Clostridioides difficile (C. diff) is a major cause of antibiotic-associated diarrhea and colitis, representing a significant healthcare challenge. The severe symptoms of a C. diff infection are primarily caused by the release of powerful protein toxins within the gut. The two main toxins responsible for the disease are Toxin A (TcdA) and Toxin B (TcdB), which are both large molecules that disrupt the integrity of the intestinal lining.
Structural and Genetic Differences
Toxin A and Toxin B are part of the large clostridial glucosylating toxin family, sharing a high degree of structural and functional similarity. Both are exceptionally large proteins, with TcdA being slightly larger at approximately 308 kilodaltons (kDa) compared to TcdB at about 270 kDa. Both toxins possess a common four-domain architecture necessary for their function: a catalytic domain, a translocation domain, a cysteine protease domain, and a C-terminal receptor-binding domain.
The genes encoding both toxins, tcdA and tcdB, are located together on a specific mobile genetic element within the bacterial chromosome called the Pathogenicity Locus (PaLoc). This locus also contains three other genes (tcdR, tcdC, and tcdE) that regulate the production and release of the toxins. The expression of both tcdA and tcdB is tightly controlled, ensuring the bacterium produces them concurrently.
While sharing an overall structure, the two toxins have significant genetic diversity, particularly in their C-terminal receptor-binding domains. They share only about 44% sequence identity, with TcdB exhibiting greater genetic variation and more distinct subtypes than TcdA. TcdA’s C-terminus is characterized by a higher number of repetitive polypeptide sequences, which form a solenoid-like structure important for binding to the host cell surface.
Cellular Damage Pathway
The core mechanism by which TcdA and TcdB damage intestinal cells is nearly identical, beginning with binding to the cell surface and subsequent internalization. After binding to their respective receptors, the toxins are taken into the cell via endocytosis, enclosed within vesicles. The acidic environment within the endosome triggers a conformational change, allowing them to form a pore and translocate their catalytic domain into the host cell’s cytosol.
Once in the cytosol, the catalytic domain functions as a glucosyltransferase, using a sugar molecule (UDP-glucose) to chemically modify specific proteins. The primary targets of both toxins are the Rho family of small guanosine triphosphatases (GTPases), including RhoA, Rac, and Cdc42. This modification, known as glucosylation, occurs at a specific threonine residue on the GTPase.
Glucosylation permanently inactivates the Rho GTPases, which regulate the assembly and organization of the cell’s internal scaffolding, the actin cytoskeleton. The inactivation of these proteins causes the cytoskeleton to collapse, leading to a characteristic change in cell shape known as “cell rounding.” This process destroys the cell-to-cell junctions, increases the permeability of the intestinal barrier, and ultimately results in cell death.
A subtle difference lies in their cellular target preference, which is linked to their distinct receptor-binding domains. TcdA was historically considered the main enterotoxin because it binds to carbohydrate receptors located on the apical (lumen-facing) side of the intestinal epithelial cells. TcdB’s receptor is less defined but appears to be located on the basolateral (tissue-facing) side of some cells, suggesting a different initial interaction point with the intestinal lining.
Clinical Impact and Virulence
Historically, TcdA was believed to be the primary driver of disease because of its ability to induce fluid secretion and inflammation, leading to its designation as the enterotoxin. Modern research has changed the understanding of their relative importance in disease severity. TcdB is now recognized as the more potent and cytotoxic of the two, often being 10 to 100 times more toxic to host cells in laboratory assays.
The dominance of TcdB is supported by the existence of clinical strains that are TcdA-negative but TcdB-positive (A-B+). These strains are fully capable of causing the entire spectrum of C. diff disease, from mild diarrhea to severe, life-threatening colitis, confirming that TcdB alone is sufficient for pathogenesis. Conversely, strains producing only TcdA are generally attenuated in animal models of infection.
The emergence of hypervirulent strains, such as the BI/NAP1/027 ribotype, further highlights the role of TcdB. These strains possess a mutation in the tcdC gene, which acts as a negative regulator of toxin production, resulting in the bacteria producing significantly higher amounts of both toxins. These hypervirulent strains can produce up to 23 times more TcdB than non-hypervirulent reference strains, often featuring a variant TcdB with a modified receptor-binding domain. This heightened production and potency is considered a major factor behind the increased incidence and severity associated with these epidemic strains.