Fig. 4

Effects of tryptophol acetate on Vibrio cholerae quorum sensing and biofilm assembly. a Tryptophol acetate concentration-dependent inhibition of the CAI-1 QS system in V. cholerae MM920. Experiments were performed in triplicate and error bars represent standard deviation of the mean. b Concentration-dependent effect of tryptophol on the CAI-1 QS system in V. cholerae MM920. Experiments were performed in triplicate and error bars represent standard deviation of the mean. c Confocal fluorescence microscopy z-stacks showing V. cholerae biofilms. Excitations were at 488 nm and 561 nm; emission 490–588 nm and 604–735 nm, respectively. V. cholerae viable cells were stained in green while dead cells were stained red with the BacLight® Dead/Live Kit. Sizes of the biofilm images are 500 μm × 500 μm. (i) V. cholerae VC1 (wild-type); (ii) V. cholerae VC1 grown in the presence of 100 μM tryptophol acetate; (iii) V. cholerae MM920 mutant; (iv) V. cholerae MM920 incubated with 900 nM CAI-1; (v) V. cholerae MM920 incubated with both 900 nM CAI-1 and 100 μM tryptophol acetate. The graph (top right) displays the means (±SD) of biofilm volume per area, generated from three independent sets of confocal fluorescence microscopy experiments calculated through the IMARIS software. d V. cholerae biofilm mass analysis at different concentrations of tryptophol acetate (μM) obtained through crystal violet staining (The concentrations in μM are indicated by the different bar colors). Biofilms were stained after 24-hr growth. Error bars indicate the standard deviations of 4 measurements. *p < 0.001, **p < 0.0001, ***p < 0.000001 versus the control calculated by ANOVA followed by Tukey’s post hoc analysis