Figure 3. Typhoid toxin and TtsA localize to the bacterial poles.

(a and b) Typhoid toxin and TtsA localize to the bacterial poles after in vitro growth or cell infection. S. Typhi strains carrying chromosomally-encoded FLAG-tagged CdtB (as a surrogate for typhoid toxin) or TtsA were grown for 24 hours in TTIM, fixed, and stained with a mouse antibody directed to the FLAG-epitope (green) (to visualize CdtB or TtsA, as indicated) and a rabbit antibody directed to S. Typhi LPS (red) (a). Alternatively, the same S. Typhi strains were grown in LB (non inducing media), applied to cultured Henle-407 human epithelial cells, and 24 hours post infection the infected cells were stained with an antibody directed to the FLAG-epitope (green) to visualize CdtB or TtsA (as indicated), a rabbit antibody directed to S. Typhi LPS (red), and DAPI for DNA detection (blue) (b) (scale bar: 5 μm). The scatter blot diagrams of each panel show fluorescence signal intensities for the corresponding FLAG-tagged protein distributed along the axes of individual bacterial cells. Twenty six measuring points were defined from the center (0) to each of the poles (1.0) of the bacterial cells and the distribution of the fluorescence intensity along the axes of bacteria were analyzed with the MicrobeJ plug-in of ImageJ (https://imagej.nih.gov/ij). The black line depicts the average of the intensities measured at each of the 26 measuring points. Data in each panel are from 1,800 individual measurements at each of the measuring points from 900 bacteria analyzed in opposite directions from the center. The bar graphs show the quantification of the signal intensities at the measuring point furthest from the center (1.0), and at the center of each bacterium (0) for each condition. Numbers represent the mean ± SEM from 1,800 measurements (**** p < 0.0001, two-sided Student’s t Test) (Supplementary data set 3).