Clostridium difficile toxin A

Clostridium difficile toxin A (TcdA) is a toxin generated by Clostridium difficile. It is similar to Clostridium difficile Toxin B. The toxins are the main virulence factors produced by the gram positive, anaerobic, Clostridium difficile bacteria. The toxins function by damaging the intestinal mucosa and cause the symptoms of C. difficile infection, including pseudomembranous colitis. Clostridium difficile toxin A (TcdA) is a toxin generated by Clostridium difficile. It is similar to Clostridium difficile Toxin B. The toxins are the main virulence factors produced by the gram positive, anaerobic, Clostridium difficile bacteria. The toxins function by damaging the intestinal mucosa and cause the symptoms of C. difficile infection, including pseudomembranous colitis. TcdA is one of the largest bacterial toxins known. With a molecular mass of 308 kDa, it is usually described as a potent enterotoxin, but it also has some activity as a cytotoxin. The toxin acts by modifying host cell GTPase proteins by glucosylation, leading to changes in cellular activities. Risk factors for C. difficile infection include antibiotic treatment, which can disrupt normal intestinal microbiota and lead to colonization of C. difficile bacteria. The gene contains an open reading frame (ORF) of 8,133 nucleotides, coding for 2,710 amino acids. TcdA and TcdB share 63% homology in their amino acid sequences. These genes are expressed during late log phase and stationary phase in response to environmental factors. Environmental stresses such as antibiotics and catabolite repression can influence toxin expression. The tcdA and tcdB genes are situated on the Clostridium difficile chromosome in a 19.6-kb pathogenicity locus (PaLoc) found only in toxigenic strains of C. difficile. Non toxigenic strains contain a 127 base pair fragment replacing the PaLoc. This locus also contains three other accessory genes tcdC, tcdR, and tcdE. TcdC expression is high during early exponential phase and declines as growth moves into stationary phase, consistent with increases in tcdA and tcdB expression. Accordingly, expression patterns have indicated tcdC as a possible negative regulator of toxin production. tcdR may serve as a positive regulator of toxin production. tcdE has been speculated to facilitate release of TcdA and TcdB through lytic activity on the bacterial cell membrane. Due to its homology with other proteins of similar function, as well as the location of the gene between tcdA and tcdB, tcdE is predicted to function as the lytic protein that facilitates release since TcdA and TcdB lack a signal peptide for secretion. The protein contains three domains. The amino N-terminal domain contains the active site, responsible for the glucosylating activity of the toxin. Both TcdA and TcdB use this highly conserved N-terminal region (74% homology between both toxins) to alter identical substrates. The carboxy C-terminal domain contains repeating units that are responsible for receptor binding on target cell surfaces. These short homologous repeating units have been termed combined repetitive oligopeptide (CROPs). A recent study demonstrates that the CROPs determine the potency of TcdA through interactions with structures on the cell surface. These CROP regions range from 21-50 residues and play a role in receptor binding. This C-terminal repetitive region is designated as the immuno-dominant region since ligand binding can be blocked by monoclonal antibodies specific to this region. This region contains the most hydrophilic portion of the molecule. A centrally located hydrophobic domain containing a cluster of 172 highly conserved hydrophobic amino acids is thought to be important for translocation of the enzymatic portion of the protein. TcdA must be internalized into the host cell via endocytosis in order to access the cytosol. Receptor binding is the first step required for entry into the cell via endocytosis in an acidic endosome. Low pH in the endosome induces structural changes such as exposure of the hydrophobic domains that are crucial for TcdA function. The N-terminal domain of TcdA functions to catalyze a glucotransferase reaction, which transfers a glucose molecule from UDP-Glucose and covalently attaches it to conserved amino acids in target molecules. Therefore, TcdA catalyzes glucosylation and the subsequent irreversible inactivation of target molecules in the Ras family of small GTPases. These target molecules include RhoA, Rac, and Cdc42, which are regulatory proteins of the eukaryotic actin cytoskeleton and modulators of many various cell signaling pathways.