Interaction of the Clostridium difficile Binary Toxin CDT and Its Host Cell Receptor, Lipolysis-stimulated Lipoprotein Receptor (LSR)

CDT (Clostridium difficile transferase) is a binary, actin ADP-ribosylating toxin frequently associated with hypervirulent strains of the human enteric pathogen C. difficile, the most serious cause of antibiotic-associated diarrhea and pseudomembranous colitis. CDT leads to the collapse of the actin cytoskeleton and, eventually, to cell death. Low doses of CDT result in the formation of microtubule-based protrusions on the cell surface that increase the adherence and colonization of C. difficile. The lipolysis-stimulated lipoprotein receptor (LSR) is the host cell receptor for CDT, and our aim was to gain a deeper insight into the interplay between both proteins. We show that CDT interacts with the extracellular, Ig-like domain of LSR with an affinity in the nanomolar range. We identified LSR splice variants in the colon carcinoma cell line HCT116 and disrupted the LSR gene in these cells by applying the CRISPR-Cas9 technology. LSR truncations ectopically expressed in LSR knock-out cells indicated that intracellular parts of LSR are not essential for plasma membrane targeting of the receptor and cellular uptake of CDT. By generating a series of N- and C-terminal truncations of the binding component of CDT (CDTb), we found that amino acids 757–866 of CDTb are sufficient for binding to LSR. With a transposon-based, random mutagenesis approach, we identified potential LSR-interacting epitopes in CDTb. This study increases our understanding about the interaction between CDT and its receptor LSR, which is key to the development of anti-toxin strategies for preventing cell entry of the toxin. Background:Clostridium difficile binary toxin CDT enters host cells via its receptor LSR. Results: Regions responsible for the CDT-LSR interaction were identified. Conclusion: An immunoglobulin-like domain of LSR and amino acids 757–866 of the binding component of CDT determine the toxin-receptor interaction. Significance: Knowledge of the toxin-receptor interaction is key to the development of anti-toxin strategies.