Article Figures & Data
Figures
- FIG 1
Apical versus basolateral invasion of enteroid monolayers. (A) Schematic representation of apical and basolateral routes of infection of enteroid monolayers grown on permeable transwell inserts. (B) Differentiated human enteroid monolayers (derived from colon) were infected either apically (Ap) or basolaterally (BL) with ∼4 × 107 CFU of S. flexneri WT strain 2457T or plasmidless strain 4243A for 2 h. After gentamicin treatment to remove extracellular bacteria, monolayers were lysed to enumerate intracellular bacteria. The data presented are pooled from three independent experiments. Each dot represents data collected from an individual monolayer (n = 7 for all groups except for apical infection with the 4243A strain, where n = 4). Error bars indicate standard deviations from the means. The asterisks above the bars indicate statistically significant differences calculated using one-way ANOVA. ***, P < 0.0001 with Bonferroni’s multiple-comparison test. (C) Transepithelial electrical resistance (TEER) of enteroid monolayers was measured prior to and after infection. The percent change in TEER is plotted (see Materials and Methods for the formula). The data presented are pooled from two independent experiments. Error bars indicate standard deviations from the means. (D) Enteroid monolayers were infected basolaterally with ∼4 × 107 CFU 2457T::GFP. Intracellular bacteria were visualized using confocal microscopy. (Top) xy projection; (bottom) xz projection. Actin was stained with an Alexa Fluor 555-conjugated phalloidin probe (red), S. flexneri::GFP was visualized by constitutive expression of green fluorescent protein (green), and nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole) (blue).
- FIG 2
Uptake of S. flexneri via M cells. (A) Enteroid monolayers derived from the ileum were differentiated (DF) with or without TNF-α and RANKL in the basolateral medium to induce M cell differentiation. Differentiated monolayers were infected apically with ∼4 × 107 CFU 2457T::GFP for 2 h, followed by gentamicin treatment to remove extracellular bacteria. Immunostaining for the human M cell marker glycoprotein 2 (GP2) confirmed induction of M cells in the enteroid monolayers. (Top) Representative xz plane; (bottom) maximum-intensity projection image. (B) Differentiated enteroid monolayers without or with M cells were infected apically with ∼4 × 107 CFU WT S. flexneri for 2 h, extracellular bacteria were removed by gentamicin treatment, and intracellular CFU were enumerated by lysis of the monolayers and serial dilution plating of monolayer lysates. The data presented are pooled from three independent experiments. Each dot represents data collected from an individual enteroid monolayer (n = 6 in each group). Error bars denote standard deviations from the means. The asterisks above the bars indicate statistically significant differences determined using two-tailed, unpaired Student’s t test. *, P < 0.05.
- FIG 3
Segmental specificity of WT S. flexneri infection. Enteroid monolayers derived from four different segments of the human intestine were infected either apically or basolaterally with ∼4 × 107 CFU WT S. flexneri 2457T for 2 h. Following gentamicin treatment to remove extracellular bacteria, the enteroid monolayers were lysed, and intracellular bacteria were enumerated. Intracellular numbers of Shigella bacteria are shown for enteroids derived from duodenum (A), jejunum (B), ileum (C), and colon (D). The data presented are pooled from 2 independent experiments. Each dot represents data collected from an individual enteroid monolayer (n = 4 in each group). Error bars indicate standard deviations from the means. The asterisks above the bars indicate statistically significant differences determined using two-tailed, unpaired Student’s t test. *, P < 0.05; **, P < 0.005.
- FIG 4
Intracellular replication of WT S. flexneri in enteroid monolayers. Enteroid monolayers derived from four different segments of the human intestine were infected basolaterally with ∼4 × 107 CFU WT S. flexneri 2457T for 2 h. The monolayers were then incubated in medium containing gentamicin to remove extracellular bacteria. Intracellular CFU were enumerated by lysing and plating the monolayers at 2.5, 5.5, 8.5, and 26.5 h postinfection. Intracellular replication is shown for enteroid monolayers derived from duodenum (A), jejunum (B), ileum (C), and colon (D). The data presented are pooled from two independent experiments for enteroids derived from duodenum and ileum. For enteroids derived from jejunum and colon, data shown are from three independent experiments. Each dot represents data collected from an individual enteroid monolayer (n is variable between different time points). Error bars denote standard deviations from the means. The asterisks above the bars indicate statistically significant differences determined using one-way ANOVA with Bonferroni’s multiple-comparison test. *, P < 0.05; ***, P < 0.0001.
- FIG 5
Effect of S. flexneri infection and intracellular replication on interleukin-8 expression. Enteroid monolayers derived from colon and ileum were infected basolaterally with ∼4 × 107 CFU WT S. flexneri 2457T for 2 h. Following infection, the monolayers were incubated in gentamicin-containing medium for 30 min to remove extracellular bacteria. Apical and basolateral supernatants were collected at 2.5, 5.5, 8.5, and 26.5 h postinfection. The mean levels of IL-8 secreted in apical and basolateral supernatants in enteroids derived from colon (A) and ileum (B) were quantified by ELISAs. The data presented are pooled from two independent experiments with at least 2 enteroid monolayers in each experiment. Error bars denote standard deviations from the means. The asterisks above the bars indicate statistically significant differences determined using two-way ANOVA with Bonferroni’s multiple-comparison test. *, P < 0.05; **, P < 0.005; ***, P = 0.0005.
- FIG 6
Effect of S. flexneri infection on mucus expression. Enteroid monolayers derived from the colon were infected either apically or basolaterally with 4 × 107 CFU of WT S. flexneri for 2 h. Following invasion, the enteroid monolayers were incubated in medium containing gentamicin for an additional 4 h to allow intracellular replication of bacteria and to remove extracellular bacteria. Undifferentiated (ND) and differentiated monolayers (both uninfected) were used as controls. Whole-cell lysates of uninfected or infected enteroids were probed for Muc2 by Western blotting. A Western blot with β-actin is shown to confirm equal loading of samples (bottom panel).
Additional Files
Supplemental material
- Supplemental file 1 -
Fig. S1. Tight junction protein ZO-1 immunostaining was visualized in enteroid monolayers differentiated to include M cells using confocal microscopy to confirm integrity of tight junctions.
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- Supplemental file 1 -
