Deletion of a 19-Amino-Acid Region in Clostridioides difficile TcdB2 Results in Spontaneous Autoprocessing and Reduced Cell Binding and Provides a Nontoxic Immunogen for Vaccination

• Published in: Infection and immunity Vol. 87; no. 8
• Main Authors: Bland, Sarah J, Larabee, Jason L, Shadid, Tyler M, Lang, Mark L, Ballard, Jimmy D
• Format: Journal Article
• Language: English
• Published: United States American Society for Microbiology 01.08.2019
• Subjects: Animals; Bacterial Proteins - chemistry; Bacterial Proteins - immunology; Bacterial Proteins - physiology; Bacterial Vaccines - immunology; CHO Cells; Clostridium difficile - immunology; Cricetulus; Glycosylation; HeLa Cells; Humans; Mice; Molecular Pathogenesis; Protein Conformation; Protein Domains; Repressor Proteins - chemistry; Repressor Proteins - immunology; Repressor Proteins - physiology; Sequence Deletion; Vaccination; Clostridioides difficile; TcdB; toxin B; immunization; Clostridium difficile; autoprocessing
• ISSN: 0019-9567; 1098-5522
• DOI: 10.1128/IAI.00210-19

toxin B (TcdB) is an intracellular toxin responsible for many of the pathologies of infection. The two variant forms of TcdB (TcdB1 and TcdB2) share 92% sequence identity but have reported differences in rates of cell entry, autoprocessing, and overall toxicity. This 2,366-amino-acid, multidomain bacterial toxin glucosylates and inactivates small GTPases in the cytosol of target cells, ultimately leading to cell death. Successful cell entry and intoxication by TcdB are known to involve various conformational changes in the protein, including a proteolytic autoprocessing event. Previous studies found that amino acids 1753 to 1852 influence the conformational states of the proximal carboxy-terminal domain of TcdB and could contribute to differences between TcdB1 and TcdB2. In the current study, a combination of approaches was used to identify sequences within the region from amino acids 1753 to 1852 that influence the conformational integrity and cytotoxicity of TcdB2. Four deletion mutants with reduced cytotoxicity were identified, while one mutant, TcdB2 , exhibited no detectable cytotoxicity. TcdB2 underwent spontaneous autoprocessing and was unable to interact with CHO-K1 or HeLa cells, suggesting a potential change in the conformation of the mutant protein. Despite the putative alteration in structural stability, vaccination with TcdB2 induced a TcdB2-neutralizing antibody response and protected against disease in a mouse model. These findings indicate that the 19-amino-acid region spanning residues 1769 to 1787 in TcdB2 is crucial to cytotoxicity and the structural regulation of autoprocessing and that TcdB2 is a promising candidate for vaccination.