Clostridioides difficile TcdB induces expression of its receptor (CSPG4) through a noncanonical Hippo signaling mechanism.

Chondroitin sulfate proteoglycan 4 (CSPG4) is a major receptor for Clostridioides difficile TcdB, but the dynamics and regulation of CSPG4 expression during C. difficile disease has not been described. Using a combination of experimental approaches, we discovered that TcdB induces CSPG4 expression through a mechanism involving small GTPase inactivation and modulation of kinases in the Hippo-signaling cascade. Treatment of HeLa cells or human pericytes with TcdB increased CSPG4 expression, and this could be mimicked by chemical inhibition of Rho. Experiments further demonstrated that TcdB-induced expression of CSPG4 is blocked by inhibitors of two core Hippo kinases (MST1/2 and LATS1/2), but the typical downstream target (YAP/TAZ) of these regulators was not required for the changes in CSPG4. Instead, data from RNA-seq and CUT&RUN experiments found CSPG4 expression was modulated by CCCTC-binding factor (CTCF), a lesser-known target of Hippo signaling. CTCF is a DNA-binding protein capable of repressing gene transcription, and our work found that reduced CTCF leads to increased CSPG4 expression. Additionally, CTCF binding at the CSPG4 gene locus is eliminated by TcdB activity. These data support a model in which TcdB upregulates CSPG4 via Rho inactivation and subsequent Hippo-mediated inactivation of the transcriptional repressor CTCF.