TY - JOUR
T1 - Human intestinal enteroids as a model of Clostridioides difficile-induced enteritis
AU - Engevik, Melinda A.
AU - Danhof, Heather A.
AU - Chang-Graham, Alexandra L.
AU - Spinler, Jennifer K.
AU - Engevik, Kristen A.
AU - Herrmann, Beatrice
AU - Endres, Bradley T.
AU - Garey, Kevin W.
AU - Hyser, Joseph M.
AU - Britton, Robert A.
AU - Versalovic, James
N1 - Funding Information:
This work was supported by grants from the National Institute of Diabetes and Digestive and Kidney Diseases (Grant P30-DK-56338 to Texas Medical Center Digestive Disease Center, Gastrointestinal Experimental Model Systems), a Young Investigator Pilot Award (to M. A. Engevik) and NIH P30-DK-56338 (to M. A. Engevik), National Institutes of Health (NIH) Grant F32AI136404 (to H. A. Danhof), NIH Grant F30-DK-112563 (to A. L. Chang-Graham), the BCM Medical Scientist Training Program (to A. L. Chang-Graham), NIH Grant T32-DK-07644 (to K. A. Engevik), Pediatric GI T32-DK-007664, NIH NIAID Grant U01-AI-124290 (to K. W. Garey and R. A. Britton), NIH Grants R01-DK-103759 and R01-AI-123278 (to R. A. Britton), and unrestricted research support from BioGaia AB (Stockholm, Sweden) (to R. A. Britton and J. Versalovic).
Publisher Copyright:
Copyright © 2020 the American Physiological Society
PY - 2020/5/1
Y1 - 2020/5/1
N2 -
Clostridioides difficile is an important nosocomial pathogen that produces toxins to cause life-threatening diarrhea and colitis. Toxins bind to epithelial receptors and promote the collapse of the actin cytoskeleton.
C. difficile toxin activity is commonly studied in cancer-derived and immortalized cell lines. However, the biological relevance of these models is limited. Moreover, no model is available for examining
C. difficile-induced enteritis, an understudied health problem. We hypothesized that human intestinal enteroids (HIEs) express toxin receptors and provide a new model to dissect
C. difficile cytotoxicity in the small intestine. We generated biopsy-derived jejunal HIE and Vero cells, which stably express LifeAct-Ruby, a fluorescent label of F-actin, to monitor actin cytoskeleton rearrangement by live-cell microscopy. Imaging analysis revealed that toxins from pathogenic
C. difficile strains elicited cell rounding in a strain-dependent manner, and HIEs were tenfold more sensitive to toxin A (TcdA) than toxin B (TcdB). By quantitative PCR, we paradoxically found that HIEs expressed greater quantities of toxin receptor mRNA and yet exhibited decreased sensitivity to toxins when compared with traditionally used cell lines. We reasoned that these differences may be explained by components, such as mucins, that are present in HIEs cultures, that are absent in immortalized cell lines. Addition of human-derived mucin 2 (MUC2) to Vero cells delayed cell rounding, indicating that mucus serves as a barrier to toxin-receptor binding. This work highlights that investigation of
C. difficile infection in that HIEs can provide important insights into the intricate interactions between toxins and the human intestinal epithelium.
NEW & NOTEWORTHY In this article, we developed a novel model of
Clostridioides difficile-induced enteritis using jejunal-derived human intestinal enteroids (HIEs) transduced with fluorescently tagged F-actin. Using live-imaging, we identified that jejunal HIEs express high levels of TcdA and CDT receptors, are more sensitive to TcdA than TcdB, and secrete mucus, which delays toxin-epithelial interactions. This work also optimizes optically clear
C. difficile-conditioned media suitable for live-cell imaging.
AB -
Clostridioides difficile is an important nosocomial pathogen that produces toxins to cause life-threatening diarrhea and colitis. Toxins bind to epithelial receptors and promote the collapse of the actin cytoskeleton.
C. difficile toxin activity is commonly studied in cancer-derived and immortalized cell lines. However, the biological relevance of these models is limited. Moreover, no model is available for examining
C. difficile-induced enteritis, an understudied health problem. We hypothesized that human intestinal enteroids (HIEs) express toxin receptors and provide a new model to dissect
C. difficile cytotoxicity in the small intestine. We generated biopsy-derived jejunal HIE and Vero cells, which stably express LifeAct-Ruby, a fluorescent label of F-actin, to monitor actin cytoskeleton rearrangement by live-cell microscopy. Imaging analysis revealed that toxins from pathogenic
C. difficile strains elicited cell rounding in a strain-dependent manner, and HIEs were tenfold more sensitive to toxin A (TcdA) than toxin B (TcdB). By quantitative PCR, we paradoxically found that HIEs expressed greater quantities of toxin receptor mRNA and yet exhibited decreased sensitivity to toxins when compared with traditionally used cell lines. We reasoned that these differences may be explained by components, such as mucins, that are present in HIEs cultures, that are absent in immortalized cell lines. Addition of human-derived mucin 2 (MUC2) to Vero cells delayed cell rounding, indicating that mucus serves as a barrier to toxin-receptor binding. This work highlights that investigation of
C. difficile infection in that HIEs can provide important insights into the intricate interactions between toxins and the human intestinal epithelium.
NEW & NOTEWORTHY In this article, we developed a novel model of
Clostridioides difficile-induced enteritis using jejunal-derived human intestinal enteroids (HIEs) transduced with fluorescently tagged F-actin. Using live-imaging, we identified that jejunal HIEs express high levels of TcdA and CDT receptors, are more sensitive to TcdA than TcdB, and secrete mucus, which delays toxin-epithelial interactions. This work also optimizes optically clear
C. difficile-conditioned media suitable for live-cell imaging.
KW - ADP Ribose Transferases/metabolism
KW - Actin Cytoskeleton/metabolism
KW - Animals
KW - Bacterial Proteins/metabolism
KW - Bacterial Toxins/metabolism
KW - Cell Shape
KW - Chlorocebus aethiops
KW - Clostridioides difficile/metabolism
KW - Clostridium Infections/metabolism
KW - Enteritis/metabolism
KW - Enterotoxins/metabolism
KW - HeLa Cells
KW - Host-Pathogen Interactions
KW - Humans
KW - Jejunum/metabolism
KW - Mucin-2/metabolism
KW - Organoids
KW - Receptors, Cell Surface/genetics
KW - Time Factors
KW - Vero Cells
KW - Virulence
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UR - http://www.scopus.com/inward/citedby.url?scp=85083545015&partnerID=8YFLogxK
U2 - 10.1152/ajpgi.00045.2020
DO - 10.1152/ajpgi.00045.2020
M3 - Article
C2 - 32223302
AN - SCOPUS:85083545015
SN - 0193-1857
VL - 318
SP - G870-G888
JO - American Journal of Physiology - Gastrointestinal and Liver Physiology
JF - American Journal of Physiology - Gastrointestinal and Liver Physiology
IS - 5
ER -