA expression levels for the proinflammatory genes IL-18 and IL-1β were determined by reverse transcription-PCR (RT-PCR) experiment (A) and quantitative real-time PCR (qPCR) analysis (B). Band intensities of RT-PCR were quantified by densitometry and normalized to the level of the GAPDH. For the qPCR data, the expression values were normalized to the GAPDH level and reported relative to the expression level in the wild-type strain. The experiments were performed on three individual samples, and the representative data are shown. The error bars indicated the means ± SD. Analysis by Student’s t test indicated that the differences were statistically significant (*, P < 0.05). (C) BMDM infected with S. Typhimurium strains were analyzed at the protein level by Western blot analysis. The expression of IL-18 as a proinflammatory gene was determined by immunoblot analysis with anti-IL-18 antibody in the cytoplasmic fraction. An anti-GAPDH antibody was used as a loading control for cytoplasmic proteins. Representative data from three independent experiments are shown.
Figure 6.
Construction and characterization of attenuated S. Typhimurium strain containing FliC and FljB.
(A) The FljB levels of recombinant attenuated S. typhimurium fljB+ fliC+ strain that contains two different flagellin genes, fljB and fliC, in the pellet and culture supernatant were determined by Western blotting using anti-FljB and anti-FliC antibodies. An anti-DnaK antibody was used as a control for cytoplasmic proteins. (B) The growth curves of the recombinant bacteria strains in LB broth containing 0.3 M NaCl at 37°C. Data are means for three independent experiments. (C) To determine the invasion rate, recombinant bacteria strains were allowed to infect INT-407 epithelial cells for 45 min, after which a gentamicin protection assay was conducted. The number of infected bacteria was confirmed by plating the serial dilutions, and the bacterial invasion rates were calculated as a percentage of wild-type, which was set as 100%. All of the experiments were performed at least three times, and representative data are shown. Error bars correspond to the means ± SD.
Figure 7.
Immune responses to LPS and flagellin in mice orally inoculated with the FljB adjuvant strain.
(A, B) IgG activity analysis of sera from S. Typhimurium-immunized mice at weeks 0, 2 and 4. The mice were immunized orally with 109 CFU of BRD509, BRD fljB+ fliC+ and boosted with BRD fljB+ fliC+. A group inoculated with PBS was used as a control. The data shown are the values for all mice in each group, and the error bars indicate the SD. *, P < 0.05 (statistically significant difference from the control group). (C) Photographic images of isolated splenic tissue from the surviving mice after the challenge.