Low Density Lipoprotein Receptor-Related Protein-1 (LRP1) Is Involved in the Uptake of Clostridioides difficile Toxin A and Serves as an Internalizing Receptor

Abstract

Toxin producing Clostridioides difficile strains cause gastrointestinal infections with the large glucosylating protein toxins A (TcdA) and B (TcdB) being major virulence factors responsible for the onset of symptoms. TcdA and TcdB enter their target cells via receptor-mediated endocytosis. Inside the cell, the toxins glucosylate and thereby inactivate small GTPases of the Rho-/Ras subfamilies resulting in actin reorganization and cell death. The receptors of TcdA are still elusive, glycoprotein 96 (gp96), the low density lipoprotein receptor family (LDLR) and sulfated glycosaminoglycans (sGAGs) have most recently been suggested as receptors for TcdA. In this study, we provide evidence on rapid endocytosis of Low density lipoprotein Receptor-related Protein-1 (LRP1) into fibroblasts and Caco-2 cells by exploiting biotinylation of cell surface proteins. In contrast, gp96 was not endocytosed either in the presence or absence of TcdA. The kinetics of internalization of TfR and LRP1 were comparable in the presence and the absence of TcdA, excluding that TcdA facilitates its internalization by triggering internalization of its receptors. Exploiting fibroblasts with a genetic deletion of LRP1, TcdA was about one order of magnitude less potent in LRP1-deficient cells as compared to the corresponding control cells. In contrast, TcdB exhibited a comparable potency in LRP1-proficient and -deficient fibroblasts. These findings suggested a role of LRP1 in the cellular uptake of TcdA but not of TcdB. Correspondingly, binding of TcdA to the cell surface of LRP1-deficient fibroblasts was reduced as compared with LRP1-proficient fibroblasts. Finally, TcdA bound to LRP1 ligand binding type repeat cluster II (amino acid 786-1,165) and cluster IV (amino acid 3332-3779). In conclusion, LRP1 appears to serve as an endocytic receptor and gp96 as a non-endocytic receptor for TcdA.

Keywords: cell surface; clostridial glycosylating toxins; clostridioides difficile infection; endocytosis; receptors.

Figure 1

Internalization of LRP1 in murine fibroblasts and human colocytes Caco-2 cells. Internalization of reversibly biotinylated cell surface proteins [transferrin receptor (TfR); glycoprotein 96 (gp96); LDL related protein 1 (LRP1)] into murine fibroblasts (A, B) or Caco-2 cells (C, D) was induced by temperature shift to 37°C. Cells were either left untreated (total) or exposed to glutathione to remove cell surface biotin (internalized). Cells were exposed to either buffer (A, C) or 333 pM TcdA (B, D) for 30 min at 37°C and biotinylated proteins were retrieved using neutravidin agarose and analyzed by immunoblotting. Representative immunoblots are shown.

Figure 2

Delayed TcdA-induced actin de-polymerization upon genetic deletion of LRP1. LRP1^-/- mouse embryonic fibroblasts (MEFs) and LRP1^+/+ MEFs were treated with 100 pM TcdA (A, B) and 3,7 pM TcdB (C) for the indicated times and the cell morphology was visualized using phase contrast microscopy. Toxin-induced actin depolymerization was quantified in terms of the number of rounded per total cells. Values represent the mean ± SD from three independent experiments. * indicates significant differences, p < 0,05 as analyzed using student´s t-test.

Figure 3

Delayed cellular uptake of TcdA upon genetic deletion of LRP1. (A) LRP1^-/- MEFs and LRP1^+/+ mouse embryonic fibroblasts (MEFs) were treated with 100 pM TcdA for the indicated times. The cellular levels of non-glucosylated Rac/Cdc42, total Rac1, pS144/141-PAK1/2, LRP1, and beta actin were analyzed by immunoblotting using the indicated antibodies. (B) Quantification of the relative level of non-glucosylated Rac/Cdc42 versus total Rac1 of time-dependent 100 pM TcdA treated MEFs (C) and time-dependent 3,7 pM TcdB treated MEFs are expressed as the mean ± SD from three independent experiments. * indicates significant differences, p < 0,05 as analyzed using student´s t-test.

Figure 4

Binding of TcdA and TcdB to the cell surface. (A) Binding of TcdA and TcdB to LRP1^-/- mouse embryonic fibroblasts (MEFs) and LRP1^+/+ MEFs was allowed at 4°C for 1 h. Unbound toxin was removed by washing with buffer. Cells with bound toxins were lysed and cell surface bound toxins were detected by immunoblotting. (B) Bound TcdA and TcdB were quantified using LabImage 1D software. Values are the mean ± SD from three independent experiments. (C) LRP1 cluster II and IV and lysates from LRP1^+/+ MEFs were immobilized on nitrocellulose and exposed to solutions of TcdA (3 nM) or TcdB (3 nM) for 1 h. Bound toxin was detected using toxin specific antitoxin antibodies. * indicates significant difference, p < 0,05 as analyzed using student´s t-test.

Figure 5

Two receptor model of TcdA and TcdB. (1) Toxin binds to (non-internalizing) binding receptors. (2) The binding receptors form a heterodimer with an internalizing receptor. (3) The toxin is released from the binding receptor, allowing internalization of the toxin internalizing receptor-complex into the early endosome. (4) The glucosyltransferase (GTD) is released into the cytosol upon acidification. The GTD mono-O-glucosylates Rho-GTPases and thereby inactivates them. Gp96, glycoprotein 96; LRP1, low density lipoproten receptor related protein 1; CSPG4, chondroitin sulfate proteoglycane; PVRL3, poliovirus receptor-like3; FZD1/2/7, Frizzled protein 1/2/7.