Thermal and structural stability of ExhC from M. sciuri

Mammaliicoccus sciuri, a commensal and pathogenic bacterium of significant clinical and veterinary relevance, expresses exfoliative toxin C (ExhC), a specific glutamyl endopeptidase belonging to the chymotrypsin family as the principal virulence factor. However, unlike most members of this family, exfoliative toxins (ETs) are inactive against a wide range of substrates and possess exquisite specificity for desmoglein-1 (Dsg1), a cadherin-like adhesion molecule that is crucial to maintaining tissue integrity, thereby preventing the separation of skin cells and the entry of pathogens. ExhC is of clinical importance since in addition to causing exfoliation in pigs and mice, it induces necrosis in multiple mammalian cell lines, a property not observed for other ETs. Before, we identified the conformation of the necrosis-linked region by crystallography resolution of the ExhC_wt (PDB: 8T3J) and ExhC_mut4 (PDB: 8T3I). Both crystal structures are dimers, this oligomeric state may play a relevant role in regulating the exfoliative activity of ETs. The comprehension of the role of ETs in pathogenesis requires the study of their moonlighting functions, however, the sites of interaction between ExhC and Dsg-1 that trigger the epidermiolytic activity are still unknown, as well as the cellular protein substrate(s) involved in the necrotic activity. In addition, searching for effective inhibitors of the functional activities of ExhC is still necessary. There are many aspects that we do not know about this toxin, which is why we consider it important to characterize his structural stability when exposed to temperature variations. In this study, thermal unfolding experiments (20-80 ℃) using circular dichroism (CD) were implemented and investigations by Small-angle X-ray scattering (SAXS) data were made to evaluate the presence of oligomers in the system and his possible changes in different temperatures (15-40 ℃). These data are being analyzed and we expect that the results contribute to understanding the underlying mechanism of necrotic and exfoliative activities observed in ExhC necessary for designing highly specific inhibitors that can potentially be used clinically and for enhancing diagnostics involving pathogens encoding exfoliative proteins.