Fig 1.
Effect of pyrogallol on Vibrio vulnificus-induced cytotoxicity in HeLa cells.
(A) HeLa cells pretreated with pyrogallol (50 μg/mL) for 1 h were infected with V. vulnificus at an MOI of 20 for 2 h. The cells were then stained with Trypan blue and the images were acquired using a microscope with a digital camera. (B) HeLa cells pretreated with pyrogallol (10–50 μg/mL) for 1 h were infected with V. vulnificus at an MOI of 20 for 2 h. LDH released into the supernatant was assayed as a cytotoxicity marker. (C) HeLa cells were cultured in a 96-well microplate with or without pyrogallol for 24 h and then incubated with MTS at 37°C for 4 h. All values are expressed as the means ± SEM (**p < 0.01; ***p < 0.001).
Fig 2.
Effects of pyrogallol on Vibrio vulnificus growth in HI broth.
A V. vulnificus suspension cultured overnight in HI broth was diluted in 1,000-fold into a 96-well microplate with or without pyrogallol and cultured in a 37°C incubator for 6 h. Bacterial growth was determined by measuring the absorbance at 600 nm using a microplate reader. The data represent the means ± SEM of three experiments (**p < 0.01; ***p < 0.001).
Fig 3.
Effect of pyrogallol and catalase on the growth of wild-type (wt) Vibrio vulnificus and the katG− mutant strain.
V. vulnificus wt (A) and the katG− mutant (B) cultured overnight in HI broth were diluted 1,000-fold into fresh HI broth with or without pyrogallol (50 μg/mL) or catalase (10 μg/mL). Bacterial growth was determined by measuring the absorbance at 600 nm using a Biophotometer. (C) V. vulnificus strains were cultured in HI broth with or without pyrogallol or catalase for 24 h and the culture suspensions were then 10-fold serially diluted with PBS. The serial dilutions (10 μL) were then loaded on HI agar plates and incubated at 37°C overnight (pyr: pyrogallol, cat: catalase).
Fig 4.
Effect of hydrogen peroxide on Vibrio vulnificus growth and HeLa cell cytotoxicity.
(A) HeLa cells were treated with H2O2 (0.25–1 mM) in DMEM for 150 min. (B) HeLa cells pretreated with H2O2 (0.25–1 mM) for 1 h were infected with V. vulnificus strains at an MOI of 20 for 150 min. LDH released into the supernatant was assayed as a cytotoxicity marker. All values are expressed as the means ± SEM (*p < 0.05; **p < 0.01; ***p < 0.001).
Table 1.
Effect of hydrogen peroxide (H2O2) on V. vulnificus growth.
Fig 5.
Effect of pyrogallol on the expression of KatG and RtxA1 proteins in Vibrio vulnificus.
(A) V. vulnificus wt or katG− mutant strains were cultured in HI broth with pyrogallol (50 μg/mL) or catalase (10 μg/mL) in a 37°C shaking incubator for 6 h. The bacterial pellets (2 × 108 CFU) were used for KatG protein detection by Western blot analysis. (B) Western blot analysis of KatG in V. vulnificus bacterial pellets (2 × 108 CFU) cultured for 6 h or 24 h. (C) Western blot analysis of RtxA1 toxin in the supernatants of V. vulnificus cultured in HI broth for 6 h (pyr: pyrogallol, cat: catalase). (C) V. vulnificus strains were cultured in HI broth for 6 h. The proteins in culture supernatants (300μl) were precipitated using cold acetone and RtxA1 protein was detected by Western blotting.