Exposure of Neutralizing Epitopes in the Carboxyl-terminal Domain of TcdB Is Altered by a Proximal Hypervariable Region

The sequence, activity, and antigenicity of TcdB varies between different strains of Clostridium difficile. As a result, ribotype-specific forms of TcdB exhibit different toxicities and are not strongly cross-neutralized. Using a combination of biochemical and immunological approaches, we compared two important variants of TcdB (TcdB(012) and TcdB(027)) to identify the mechanisms through which sequence differences alter epitopes and activity of the toxin. These analyses led to the discovery of a critical variation in the 1753-1851 (B2') region of Taal, which affects the exposure of neutralizing epitopes in the toxin, Sequence comparisons found that the B2' region exhibits only 77% identity and is the most variable sequence between the two forms of TcdB. A combination of biochemical, analytical, and rnutagenesis experiments revealed that the B2' region promotes protein-protein interactions. These interactions appear to shield neutralizing epitopes that would otherwise be exposed in the toxin, an event found to be less prominent in TcdB012 due to sequence differences in the 1773-1780 and 1791-1798 regions of the B2' domain. When the carboxyl-terminal domains of TcdB(027) and TcdB, are swapped, neutralization experiments suggest that the amino terminus of TcdB interacts with the B2' region and impacts the exposure of neutralizing epitopes in the carboxyl terminus. Collectively, these data suggest that variations in the B2' region affect protein-protein interactions within TcdB and that these interactions influence the exposure of neutralizing epitopes.