CRYO-EM CHARACTERIZATION OF HOST-SPECIFIC Staphylococcus aureus EXFOLIATIVE TOXINS AND THEIR INTERACTION WITH DESMOGLEIN 1

Staphylococcus aureus is a commensal and opportunistic bacterium capable of infecting a wide range of hosts, including humans and ruminants. It represents a global public health concern and causes substantial economic losses in livestock production. Exfoliative toxins (ETs) are key virulence factors that promote bacterial dissemination by inducing detachment of the epidermal granular layer through specific cleavage of Desmoglein 1 (Dsg1) at the Glu-Gly peptide bond. However, the underlying molecular mechanisms of this interaction remain understood. This study aims to elucidate the molecular and structural basis of ET-Dsg1 interactions at atomic resolution, focusing on native ETs produced by S. aureus. Four ET serotypes have been identified: ETA, ETB, and ETD (primarily human-associated), and ETE (specific to ovines and caprines). In this work, ET proteins will be expressed in E. coli BL21 (DE3)-T1R, while EC3–EC4 ectodomains of human and ovine Dsg1 will be produced using a baculovirus expression system. Proteins will be purified via gel filtration chromatography on a Superdex® 75 10/300 GL column using 20 mM Tris, 150 mM NaCl, pH 7.4. Secondary structures will be assessed by circular dichroism (CD), and thermal stability will be evaluated via differential scanning calorimetry (DSC). Protein complexes will be formed by incubating ETs with Dsg1, followed by structural analysis using time-resolved cryo-electron microscopy (cryo-EM) on a Talos Arctica G2 microscope (Thermo Fisher Scientific). Single-particle analysis (SPA) will be employed for high-resolution structure determination, conducted at LNNano (CNPEM, Campinas, Brazil). To date, ETE has been successfully expressed and purified. CD analysis revealed a predominantly disordered structure, and thermal denaturation experiments showed irreversible unfolding between 35 and 64 °C, with an endothermic transition at 46.4 ± 1.3 °C. Melting temperature was determined as 49.0 ± 1.4 °C, with calorimetric and Van’t Hoff enthalpies of 220 ± 50 and 270 ± 70 kcal/mol, respectively. Other ETs are currently under cloning and expression. The structural characterization of ETD and the EC3–EC4 domains of Dsg1 will enable detailed analysis of protein–protein interactions, supported by comparison with crystallographic data, docking, and molecular dynamics simulations.