The glucosyltransferase activity of C. difficile Toxin B is required for disease pathogenesis
| Autor: | Nigel P. Minton, Roman A. Melnyk, Michelle L. Kelly, Megan Garland, Sarah A. Kuehne, Matthew Bogyo, Terry W. Bilverstone, Martina Tholen, Philip Kaye, Rory J. Cave, Donna M. Bouley |
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| Přispěvatelé: | McClane, Bruce A. |
| Rok vydání: | 2020 |
| Předmět: |
Male
Mutant Artificial Gene Amplification and Extension Pathogenesis Pathology and Laboratory Medicine Toxicology medicine.disease_cause Polymerase Chain Reaction Mice Cricetinae Medicine and Health Sciences Toxins Biology (General) Enterocolitis Pseudomembranous Mammals 0303 health sciences 030302 biochemistry & molecular biology Eukaryota Animal Models Clostridium difficile 3. Good health Mutant Strains Experimental Organism Systems Glucosyltransferases Vertebrates Hamsters Female Glucosyltransferase Research Article QH301-705.5 Clostridium Difficile Bacterial Toxins Toxic Agents Immunology Hamster Mouse Models Clostridium difficile toxin B Biology Research and Analysis Methods Rodents Microbiology 03 medical and health sciences Model Organisms Bacterial Proteins In vivo Virology Genetics medicine Animals Animal Models of Disease Molecular Biology Techniques Molecular Biology 030304 developmental biology Bacteria Clostridioides difficile Toxin Gut Bacteria Organisms Biology and Life Sciences RC581-607 Disease Models Animal Animal Models of Infection Amniotes Mutation Animal Studies biology.protein Parasitology Immunologic diseases. Allergy Zoology Gene Deletion |
| Zdroj: | PLoS Pathogens PLoS Pathogens, Vol 16, Iss 9, p e1008852 (2020) |
| ISSN: | 1553-7374 1553-7366 |
| Popis: | Enzymatic inactivation of Rho-family GTPases by the glucosyltransferase domain of Clostridioides difficile Toxin B (TcdB) gives rise to various pathogenic effects in cells that are classically thought to be responsible for the disease symptoms associated with C. difficile infection (CDI). Recent in vitro studies have shown that TcdB can, under certain circumstances, induce cellular toxicities that are independent of glucosyltransferase (GT) activity, calling into question the precise role of GT activity. Here, to establish the importance of GT activity in CDI disease pathogenesis, we generated the first described mutant strain of C. difficile producing glucosyltransferase-defective (GT-defective) toxin. Using allelic exchange (AE) technology, we first deleted tcdA in C. difficile 630Δerm and subsequently introduced a deactivating D270N substitution in the GT domain of TcdB. To examine the role of GT activity in vivo, we tested each strain in two different animal models of CDI pathogenesis. In the non-lethal murine model of infection, the GT-defective mutant induced minimal pathology in host tissues as compared to the profound caecal inflammation seen in the wild-type and 630ΔermΔtcdA (ΔtcdA) strains. In the more sensitive hamster model of CDI, whereas hamsters in the wild-type or ΔtcdA groups succumbed to fulminant infection within 4 days, all hamsters infected with the GT-defective mutant survived the 10-day infection period without primary symptoms of CDI or evidence of caecal inflammation. These data demonstrate that GT activity is indispensable for disease pathogenesis and reaffirm its central role in disease and its importance as a therapeutic target for small-molecule inhibition. Author summary Novel non-antibiotic therapies are required for the treatment of Clostridioides difficile infection (CDI). An emerging class of promising therapeutics for CDI are antivirulence agents that block the actions of C. difficile Toxin B (TcdB), the primary determinant of virulence. In order to develop such treatments, molecular targets and mechanisms must be identified and validated. Historically the glucosyltransferase domain (GTD) represented an ideal target owing to its perceived importance for disease pathogenesis. However, studies capitalizing on recent advances in recombinant TcdB production have unveiled GTD-independent mechanisms of toxicity when applied at high concentrations in vitro, thus questioning the role of the GTD. Here we generate the first-reported mutant strain of C. difficile expressing glucosyltransferase-defective TcdB. Application thereof demonstrates that the GTD is essential for disease in mice and hamsters, thus reoffering the GTD as an ideal candidate for small-molecule inhibitor (SMI) development. |
| Databáze: | OpenAIRE |
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