Intrinsic Toxin-Derived Peptides Destabilize and Inactivate Clostridium difficile TcdB

Abstract

Clostridium difficile infection (CDI) is a major cause of hospital-associated, antibiotic-induced diarrhea, which is largely mediated by the production of two large multidomain clostridial toxins, TcdA and TcdB. Both toxins coordinate the action of specific domains to bind receptors, enter cells, and deliver a catalytic fragment into the cytosol. This results in GTPase inactivation, actin disassembly, and cytotoxicity. TcdB in particular has been shown to encode a region covering amino acids 1753 to 1851 that affects epitope exposure and cytotoxicity. Surprisingly, studies here show that several peptides derived from this region, which share the consensus sequence 1769 NVFKGNTISDK 1779 , protect cells from the action of TcdB. One peptide, PepB2, forms multiple interactions with the carboxy-terminal region of TcdB, destabilizes TcdB structure, and disrupts cell binding. We further show that these effects require PepB2 to form a higher-order polymeric complex, a process that requires the central GN amino acid pair. These data suggest that TcdB 1769 -1779 interacts with repeat sequences in the proximal carboxy-terminal domain of TcdB (i.e., the CROP domain) to alter the conformation of TcdB. Furthermore, these studies provide insights into TcdB structure and functions that can be exploited to inactivate this critical virulence factor and ameliorate the course of CDI.IMPORTANCE Clostridium difficile is a leading cause of hospital-associated illness that is often associated with antibiotic treatment. To cause disease, C. difficile secretes toxins, including TcdB, which is a multidomain intracellular bacterial toxin that undergoes conformational changes during cellular intoxication. This study describes the development of peptide-based inhibitors that target a region of TcdB thought to be critical for structural integrity of the toxin. The results show that peptides derived from a structurally important region of TcdB can be used to destabilize the toxin and prevent cellular intoxication. Importantly, this work provides a novel means of toxin inhibition that could in the future develop into a C. difficile treatment. KEYWORDS Clostridium difficile, TcdB, exotoxins, peptidesT cdB is an intracellular bacterial toxin produced by the spore-forming, anaerobic, human pathogen Clostridium difficile (1). This 2,366-amino-acid (aa) protein binds to cell surface receptors (CSPG4, PVRL3, and FZD), undergoes endocytosis, and translocates into the cytosol, where it glucosylates small GTPases (2-5). Small-angle X-ray scattering, electron microscopy, and biochemical analyses indicate that TcdB is organized into 4 domains, with an amino-terminal glucosylation domain (aa 1 to 543), a cysteine-protease domain (aa 544 to 801), a cell entry/translocation domain (aa 802 to 1650), and a carboxyl-terminal cell-binding domain (aa 1651 to 2366) that includes combined repetitive oligopeptide repeats (CROP) from aa 1830 to 2366 (6-15). More recently, the crystal structure of TcdA, a protein that shares sequence and functional identity w...

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

“…Treatment with TcdB2 Δ1773-1774 and TcdB2 Δ2213-2223 or TcdB2 alone resulted in an EC 50 of between 10 and 100 fM. Of the mutants tested, only cells treated with TcdB2 Δ1769 -1787 , the deletion region corresponding to TcdB-derived inhibitory peptide PepB2 (16), showed no loss of cell viability at the highest concentration tested. This observation held true even when cells were exposed to TcdB2 Δ1769 -1787 at a final concentration of 1 M (data not shown).…”

“…The TcdB2 deletion mutant retains glucosylation activity. Previous work has suggested that the efficiency of glucosylation of TcdA and TcdB is hindered by conformational restrictions within the proteins (16,20). It was therefore predicted that deletions which remove conformational constraints in TcdB could result in forms of the protein with altered glucosylation activity.…”

“…Interrupting this structural stability could result in a loss of the CROP domain chaperone effect on the APD, allowing spontaneous autoprocessing. Indeed, in this regard, it is notable that we previously reported that a synthetic peptide of the region from amino acids 1769 to 1787 binds to the CROP region in TcdB2 (16).…”

“…Moreover, a deletion mutant of TcdA containing only the first 1,056 amino acids undergoes more efficient and nearly complete autoprocessing compared with that for the full-length toxin (23). In studies using a peptide based on the region from amino acids 1769 to 1787, the peptide destabilized TcdB2 and enhanced autoprocessing in the presence of IP6 (16). In TcdB2 Δ1769 -1787 , we were able to confirm that the spontaneous autoprocessing event involved the APD by showing a loss in autoprocessing when C698 was mutated in the mutant.…”

“…The region from amino acids 1753 to 1852 influences the exposure of carboxyterminal epitopes in the CROP domain and contributes to solution multimerization of carboxy-terminal fragments of the toxin (15). Peptides derived from the region from amino acids 1753 to 1852 destabilize and inactivate TcdB through interactions with repeating sequences in the CROP domain (16). This stretch of 99 amino acids also affects the overall activity of TcdB; sequence differences present in this region have been seen to influence the efficiency with which TcdB1 and TcdB2, the two major variants of the toxin, enter cells (17).…”