Fig. 6

Effect of tryptophol acetate on V. cholerae toxin secretion. a Cholerae Toxin B (CTB) level expression analyzed by anti-CTB western blotting. V. cholerae bacterial cells (VC1 WT strain) were cultured for 16 h at 30 ^oC without tryptophol acetate, and in the presence of 100 μM tryptophol acetate. The treated and untreated bacterial cultures were used to isolate the CTB (see “Methods” section). The samples were immunoblotted for CTB protein levels. Beta-actin used as loading control. b V. cholerae toxin production upon adding different concentrations of tryptophol acetate to the V. cholerae growing medium quantified with ELISA assay for CTX using GM-1 (the CTX receptor). The Y axis corresponds to percentage of CTX production in the growing medium. Error bars indicate standard deviations of four independent cultures. *p < 0.0001, versus the untreated bacteria calculated by ANOVA followed by Tukey’s post hoc analysis. c Confocal fluorescence microscopy images of HeLa cells grown for 16 h and exposed to extraction from VC1 WT cell lysate in the absence (i) and presence (ii) of 100 μM tryptophol acetate for 2 h. Cells were fixed and labeled with monoclonal anti-cholerae toxin, subunit B antibodies (red-excitations were at 633; emission 681 nm) and imaged. Nuclei were visualized upon co-staining of the cells with Hoechst 33342 (blue-excitations were at 405 nm; emission 445 nm). Individual channels and merged confocal images are shown. Scale bar: 50 μm. d Confocal fluorescence microscopy images showing the effects of CT extracted from VC1 WT cell lysate; cells were grown in the absence and presence of 100 μM tryptophol acetate. The HeLa cells were treated with CT for 16 h at 37 ^oC and CO₂ conditions. Propidium iodide staining (red) indicates dead cells and Syto 9 staining (green) indicates viable cells. Excitations were at 488 nm and 561 nm; emission 490–588 nm and 604–735 nm, respectively. (i) Viability staining of HeLa cells exposed to CT. (ii) Viability staining of HeLa cells exposed to CT under treatment of 100 μM tryptophol acetate. Scale bar: 100 μm