Dissertation supervisor: Professor Michael Baldwin. ; Includes vita. ; Clostridium difficile (C. difficile) has recently emerged as a worldwide nosocomial and iatrogenic infection. C. difficile is a gram-positive, motile bacillus whose sporulation ability significantly contributes to its persistence in clinical settings. C. difficile secretes toxins, two of which have been confirmed as the major virulence factors for disease in a real world setting, TcdA and TcdB. Recent evidence, significantly from the increasingly prevalent hypervirulent strains of C. difficile, has highlighted the importance of TcdB specifically, so we set out to investigate the structural and functional mechanisms underlying the toxicity of TcdB. We utilized a plethora of truncated and modified recombinant TcdB proteins for a wide array of biochemical assays intending to more fully understand the role of TcdB in disease pathology, both in the classic (630) and various, newly characterized, hypervirulent strains of C. difficile. Immunofluorescence and immunoprecipitation techniques strongly suggested that there are 3 separate domains of TcdB which are each able to bind to cell surfaces. We demonstrated, for the first time, that TcdB aa 1494-2366 are dispensable for toxicity in three cell lines. We found that TcdB aa 1372-1493, which we have suggested calling RBD3, are actually an independent, novel binding domain that previously had gone uninvestigated and uncharacterized in the literature. Using both in vitro and cell based assays, we also identified RBD3 to be the interacting domain for the previously suggested binding partner for TcdB: PVRL3. We have also shown that, in isolation, RBD3 interacts with PVRL3 on cellular membranes, and that this interaction itself is sufficient for dynamin-dependent endocytosis and processing through to the lysosome. Data also indicate that while TcdB RBD3 has similar interaction with PVRL3 and similar cellular processing between the classic and hypervirulent strains, the CROPs domain, classically viewed as ...