Establishing Human Ileal Enteroids as an Alternative Model to Study Vibrio cholerae Pathogenesis.
Hahm, Mary.
2019
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Cholera is the
secretory diarrheal disease caused by the bacterium Vibrio cholerae. Current laboratory
models for studying V. cholerae infection include the infant mouse, the infant rabbit,
and Caco-2 cell culture models. While these existing models are useful, they all have
their limitations and prevent us from fully understanding the pathogenesis of V.
cholerae. For example, infant mice do ... read morenot experience watery diarrheal symptoms as humans
do. Caco-2 cells are derived from colon adenocarcinoma, but V. cholerae is known to
infect the small intestine. Thus, there is a need for an alternative model to study V.
cholerae in a system that is more physiologically relevant to human infection. Enteroids
are a promising cell culture technology to study host-pathogen interactions and have
been successfully used to study several other gastrointestinal pathogens. Enteroids are
derived from isolated crypts of the human small intestine and represent major
differentiated cell types. Here, I use ileal enteroids seeded on 2D transwells to mimic
the polarized epithelial monolayers of the small intestine as a novel system for
studying early association and infection dynamics of V. cholerae. Wild-type V. cholerae
associates and replicates on differentiated enteroid monolayers. Importantly, the
association appears to be specific. A well-characterized mutant strain lacking the
master quorum-sensing regulator LuxO, which is severely deficient for colonization in
the infant mouse model, is also deficient for association and replication with the
enteroid monolayers. Confocal immunofluorescence microscopy was used to quantify and
localize the associated bacteria. Wild-type V. cholerae associates with monolayers as
individual bacteria and small clusters resembling microcolonies, which have been
observed inside animal hosts. Heterogeneity is observed in the infecting population.
Previously reported cholera toxin-induced damage to tight junctions was not observed
post-infection with V. cholerae at early time points. In 3D enteroids, cholera toxin
induces enteroid swelling, likely via chloride ion movement through the CFTR channel,
and dramatically induces Mucin-2 levels. These results suggest that 2D and 3D enteroid
systems can be used to help fill gaps in knowledge about early infection dynamics and
human small intestinal epithelial cell damage induced by V.
cholerae.
Thesis (M.S.)--Tufts University, 2019.
Submitted to the Dept. of Molecular Microbiology.
Advisor: Wai-Leung Ng.
Committee: Carol Kumamoto, Aimee Shen, and Shumin Tan.
Keyword: Microbiology.read less - ID:
- qz20t592r
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