Abstract
TcdB is an intracellular bacterial toxin indispensable to Clostridioides difficile infections. The ability to use chondroitin sulfate proteoglycan 4 (CSPG4) as a primary cell surface receptor is evolutionarily conserved by the two major variants of TcdB. As CSPG4 does not typically undergo receptor-mediated endocytosis, we sought to identify environmental factors that stabilize interactions between TcdB and CSPG4 to promote cell binding and entry into the cytosol. Using a series of TcdB receptor-binding mutants and cell lines with various receptor expression profiles, we discovered that extracellular Ca2+ promotes receptor-specific interactions with TcdB. Specifically, TcdB exhibits preferential binding to CSPG4 in the presence of Ca2+, with the absence of Ca2+ resulting in CSPG4-independent cell surface interactions. Furthermore, Ca2+ did not enhance TcdB binding to chondroitin sulfate (CS), the sole glycosaminoglycan of CSPG4. Instead, CS was found to impact the rate of cell entry by TcdB. Collectively, results from this study indicate that Ca2+ enhances cell binding by TcdB and CS interactions contribute to subsequent steps in cell entry. Importance: Clostridioides difficile is a leading cause of antibiotic-associated gastrointestinal illness, and many disease pathologies are caused by the toxin TcdB. TcdB engages multiple cell surface receptors, with receptor tropisms differing among the variants of the toxin. Chondroitin sulfate proteoglycan 4 (CSPG4) is a critical receptor for multiple forms of TcdB, and insights into TcdB-CSPG4 interactions are applicable to many disease-causing strains of C. difficile. CSPG4 is modified by chondroitin sulfate (CS) and contains laminin-G repeats stabilized by Ca2+, yet the relative contributions of CS and Ca2+ to TcdB cytotoxicity have not been determined. This study demonstrates distinct roles in TcdB cell binding and cell entry for Ca2+ and CS, respectively. These effects are specific to CSPG4 and contribute to the activities of a prominent isoform of TcdB that utilizes this receptor. These findings advance an understanding of factors contributing to TcdB's mechanism of action and contribution to C. difficile disease.