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 LRP1deficient 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.

Sulfated glycosaminoglycans and low-density lipoprotein receptor mediate the cellular entry of Clostridium novyi alpha-toxin

“…Recent studies showed that TcdA uses sulfated glycosaminoglycans as the cell surface attachment factors and LDLR family proteins as the entry receptors. 8 , 12 The primary sequences between Tcnα and TcdA are divergent, with only ~30% identity. In the LCT family, TcdA is most similar to TcsH (~78% identity).…”

LDLR, LRP1, and Megalin redundantly participate in the uptake of Clostridium novyi alpha-toxin

“…Although LRP1 and Megalin can mediate the cellular entry of Tcnα, they were not found in the candidate list of our previous CRISPR screen for Tcnα 13 . Likewise, LRP1 was demonstrated as an entry receptor for TcdA 30 but it did not stand out from the previous genome-wide screen 27 . We noticed that HeLa cells were employed in both genetic screens for TcdA and Tcnα, as well as the following validation experiments.…”

“…We noticed that HeLa cells were employed in both genetic screens for TcdA and Tcnα, as well as the following validation experiments. On the other hand, previously Schottelndreier et al used mouse embryonic fibroblasts (MEFs) for studying the role of LRP1 in TcdA entry 30 . According to a public protein profiling database ( http://www.proteinatlas.org ) 31 , 32 , LDLR and LRP1 have contrasting mRNA expression profiles in many different cell lines, while Megalin is absent in most cell lines (Fig.…”

“…4b ). MEFs were previously used to study the role of Lrp1 in mediating the entry of TpeL and TcdA but these are mouse cells 30 , 33 . Therefore, we chose U-87 MG, a human glioma cell line that expresses both LDLR and LRP1, to generate LDLR and LRP1 knockout cells using the CRISPR/Cas9 approach (Fig.…”

Genome wide CRISPR screen for Pasteurella multocida toxin (PMT) binding proteins reveals LDL Receptor Related Protein1 (LRP1) as crucial cellular receptor

“…On the cell surface the two parts remain non-covalently linked. More than 40 distinct ligands have been identified to interact with LRP1 including apolipoproteins and cholesteryl esters in remnants (14, 15), extracellular matrix (ECM) components such as fibronectin (16, 17), a minor-group common cold virus (18) and Rift Valley fever virus (19) as well as bacterial toxins like Clostridium perfringens TpeL (13), Pseudomonas Exotoxin A (ExoA, (20)), TcdA produced by Clostridoides difficile (21) and the vacuolating toxin (VacA) from Helicobacter pylori (22). These diverse ligands do not contain a common binding motif.…”