The spore-forming bacterial pathogen Clostridioides difficile caused ~450,000 diarrheal infections and ~30,000
deaths in 2017, making it the leading cause of healthcare-associated infections in the US. C. difficile thrives in
the dysbiotic gut because an intact resident microflora antagonizes its growth. As C. difficile grows in the
colon, it makes the glucosylating toxins responsible for causing disease pathology and the spores necessary to
transmit disease. Recent work has shown that toxin production promotes C. difficile growth in the gut by inducing
inflammation, which generates host-derived metabolites that C. difficile specifically exploits. For example, toxin-
mediated inflammation stimulates the host to degrade collagen and produce sorbitol. C. difficile then catabolizes
the resulting metabolites, allowing it to grow to higher levels in the gut. Since these metabolites are likely
concentrated close to the gut epithelium where they are produced, proximity to this cell layer may benefit C.
difficile infection. Consistent with this hypothesis, C. difficile chemotaxes towards mucin-derived sugars, and its
utilization of these sugars enhances its growth during infection. The mucus layer also impedes toxin binding to
target cells, so the growth of C. difficile near the epithelial layer may promote more damage to this layer than
luminal bacteria.
While these studies suggest that an epithelium-proximal C. difficile sub-population may play key roles during
infection, C. difficile is primarily thought of as a gut luminal pathogen. For example, the authors of a previous
fluorescent in situ hybridization study directed at localizing C. difficile during murine infection concluded that it
rarely associates with the gut epithelium. However, using novel C. difficile constitutive fluorescent reporter strains
we find that C. difficile frequently grows in close proximity to the gut epithelium, even though most of the
population is luminal. Furthermore, toxin production appears to promote C. difficile growth close to the epithelium.
Combined with our finding that gene expression levels in epithelium-associated, but not luminal, C.
difficile correlates with disease outcome, we hypothesize that toxin gene expression in the epithelium-proximal
sub-population may drive disease prognosis in mice. To test this hypothesis, we are using transcriptional reporter
strains to localize C. difficile and quantify its toxin gene expression at the single-cell level during infection of both
mice and a colonoid-derived monolayer system. By analyzing these reporters in C. difficile mutants that make
varying amounts of toxin or have metabolic defects, we will determine the relationship between epithelium-
proximal growth, host metabolite utilization, and damage. Since our preliminary data further suggest that toxin
gene expression is inversely regulated with sporulation gene expression, we will also test the hypothesis that C.
difficile establishes a “division of labor” during infection between toxin-producing vs. sporulating cells in mutants
with altered distributions of these two processes. Collectively, these analyses will fundamentally advance our
understanding of how C. difficile establishes infection and may identify novel determinants of disease severity.
