Alessandra di Masi1, Loris Leboffe1, Fabio Polticelli1,2, Federica Tonon3, Cristina Zennaro3, Marianna Caterino4,5, Pasquale Stano1, Stephan Fischer6, Marlen Hägele7, Martin Müller7, Alexander Kleger7, Panagiotis Papatheodorou6,8, Giuseppina Nocca9,10, Alessandro Arcovito9, Andrea Gori11, Margherita Ruoppolo4,5,12, Holger Barth6, Nicola Petrosillo13, Paolo Ascenzi1, Stefano Di Bella13. 1. Department of Sciences, Roma Tre University, Roma, Italy. 2. National Institute of Nuclear Physics, Roma Tre Section, Roma, Italy. 3. Department of Medical, Surgery and Health Sciences, University of Trieste, Trieste, Italy. 4. Department of Molecular Medicine and Medical Biotechnology, University of Napoli "Federico II", Napoli, Italy. 5. Associazione Culturale DiSciMuS RCF, Casoria, Napoli, Italy. 6. Institute of Pharmacology and Toxicology, University of Ulm Medical Center, Ulm, Germany. 7. Department of Internal Medicine I, University of Ulm Medical Center, Germany. 8. Institute of Experimental and Clinical Pharmacology and Toxicology, University of Freiburg, Freiburg, Germany. 9. Institute of Biochemistry and Clinical Biochemistry, Catholic University of Sacred Heart, Roma, Italy. 10. Institute of Chemistry of Molecular Recognition, CNR, Roma, Italy. 11. Clinic of Infectious Diseases, San Gerardo Hospital, University of Milano-Bicocca, Monza, Italy. 12. CEINGE Biotecnologie Avanzate, Napoli, Italy. 13. 2nd Infectious Diseases Division, National Institute for Infectious Diseases "L. Spallanzani", Roma, Italy.
Abstract
Background: The pathogenic effects of Clostridium difficile are primarily attributable to the production of the large protein toxins (C difficile toxins [Tcd]) A (TcdA) and B (TcdB). These toxins monoglucosylate Rho GTPases in the cytosol of host cells, causing destruction of the actin cytoskeleton with cytotoxic effects. Low human serum albumin (HSA) levels indicate a higher risk of acquiring and developing a severe C difficile infection (CDI) and are associated with recurrent and fatal disease. Methods: We used a combined approach based on docking simulation and biochemical analyses that were performed in vitro on purified proteins and in human epithelial colorectal adenocarcinoma cells (Caco-2), and in vivo on stem cell-derived human intestinal organoids and zebrafish embryos. Results: Our results show that HSA specifically binds via its domain II to TcdA and TcdB and thereby induces their autoproteolytic cleavage at physiological concentrations. This process impairs toxin internalization into the host cells and reduces the toxin-dependent glucosylation of Rho proteins. Conclusions: Our data provide evidence for a specific HSA-dependent self-defense mechanism against C difficile toxins and provide an explanation for the clinical correlation between CDI severity and hypoalbuminemia.
Background: The pathogenic effects of Clostridium difficile are primarily attributable to the production of the large protein toxins (C difficile toxins [Tcd]) A (TcdA) and B (TcdB). These toxins monoglucosylate Rho GTPases in the cytosol of host cells, causing destruction of the actin cytoskeleton with cytotoxic effects. Low humanserum albumin (HSA) levels indicate a higher risk of acquiring and developing a severe C difficile infection (CDI) and are associated with recurrent and fatal disease. Methods: We used a combined approach based on docking simulation and biochemical analyses that were performed in vitro on purified proteins and in human epithelial colorectal adenocarcinoma cells (Caco-2), and in vivo on stem cell-derived human intestinal organoids and zebrafish embryos. Results: Our results show that HSA specifically binds via its domain II to TcdA and TcdB and thereby induces their autoproteolytic cleavage at physiological concentrations. This process impairs toxin internalization into the host cells and reduces the toxin-dependent glucosylation of Rho proteins. Conclusions: Our data provide evidence for a specific HSA-dependent self-defense mechanism against C difficile toxins and provide an explanation for the clinical correlation between CDI severity and hypoalbuminemia.
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