The Binary Toxin CDT of Clostridium difficile as a Tool for Intracellular Delivery of Bacterial Glucosyltransferase Domains

Beer, Lara-Antonia; Tatge, Helma; Schneider, Carmen; Ruschig, Maximilian; Hust, Michael; Barton, Jessica; Thiemann, Stefan; Fühner, Viola; Russo, Giulio; Gerhard, Ralf

doi:10.3390/toxins10060225

Cited by 19 publications

(19 citation statements)

References 49 publications

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“…Although simultaneous binding of FZD and CSPG4 to TcdB was shown in a bio-layer interferometry (BLI) assay [9], it is not clear whether binding of TcdB to the cell surface happens the same way. For instance, we also observed binding of the glucosyltransferase domain (GTD) of TcdB to the cell surface [19], which might directly or indirectly also contribute to cell adhesion or receptor recognition. For this reason, we omitted binding assays for different toxins but focused on cell rounding as an acknowledged surrogate marker for a rather complex uptake process.…”

Section: Discussionmentioning

confidence: 90%

Receptor Binding Domains of TcdB from Clostridioides difficile for Chondroitin Sulfate Proteoglycan-4 and Frizzled Proteins Are Functionally Independent and Additive

Henkel

Tatge

Schöttelndreier

et al. 2020

Toxins

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Toxin B (TcdB) produced by Clostridioides difficile is a main pathogenicity factor that affects a variety of different cell types within the colonic mucosa. TcdB is known to utilize frizzled-1,2,7 and chondroitin sulfate proteoglycan-4 (CSPG4) as protein receptors. By using human cervical cancer cell line HeLa CSPG4 knockout (CSPG4−/−) cells as well as TcdB mutants which do not bind to either CSPG4 or frizzled-1,2,7, or both, we evaluated the impact of the individual receptors for cytopathic and cytotoxic effects of TcdB. We compared TcdB from the reference strain VPI10463 (TcdBVPI) and the endemic strain R20291 (TcdBR20) which does not interact with frizzled-1,2,7. TcdBVPI devoid of CSPG4 binding (TcdBVPI ΔCROP) shows identical cytopathic potency as full-length TcdB in HeLa CSPG4−/− cells, indicating that interaction with frizzled proteins is not affected in the presence of the C-terminal CROP domain. We validated CSPG4 as cellular receptor for both TcdB toxinotypes in HeLa and HEp-2 cells. By exchange of a single phenylalanine residue, 1597 with serine, we generated a mutated TcdBVPI variant (TcdBVPI F1597S) that in accordance with TcdBR20 lacks binding to frizzled-1,2,7 and showed identical potency as TcdBR20 on HeLa cells. This enabled us to estimate the respective share of CSPG4 and frizzled-1,2,7 in the cytotoxic and cytopathic effect induced by TcdB. Our data reveal that binding to frizzled-1,2,7 and to CSPG4 occurs independently and in an additive manner.

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Section: Discussionmentioning

confidence: 90%

Receptor Binding Domains of TcdB from Clostridioides difficile for Chondroitin Sulfate Proteoglycan-4 and Frizzled Proteins Are Functionally Independent and Additive

Henkel

Tatge

Schöttelndreier

et al. 2020

Toxins

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“…Two days before toxin treatment, 7.5 × 10 5 cells were seeded in 10 cm dishes to achieve a 75% confluency on the day of toxin treatment. CDTa and CDTb were generated using an Escherichia coli expression system as previously described ( Beer et al, 2018 ). Cells were treated with 1.5 μg/mL CDTa and 3 μg/mL CDTb.…”

Section: Methodsmentioning

confidence: 99%

The Binary Toxin of Clostridioides difficile Alters the Proteome and Phosphoproteome of HEp-2 Cells

2021

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Clostridioides difficile is a major cause of nosocomial infection worldwide causing antibiotic-associated diarrhea and some cases are leading to pseudomembranous colitis. The main virulence factors are toxin A and toxin B. Hypervirulent strains of C. difficile are linked to higher mortality rates and most of these strains produce additionally the C. difficile binary toxin (CDT) that possesses two subunits, CDTa and CDTb. The latter is responsible for binding and transfer of CDTa into the cytoplasm of target cells; CDTa is an ADP ribosyltransferase catalyzing the modification of actin fibers that disturbs the actin vs microtubule balance and induces microtubule-based protrusions of the cell membrane increasing the adherence of C. difficile. The underlying mechanisms remain elusive. Thus, we performed a screening experiment using MS-based proteomics and phosphoproteomics techniques. Epithelial Hep-2 cells were treated with CDTa and CDTb in a multiplexed study for 4 and 8 h. Phosphopeptide enrichment was performed using affinity chromatography with TiO2 and Fe-NTA; for quantification, a TMT-based approach and DDA measurements were used. More than 4,300 proteins and 5,600 phosphosites were identified and quantified at all time points. Although only moderate changes were observed on proteome level, the phosphorylation level of nearly 1,100 phosphosites responded to toxin treatment. The data suggested that CSNK2A1 might act as an effector kinase after treatment with CDT. Additionally, we confirmed ADP-ribosylation on Arg-177 of actin and the kinetic of this modification for the first time.

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“…The coding sequence for mature CDTa, lacking the export sequence, was cloned into pQE30 vector for expression as N-terminally His6-tagged protein. Mature CDTb was expressed in E. coli as GST fusion protein using the pGEX2T vector (GE Healthcare) as described earlier ( Beer et al, 2018 ). GST-CDTb was purified via glutathione (GSH)-sepharose columns according to standard protocol.…”

Section: Methodsmentioning

confidence: 99%

Clostridioides difficile Toxin CDT Induces Cytotoxic Responses in Human Mucosal-Associated Invariant T (MAIT) Cells

et al. 2021

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Clostridioides difficile is the major cause of antibiotic-associated colitis (CDAC) with increasing prevalence in morbidity and mortality. Severity of CDAC has been attributed to hypervirulent C. difficile strains, which in addition to toxin A and B (TcdA, TcdB) produce the binary toxin C. difficile transferase (CDT). However, the link between these toxins and host immune responses as potential drivers of immunopathology are still incompletely understood. Here, we provide first experimental evidence that C. difficile toxins efficiently activate human mucosal-associated invariant T (MAIT) cells. Among the tested toxins, CDT and more specifically, the substrate binding and pore-forming subunit CDTb provoked significant MAIT cell activation resulting in selective MAIT cell degranulation of the lytic granule components perforin and granzyme B. CDT-induced MAIT cell responses required accessory immune cells, and we suggest monocytes as a potential CDT target cell population. Within the peripheral blood mononuclear cell fraction, we found increased IL-18 levels following CDT stimulation and MAIT cell response was indeed partly dependent on this cytokine. Surprisingly, CDT-induced MAIT cell activation was found to be partially MR1-dependent, although bacterial-derived metabolite antigens were absent. However, the role of antigen presentation in this process was not analyzed here and needs to be validated in future studies. Thus, MR1-dependent induction of MAIT cell cytotoxicity might be instrumental for hypervirulent C. difficile to overcome cellular barriers and may contribute to pathophysiology of CDAC.

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The Binary Toxin CDT of Clostridium difficile as a Tool for Intracellular Delivery of Bacterial Glucosyltransferase Domains

Cited by 19 publications

References 49 publications

Receptor Binding Domains of TcdB from Clostridioides difficile for Chondroitin Sulfate Proteoglycan-4 and Frizzled Proteins Are Functionally Independent and Additive

Receptor Binding Domains of TcdB from Clostridioides difficile for Chondroitin Sulfate Proteoglycan-4 and Frizzled Proteins Are Functionally Independent and Additive

The Binary Toxin of Clostridioides difficile Alters the Proteome and Phosphoproteome of HEp-2 Cells

Clostridioides difficile Toxin CDT Induces Cytotoxic Responses in Human Mucosal-Associated Invariant T (MAIT) Cells

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