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. 2015 Jan;83(1):138-45.
doi: 10.1128/IAI.02561-14. Epub 2014 Oct 13.

Persistence and Toxin Production by Clostridium Difficile Within Human Intestinal Organoids Result in Disruption of Epithelial Paracellular Barrier Function

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Free PMC article

Persistence and Toxin Production by Clostridium Difficile Within Human Intestinal Organoids Result in Disruption of Epithelial Paracellular Barrier Function

Jhansi L Leslie et al. Infect Immun. .
Free PMC article

Abstract

Clostridium difficile is the leading cause of infectious nosocomial diarrhea. The pathogenesis of C. difficile infection (CDI) results from the interactions between the pathogen, intestinal epithelium, host immune system, and gastrointestinal microbiota. Previous studies of the host-pathogen interaction in CDI have utilized either simple cell monolayers or in vivo models. While much has been learned by utilizing these approaches, little is known about the direct interaction of the bacterium with a complex host epithelium. Here, we asked if human intestinal organoids (HIOs), which are derived from pluripotent stem cells and demonstrate small intestinal morphology and physiology, could be used to study the pathogenesis of the obligate anaerobe C. difficile. Vegetative C. difficile, microinjected into the lumen of HIOs, persisted in a viable state for up to 12 h. Upon colonization with C. difficile VPI 10463, the HIO epithelium is markedly disrupted, resulting in the loss of paracellular barrier function. Since similar effects were not observed when HIOs were colonized with the nontoxigenic C. difficile strain F200, we directly tested the role of toxin using TcdA and TcdB purified from VPI 10463. We show that the injection of TcdA replicates the disruption of the epithelial barrier function and structure observed in HIOs colonized with viable C. difficile.

Figures

FIG 1
FIG 1
Assessment of HIO epithelial barrier function. (A) Representative images of FD4 dynamics in HIOs following injection. HIOs (n = 5 per treatment) were injected with FD4 and imaged at 0, 2, 10, 12, 14, 16, and 18 h postinjection. HIOs retained the majority of injected FD4 within the lumen over 18 h. The addition of EGTA to the media 12 h after injection resulted in the rapid loss of FD4 from the lumen, indicating the loss of epithelial paracellular barrier function. Images represent the results from at least three independent experiments. (B) Quantitation of barrier disruption by determination of the fraction of initial FD4 fluorescence retained over time. Points represent the medians and bars represent the interquartile ranges. Eighteen hours after injection, control HIOs retained significantly more FD4 than the EGTA-treated HIOs (P = 0.0079 by Mann-Whitney test).
FIG 2
FIG 2
Vegetative C. difficile persists in the HIO lumen. (A) HIOs injected with either C. difficile strain VPI 10463 or F200 were collected at 0, 2, and 12 h postinjection and plated on BHI with cysteine to quantitate the number of vegetative CFU per HIO. C. difficile was able to persist in the lumen of HIOs for 12 h. Points on the graph represent individual HIOs, and the dashed line represents the limit of detection. Bars indicate the means and SEM. (B) Twelve hours after colonization, HIOs injected with C. difficile were fixed and stained using hematoxylin and eosin. The epithelium of the HIO colonized with strain VPI 10463 is severely disrupted, and large rods with what appear to be subterminal spores are visible (inset) within the HIO (inset is a 3.25× digital zoom of the boxed region). (C) An HIO colonized with the nontoxigenic strain F200 has an intact epithelium despite the presence of large rod-shaped bacteria in the lumen of the HIO.
FIG 3
FIG 3
Injection of toxigenic but not nontoxigenic C. difficile results in loss of HIO barrier function. (A) HIOs were injected with FD4 alone (control) (n = 5), a nontoxigenic strain (F200) of C. difficile (n = 5), filtered culture supernatant from that strain (n = 6), a toxigenic C. difficile strain (VPI 10463) (n = 5), or filtered culture supernatant from the toxigenic strain (n = 5). Only VPI 10463-injected HIOs lost barrier function. Images are representative of three independent experiments using 5 to 6 HIOs per group. (B) Quantification of epithelial barrier disruption by measuring retention of injected FD4 after 12 h. Bars represent the medians and the interquartile ranges. Only the HIOs colonized with the toxigenic strain (VPI 10463) lost a significantly greater amount of FD4 from the lumen, indicating significant epithelial damage (P = 0.0079 by Mann-Whitney test).
FIG 4
FIG 4
Purified TcdA and TcdB injected into the lumen of HIOs disrupt paracellular barrier function. (A) Representative images of FD4 leakage from the lumen of HIOs treated with C. difficile toxin. HIOs (n = 5 per treatment) were injected with FD4 alone (top), with purified TcdA (middle), or with TcdB (bottom). In this system, TcdA is more potent than TcdB. Images are representative of at least three independent experiments using 5 to 6 HIOs per group. (B) Quantitation of fluorescence of each HIO relative to time zero. Injection of purified TcdA into HIOs causes significantly greater loss of paracellular barrier function than injection with either TcdB (blue asterisk, P = 0.0159) or FD4 alone (red asterisks, P = 0.0079). Points represent the median percentages, and bars represent the interquartile ranges. The data were analyzed using the Mann-Whitney test. Control data presented in panels A and B are the same as those used in Fig. 1, as these assays were preformed at the same time.
FIG 5
FIG 5
Cellular effects of injection of HIOs with TcdA or TcdB. HIOs were injected with FD4 alone (control), TcdA, or TcdB and monitored for disruption of barrier function. At 18 h postinjection, the HIOs were fixed and stained. (A) The normal basolateral distribution of the adherens junction protein E-cadherin (ECAD) is disrupted following injection of TcdA. In these HIOs, ECAD is redistributed and can be seen on the apical surface of the epithelium. HIOs injected with TcdB maintain a basolateral distribution of ECAD similar to that of the controls. (B) Altered localization of TJ proteins ZO-1 and OCLN following injection with TcdA. In control HIOs, ZO-1 is present at the apical surface of the epithelium, whereas OCLN is seen at the lateral surface of the cell. In HIOs injected with TcdA, apical ZO-1 at TJs is lost and OCLN no longer is restricted to the lateral surface, while TcdB-injected HIOs have ZO-1 and OCLN immunofluorescence similar to those of the control. DAPI, 4′,6-diamidino-2-phenylindole. (C) HIOs were stained with phalloidin to assess the organization of F-actin and acetylated alpha tubulin (AcTub) to visualize stabilized microtubules. In control HIOs, F-actin is strongly localized to the apical surface of the epithelium, while AcTub immunofluorescence is strongest on the apical and basal sides of the cell. Compared to control HIOs injected with TcdA, epithelial cells displayed a reduction of F-actin staining, with areas where staining was undetectable, and showed a severe disruption of AcTub at the apical border. In contrast, in HIOs injected with TcdB, phalloidin staining was similar to that of the controls and AcTub immunofluorescence was mostly similar to that of the controls, with a mild reduction of immunofluorescence on the basal side of the cells.
FIG 6
FIG 6
Basolateral exposure to purified toxins causes loss of barrier function. Purified TcdA or TcdB was added to tissue culture media containing HIOs, followed by the addition of FD4. In this assay, the loss of paracellular barrier function was indicated by diffusion of FD4 into the lumen of HIOs, while HIOs with an intact epithelium excluded FD4. Bright-field images show the location of the HIOs. Images taken under fluorescent light indicate barrier function status. None (0/10) of the untreated HIOs lost barrier function, whereas 100% (13/13) of HIOs treated with TcdA lost barrier function, while only 23% of HIOs treated with TcdB lost barrier function. The results shown are representative of five independent experiments.

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