Clostridium difficile Binary Toxin CDT Induces Clustering of the Lipolysis-Stimulated Lipoprotein Receptor into Lipid Rafts

Abstract

Clostridium difficile is the leading cause of antibiotics-associated diarrhea and pseudomembranous colitis. Hypervirulent C. difficile strains produce the binary actin-ADP-ribosylating toxin CDT (C. difficile transferase), in addition to the Rho-glucosylating toxins A and B. We recently identified the lipolysis-stimulated lipoprotein receptor (LSR) as the host receptor that mediates uptake of CDT into target cells. Here we investigated in H1-HeLa cells, which ectopically express LSR, the influence of CDT on the plasma membrane distribution of the receptor. We found by fluorescence microscopy that the binding component of CDT (CDTb) induces clustering of LSR into subcompartments of the plasma membrane. Detergent extraction of cells treated with CDTb, followed by sucrose gradient fractionation, uncovered accumulation of LSR in detergent-resistant membranes (DRMs) that contained typical marker proteins of lipid rafts. Membrane cholesterol depletion with methyl-β-cyclodextrin inhibited the association of LSR with DRMs upon addition of CDTb. The receptor-binding domain of CDTb also triggered LSR clustering into DRMs. CDTb-triggered clustering of LSR into DRMs could be confirmed in Caco-2 cells. Our data suggest that CDT forces its receptor to cluster into lipid rafts and that oligomerization of the B component might enhance but is not essential for this process.

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

“…We then used LSR Ig -His 6 as prey protein in a GST pulldown assay and a GST fusion of the RBD from CDTb (amino acids 677-876) as bait. LSR-dependent cell surface-binding of GST-RBD was shown previously (31). As shown in Fig.…”

“…It is conceivable that the endocytosis of LSR is triggered also by other membrane proteins, which might form a complex with LSR. We found recently that LSR accumulates in lipid rafts upon binding of CDTb (31). Interestingly, Wigelsworth et al (43) found that the lipid raft-associated protein CD44 (cluster of differentiation 44 protein) is crucial for CDT uptake into host cells.…”

“…The plasmid pEGFP-N1/LSR was used for the ectopic expression of LSR-EGFP in mammalian cells and is described elsewhere (31). The pEGFP-N1 vector backbone was also used for the ectopic expression of LSR 1-240 -EGFP and LSR ⌬CR -EGFP.…”

“…The cloning, expression, and purification of the RBD of CDTb as a GST fusion protein (GST-RBD) has been described elsewhere (31). N-and C-terminal truncations of RBD were expressed and purified from E. coli strain BL21 (DE3) as GST fusion proteins in the same manner as GST-RBD.…”

Development and Optimization of a High-Throughput Assay To Measure Neutralizing Antibodies against Clostridium difficile Binary Toxin

“…It has also been reported that in monolayers of Caco-2 cells, transepithelial resistance (TER) is decreased by the formation of pores by Ib oligomer insertion into the cell membrane (34). Papatheodorou et al (35) have shown by fluorescence microscopy that a_CDTb can induce the clustering of LSR into subcompartments of the plasma membrane (lipid rafts), but they were not able to conclude whether the activated B component forms a heptamer that subsequently binds to the LSR receptor or whether the monomer first binds to the LSR receptor and then oligomerizes. We speculate that a_CDTb causes a high degree of LSR clustering on the HT-29 cell membrane, which might cause cytotoxicity through necrosis or disruption of the cell membrane similar to the cytotoxicity caused by Ib toxin.…”