ClostridiumdifficiletoxinB(TcdB)isamajorvirulentfactorresponsiblefordiseasesassociated with C. difficile infection. Our understanding of TcdB pathogenesis remains limited, owing in large part to a lack of established receptors through which to understand the in vivo targeting anddiseaseprogressionincolonictissues.Werecentlycarriedoutgenome-widescreensusing the CRISPR-Cas9 approach and identified two distinct candidate receptors: Frizzled family proteins(FZDs)andchondroitinsulfateproteoglycan4(CSPG4).Wefurtherdemonstratedthat both FZDs and CSPG4 are functional receptors for TcdB, and each can mediate binding and entryofTcdBindependently.However,howFZDsandCSPG4contributetoTcdBpathogenesis incolonictissuesduringC.difficileinfectionremainstobeestablished.Hereweproposethree aimstoaddressthiskeyknowledgebarrier. First,wefoundthatFZDsandCSPG4aredifferentiallyexpressedincolonictissues,withFZDs mainlyinthecolonicepitheliumandCSPG4largelyexpressedinsub-epithelialmyofibroblasts. We thus propose a novel ?two-receptor, two-stage? hypothesis to explain TcdB pathogenesis: TcdB first targets and enter the colonic epithelium via binding to FZDs. Disruption of the epithelium allows the toxin to gain access to sub-epithelial myofibroblasts, where TcdB utilizes CSPG4 as an alternative receptor.
Aims 1 and 2 will examine this hypothesis, with Aim 1 focusing on the role of FZDs for TcdB binding and entry into the colonic epithelium during C. difficileinfectioninanimalmodels,andAim2focusingontheroleofCSPG4andmyofibroblasts in TcdB pathogenesis during C. difficile infection. Furthermore, we also found that binding of TcdB to FZDs inhibits Wnt signaling, which is essential for colonic stem cells. We thus hypothesize a novel mechanism contributing to TcdB pathogenesis: TcdB may disrupt colonic stemcellsthroughtoxin-receptorengagement,independentofitsenzymaticactivityinsidecells.
Aim 3 will test this hypothesis by examining the biological consequences and pathological relevance of Wnt signaling inhibition utilizing colonic organoid models and by infection experiments in animal models with engineered C. difficile that expressing the receptor-binding domain of TcdB. We will also explore the therapeutic benefit of GSK-3 inhibitors, which modulate Wnt signaling, for reducing the damage to the colonic epithelium by TcdB during C. difficile infection. Together, these studies will provide a molecular understanding of disease progressionduringC.difficileinfectionandpotentiallyleadtonoveltherapeuticinterventions.

Public Health Relevance

Clostridium difficile toxin B (TcdB) is a critical virulence factor causing diseases associated with C. difficile infection (CDI). Here we seek to establish the pathologically relevant receptors for TcdB in the colonic tissues during CDI and explore novel therapeutic approaches targeting toxin-receptor interactions. These studies will provide a molecular understanding of disease progression during CDI and potentially lead to novel therapeutic interventions.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
Project #
Application #
Study Section
Bacterial Pathogenesis Study Section (BACP)
Program Officer
Ranallo, Ryan
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Boston Children's Hospital
Independent Hospitals
United States
Zip Code
Zhang, Sicai; Lebreton, Francois; Mansfield, Michael J et al. (2018) Identification of a Botulinum Neurotoxin-like Toxin in a Commensal Strain of Enterococcus faecium. Cell Host Microbe 23:169-176.e6
Masuyer, Geoffrey; Zhang, Sicai; Barkho, Sulyman et al. (2018) Structural characterisation of the catalytic domain of botulinum neurotoxin X - high activity and unique substrate specificity. Sci Rep 8:4518
Chen, Peng; Tao, Liang; Wang, Tianyu et al. (2018) Structural basis for recognition of frizzled proteins by Clostridium difficile toxin B. Science 360:664-669
Zhang, Sicai; Berntsson, Ronnie P-A; Tepp, William H et al. (2017) Structural basis for the unique ganglioside and cell membrane recognition mechanism of botulinum neurotoxin DC. Nat Commun 8:1637