Fig. 4. (A–C) HD5 potentiates destruction of the epithelium by Shigella.

(A) Trans-epithelial electrical resistance (TEER) analysis of epithelial integrity of the monolayer of polarized Caco-2 cells infected with Sf301 in either the absence or presence of HD5 (8 μM). Percent TEER values were normalized against values of each treatment group at time 0. Data are mean ± SD from at least three independent experiments. Statistical significance was determined using a two-way ANOVA, ^**, p < 0.01; ^***, p < 0.001. (B) Intracellular CFU of the polarized Caco-2 monolayer 6 h post-inoculation of Sf301 in either the absence or presence of HD5 (8 μM). Data are mean ± SD from at least three independent experiments. Statistical significance was determined using a t test ^****, p < 0.0001. (C) Disruption of the tight junction (as shown by immunofluorescence) and impairment of the integrity (as shown by SEM) of the epithelium. Polarized Caco-2 cells grown in transwell inserts were infected with Sf301 in either the absence or presence of HD5 (8 μM) for 6 h, followed by immunostaining with antibody against the tight junction marker ZO-1 (green). Nuclei are stained with DAPI (blue). The scale bars represent 50 μm. Invading bacteria in the polarized Caco-2 monolayer are indicated by red arrows in the SEM image. The scale bars represent 2 μm. (D, E) Effects of concentrated ileal fluid aspirates on Shigella Sf301 adhesion (D) and invasion (E) in comparison with DMEM either without or with HD5 (4 μM). Anti-HD5 antiserum was added to ileal fluids and HD5-containing DMEM at a dilution titer of 1:100 to examine its neutralizing activity against Shigella infection promoted by endogenous HD5. Data are shown as mean ± SD of at least three independent experiments. Statistical significance between indicated groups was determined using a one-way ANOVA (Tukey’s multiple comparison test), and p values are as follows: ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, and ^****p < 0.0001.