Fig 1.
Transcriptional analyses of structural flp genes in three tad operons of V. vulnificus.
RNA was isolated from bacteria grown in the rat peritoneal cavity (IV, in vivo) or in 2.5% NaCl HI broth (IT, in vitro) and the transcript levels of the structural flp genes were analyzed via real-time (A) and conventional RT-PCR (B). RNA was isolated from bacteria grown under 2.5% NaCl HI broth, iron-limited, solid surface and in vivo conditions and the transcript levels of the structural flp genes were analyzed via real-time (C) and conventional RT-PCR (D). DP (80 μM) was added to 2.5% NaCl HI broth for iron limitation. The real-time RT-PCR data were normalized to gyrA and expression relative to the in vitro level. Data shown represent the mean ± SEM of three independent experiments performed in triplicate. Statistical analysis was carried out using Student’s t test (*, P < 0.05; ***, P < 0.001).
Table 1.
Effect of the mutation of tad operons on the lethality for mice.
Fig 2.
Significantly decreased adhesion to host cells by the Δtad123 mutant (A) and time-dependent recovery (B).
HeLa cells were treated with log-phase V. vulnificus cells at an MOI of 250 bacteria in the presence of 2 μg/ml tetracycline, and the bacterial cells that adhered to HeLa cells were counted at appropriate time points. The morphology of the infected HeLa cells was observed after Giemsa staining at ×1,000 magnification. Data shown represent the mean ± SEM of five independent experiments performed with six (A) or seventeen to forty-four replicates (B). WT (pLAFR3), wild type harboring pLAFR3; Δtad123 (pLAFR3), Δtad123 mutant harboring pLAFR3; Δtad123 (pLAFR3::tad1), Δtad123 mutant in trans complemented with pLAFR3::tad1 locus; Δtad123 (pLAFR3::tad2), Δtad123 mutant in trans complemented with pLAFR3::tad2 locus); Δtad123 (pLAFR3::tad3), Δtad123 mutant in trans complemented with pLAFR3::tad3 locus. Statistical analysis was carried out using Student’s t test (**, P < 0.01).
Fig 3.
Ultrastructural observation of V. vulnificus.
Bacteria were grown in vivo using a rat peritoneal infection model. The specimens were examined by SEM.
Fig 4.
(A) Dot blot analysis. Bacteria were grown on a 2.5% NaCl HI-Amp agar plate supplemented with (induced) or without (non-induced) 0.1% arabinose for 4 h. The Flp-V5 fusion protein was detected using an anti-V5 polyclonal antibody. Δtad-FlpV5 indicates the Δtad123 mutant strain carrying pBAD24 expressing the Flp-V5 fusion protein, and WT-FlpV5 indicates the wild-type strain carrying pBAD24 expressing the Flp-V5 fusion protein. (B) Immunogold labeling and TEM analysis. Bacteria were fixed and incubated with an anti-V5 polyclonal antibody and a 5-nm colloidal gold conjugated goat anti-rabbit IgG secondary antibody. Only V. vulnificus cells expressing the Flp-V5 fusion protein showed positivity for immunogold particles on the cell surface. (C) A quantification of immunogold particles. The number of immunogold particles were determined from the representative 5 bacterial cells per group under x10,000 magnification. Data shown represent the mean ± SEM. Statistical analysis was carried out Student’s t test. *, P < 0.05; **, P < 0.01. (D) Immunofluorescent detection of Flp-V5. Bacterial cells were visualized by staining their DNA with DAPI (blue), and anti-V5 detection appears in red (white arrowheads).
Fig 5.
Delayed RtxA1 secretion (A) and cytotoxicity (B) by the Δtad123 mutant cells.
(A) For RtxA1 detection, log-phase V. vulnificus cells were incubated with HeLa cells in 6-well plates at an MOI of 100 bacteria in the presence of 2 μg/ml tetracycline for 35 or 45 min. The cells in each well were lysed with lysis buffer, followed by concentration using the Amicon Ultra-0.5 centrifugal filter apparatus. The RtxA1 toxin was detected by Western blot analysis using an anti-GD domain antibody (RtxA1-C, a band of approximately 130 kDa). (B) Effect of Δtad123 mutations on cytotoxicity against HeLa cells. (C) Restoration of cytotoxicity in tad-complemented strains in the presence of antibiotics. Data shown represent the mean ± SEM of three independent experiments performed with five replicates. WT (pLAFR3), wild type harboring pLAFR3; Δtad123 (pLAFR3), Δtad123 mutant harboring pLAFR3; Δtad123 (pLAFR3::tad1), Δtad123 mutant in trans complemented with pLAFR3::tad1 locus; Δtad123 (pLAFR3::tad2), Δtad123 mutant in trans complemented with pLAFR3::tad2 locus); Δtad123 (pLAFR3::tad3), Δtad123 mutant in trans complemented with pLAFR3::tad3 locus. Statistical analysis was carried out Student’s t test. **, P < 0.01; ***, P < 0.001; ns, not significant.
Fig 6.
Decreased biofilm formation in the Δtad123 mutant cells.
(A) For biofilm formation, log-phase V. vulnificus cells (5x105 CFU/ml) was applied into each well of 24 well plate. The plates were further incubated at 37°C for 24 hours. After gentle washing with PBS, the wells were stained with 200 μl of 0.3% crystal violet for 15 min and gently washed with PBS. Data shown represent the mean ± SEM of three independent experiments performed with six replicates. The stained biofilm was extracted with 100% ethanol and diluted (two-fold) with PBS to measure the absorbance at 595 nm. (B) Confocal microscopic observation of the acridine orange-stained biofilm. WT (pLAFR3), wild type harboring pLAFR3; Δtad123 (pLAFR3), Δtad123 mutant harboring pLAFR3; Δtad123 (pLAFR3::tad1), Δtad123 mutant in trans complemented with pLAFR3::tad1 locus; Δtad123 (pLAFR3::tad2), Δtad123 mutant in trans complemented with pLAFR3::tad2 locus); Δtad123 (pLAFR3::tad3), Δtad123 mutant in trans complemented with pLAFR3::tad3 locus. Statistical analysis was carried out using Student’s t test. ***, P < 0.001. WT, wild-type V. vulnificus.
Fig 7.
Effect of the triple tad operon mutation on in vivo and in vitro invasion.
(A) In vivo invasion. Log-phase bacteria were inoculated into ligated ileal loops of mice. Blood samples were acquired from infected mice by cardiac puncture at the indicated times. Viable cells were counted by plating on 2.5% NaCl HI agar plates. Data shown represent the mean ± SEM of three independent experiments performed in five mice. (B) In vitro invasion. HCA-7 cells grown on Transwell filters were apically exposed to log-phase bacteria. Invasiveness was determined by measuring the number of bacterial cells that translocated from the apical to the basolateral compartment of the Transwells. WT (pLAFR3), wild type harboring pLAFR3; Δtad123 (pLAFR3), Δtad123 mutant harboring pLAFR3; Δtad123 (pLAFR3::tad1), Δtad123 mutant in trans complemented with pLAFR3::tad1 locus; Δtad123 (pLAFR3::tad2), Δtad123 mutant in trans complemented with pLAFR3::tad2 locus); Δtad123 (pLAFR3::tad3), Δtad123 mutant in trans complemented with pLAFR3::tad3 locus. Data shown represent the mean ± SEM of three independent experiments performed with triplicates. (C) Restoration of in vitro invasion in tad-complemented strains at 6 h post-incubation in the presence of 2 μg/ml tetracycline. Viable bacterial cells were counted by plating on 2.5% NaCl HI agar plates. Data shown represent the mean ± SEM of three independent experiments performed with triplicates. Statistical analysis was carried out using Student’s t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ND, not detected (detection limit; 1x103 CFU/mL).
Fig 8.
Recovery of V. vulnificus from blood after intravenous or intraperitoneal mouse infection.
Each mouse was intravenously (A) or intraperitoneally (B) injected with bacteria that had been previously incubated in the rat peritoneal cavity for 6 hours for in vivo adaptation. Blood samples were acquired from infected mice via cardiac puncture at the indicated times. Viable bacterial cells were counted by plating on 2.5% NaCl HI agar plates. Very low numbers of Δtad123 mutant cells were recovered from the blood of infected mice. Data shown represent the mean ± SEM of three independent experiments performed in seven mice. Statistical analysis was carried out using Student’s t test (*, P < 0.05; **, P < 0.01; ***, P < 0.001).
Fig 9.
Survival of V. vulnificus against human serum bactericidal killing.
(A) Log-phase bacteria grown in the presence of 2 μg/ml tetracycline were washed with PBS and then incubated with different NHS concentrations at 37 °C for 2 h and the numbers of viable cells were determined. (B) Log-phase bacteria grown in the presence of 2 μg/ml tetracycline were washed with PBS were incubated with 60% NHS and the numbers of viable cell were determined in a time-dependent manner. (C) Log-phase bacteria grown in the presence of 2 μg/ml tetracycline were washed with PBS were incubated with HIS, undiluted NHS, EGTA/Mg2+-treated NHS, MBL-depleted NHS or bentonite-treated NHS for 2 h. Viable bacterial cells were counted by plating on 2.5% NaCl HI agar plates. Bacteria were incubated in PBS as a control. WT (pLAFR3), wild type harboring pLAFR3; Δtad123 (pLAFR3), Δtad123 mutant harboring pLAFR3; Δtad123 (pLAFR3::tad1), Δtad123 mutant in trans complemented with pLAFR3::tad1 locus; Δtad123 (pLAFR3::tad2), Δtad123 mutant in trans complemented with pLAFR3::tad2 locus); Δtad123 (pLAFR3::tad3), Δtad123 mutant in trans complemented with pLAFR3::tad3 locus. Data shown represent the mean ± SEM of four independent experiments performed in triplicates. Statistical analysis was carried out using Student’s t test (C). ***, P < 0.001. ns, non-significant (detection limit; 1x103CFU/mL). (D) Viable V. vulnificus cells in bloodstream of anti-C5 monoclonal antibody pretreated mice. Groups of mice were administered with 40mg/kg mouse anti-C5 two times as described Materials and Methods and then the mice were intraperitoneally infected with 5x105 wild type or Δtad123 mutant cells previously incubated in the rat peritoneal cavity for 6 hours for in vivo adaptation. Blood samples were acquired by eye bleeding. Viable bacterial cells were counted by plating on 2.5% NaCl HI agar plates. Data are shown as the mean ± SEM (n = 5). Statistical analysis was done using Student’s t test (*, P < 0.05; **, P < 0.01; ***, P < 0.001). IC, isotype control.
Table 2.
Strains and plasmids used in this study.