Abstract
ABSTRACTClostridium 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 1769NVFKGNTISDK1779, 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 TcdB1769–1779 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.
Highlights
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
TcdB can be deactivated using 17- to 20-amino-acid peptides derived from a region of the toxin that is necessary for forming intramolecular contacts
TcdB is inhibited by PepB2 through a mechanism that depends on combined repetitive oligopeptide repeats (CROP) domain targeting that results in toxin destabilization and delayed cell binding
Summary
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. 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 TcdB1769–1779 interacts with repeat sequences in the proximal carboxy-terminal domain of TcdB (i.e., the CROP domain) to alter the conformation of TcdB. TcdB 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 [2,3,4,5]. Experimental results indicate that PepB2 blocks TcdB toxicity by binding the CROP domain, forming a polymeric complex, and destabilizing the toxin
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