Mapping C. difficile TcdB interactions with host cell-surface and intracellular factors using proximity-dependent biotinylation labeling
ABSTRACT Many bacterial toxins exert their cytotoxic effects by enzymatically inactivating one or more cytosolic targets in host cells. To reach their intracellular targets, these toxins possess functional domains or subdomains that interact with and exploit various host factors and biological proce...
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American Society for Microbiology
2025-02-01
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| Series: | mBio |
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| Online Access: | https://journals.asm.org/doi/10.1128/mbio.03336-24 |
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| author | Jennifer S. Ward Karl J. Schreiber John Tam Ji-Young Youn Roman A. Melnyk |
| author_facet | Jennifer S. Ward Karl J. Schreiber John Tam Ji-Young Youn Roman A. Melnyk |
| author_sort | Jennifer S. Ward |
| collection | DOAJ |
| description | ABSTRACT Many bacterial toxins exert their cytotoxic effects by enzymatically inactivating one or more cytosolic targets in host cells. To reach their intracellular targets, these toxins possess functional domains or subdomains that interact with and exploit various host factors and biological processes. Despite great progress in identifying many of the key host factors involved in the uptake of toxins, significant knowledge gaps remain as to how partially characterized and newly discovered microbial toxins exploit host factors or processes to intoxicate target cells. Proximity-dependent biotinylation (e.g., BioID) is a powerful method to identify nearby host factors in living cells, offering the potential to identify host targets of microbial toxins. Here, we used BioID to interrogate proximal interactors of the multi-domain Clostridioides difficile TcdB toxin. Expressed fusions of TurboID to different fragments of TcdB identified several high-confidence proteins in the cytosol, including members of the Rho GTPase signaling network and the actin cytoskeletal network. Additionally, we developed an extracellular proximity labeling method using recombinant TurboID-toxin chimeras, which uncovered a limited number of cell-surface targets including LRP1, which was previously identified as a cell-surface receptor of TcdB. Our work reveals surface receptors and intracellular components exploited by bacterial toxins, highlighting key vulnerabilities in host cells.IMPORTANCEBacterial toxins are the causative agents of many human diseases. Further characterizing the intoxication mechanisms of these proteins is important for the development of vaccines and treatments for toxin-mediated disease. Proximity-dependent biotinylation approaches offer an orthogonal approach to complement genetic screens. Here, we evaluate the potential of this method to identify host-toxin interactions on the cell surface and in the cytosol, where the toxin modifies essential host targets. Critically, we have highlighted several limitations of this method as applied to protein toxins, which are important for researchers to weigh when considering this technique for exotoxin studies. |
| format | Article |
| id | doaj-art-4a8719d8d0064800b25cfa9893c1d28f |
| institution | Kabale University |
| issn | 2150-7511 |
| language | English |
| publishDate | 2025-02-01 |
| publisher | American Society for Microbiology |
| record_format | Article |
| series | mBio |
| spelling | doaj-art-4a8719d8d0064800b25cfa9893c1d28f2025-02-05T14:00:48ZengAmerican Society for MicrobiologymBio2150-75112025-02-0116210.1128/mbio.03336-24Mapping C. difficile TcdB interactions with host cell-surface and intracellular factors using proximity-dependent biotinylation labelingJennifer S. Ward0Karl J. Schreiber1John Tam2Ji-Young Youn3Roman A. Melnyk4Department of Biochemistry, University of Toronto, Toronto, Ontario, CanadaMolecular Medicine Program, The Hospital for Sick Children Research Institute, Toronto, Ontario, CanadaMolecular Medicine Program, The Hospital for Sick Children Research Institute, Toronto, Ontario, CanadaMolecular Medicine Program, The Hospital for Sick Children Research Institute, Toronto, Ontario, CanadaDepartment of Biochemistry, University of Toronto, Toronto, Ontario, CanadaABSTRACT Many bacterial toxins exert their cytotoxic effects by enzymatically inactivating one or more cytosolic targets in host cells. To reach their intracellular targets, these toxins possess functional domains or subdomains that interact with and exploit various host factors and biological processes. Despite great progress in identifying many of the key host factors involved in the uptake of toxins, significant knowledge gaps remain as to how partially characterized and newly discovered microbial toxins exploit host factors or processes to intoxicate target cells. Proximity-dependent biotinylation (e.g., BioID) is a powerful method to identify nearby host factors in living cells, offering the potential to identify host targets of microbial toxins. Here, we used BioID to interrogate proximal interactors of the multi-domain Clostridioides difficile TcdB toxin. Expressed fusions of TurboID to different fragments of TcdB identified several high-confidence proteins in the cytosol, including members of the Rho GTPase signaling network and the actin cytoskeletal network. Additionally, we developed an extracellular proximity labeling method using recombinant TurboID-toxin chimeras, which uncovered a limited number of cell-surface targets including LRP1, which was previously identified as a cell-surface receptor of TcdB. Our work reveals surface receptors and intracellular components exploited by bacterial toxins, highlighting key vulnerabilities in host cells.IMPORTANCEBacterial toxins are the causative agents of many human diseases. Further characterizing the intoxication mechanisms of these proteins is important for the development of vaccines and treatments for toxin-mediated disease. Proximity-dependent biotinylation approaches offer an orthogonal approach to complement genetic screens. Here, we evaluate the potential of this method to identify host-toxin interactions on the cell surface and in the cytosol, where the toxin modifies essential host targets. Critically, we have highlighted several limitations of this method as applied to protein toxins, which are important for researchers to weigh when considering this technique for exotoxin studies.https://journals.asm.org/doi/10.1128/mbio.03336-24toxinBioIDC. difficilediphtheria toxinreceptorbiotin |
| spellingShingle | Jennifer S. Ward Karl J. Schreiber John Tam Ji-Young Youn Roman A. Melnyk Mapping C. difficile TcdB interactions with host cell-surface and intracellular factors using proximity-dependent biotinylation labeling mBio toxin BioID C. difficile diphtheria toxin receptor biotin |
| title | Mapping C. difficile TcdB interactions with host cell-surface and intracellular factors using proximity-dependent biotinylation labeling |
| title_full | Mapping C. difficile TcdB interactions with host cell-surface and intracellular factors using proximity-dependent biotinylation labeling |
| title_fullStr | Mapping C. difficile TcdB interactions with host cell-surface and intracellular factors using proximity-dependent biotinylation labeling |
| title_full_unstemmed | Mapping C. difficile TcdB interactions with host cell-surface and intracellular factors using proximity-dependent biotinylation labeling |
| title_short | Mapping C. difficile TcdB interactions with host cell-surface and intracellular factors using proximity-dependent biotinylation labeling |
| title_sort | mapping c difficile tcdb interactions with host cell surface and intracellular factors using proximity dependent biotinylation labeling |
| topic | toxin BioID C. difficile diphtheria toxin receptor biotin |
| url | https://journals.asm.org/doi/10.1128/mbio.03336-24 |
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