Identification of potential inhibitors of necrotic and epidermolytic activity of exfoliative toxin C from Staphylococcus sciuri through secondary sites mapping

Abstract

Staphylococcus sciuri is a commensal and pathogenic bacterium of significant clinical and veterinary relevance. Recently, a strain of S. sciuri has been described as the etiologic agent of exudative epidermitis (EE) in pigs and the main virulence factor involved in this clinical manifestation was the exfoliative protein C (ExhC). This enzyme belongs to the group of exfoliative toxins (ETs) that are commonly described as glutamyl endopeptidases belonging to the chymotrypsin family. However, unlike most members of this family, ETs are inactive against a wide range of substrates, showing exquisite specificity for desmoglein-1 (Dsg-1), a skin protein responsible for cell-to-cell adhesion. The cleavage of Dsg-1 causes exfoliation of the mammalian epidermis and increases the efficiency of invasion and infection of the host by pathogenic bacteria. This fine-tuned substrate specificity suggests that, in addition to the catalytic triad of serine-proteinases, other unknown surface regions of ETs and Dsgs-1 may be essential to trigger the exfoliative activity. ExhC has additional importance because, besides causing EE in pigs and mice, it is also capable to induce necrosis in multiple mammalian cell lines, a property not observed for other ETs. During the development of the national research project, essential residues for necrotic activity were identified and the resolution of the crystallographic structures of wild-type and mutant ExhC indicated the possible location of the binding site responsible for triggering death in mammalian cells. 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, a search for effective inhibitors of the functional activities of ExhC is still necessary. In this research project, phage display assays, together with computational biology and biophysics techniques, will be extensively explored to unravel the main mechanisms of action of ExhC, using peptide sequences with high interaction affinity, not only to inhibit epidermiolytic and/or necrotic activity (1), but also to identify ExhC exosites and allosteric sites (2), critical domains of interaction between the ExhC-Dsg-1 for epidermolytic activity (3) and in the search for cellular protein(s) involved in necrotic activity (4). The strategy is to use the TriCo-16 library to screen for ExhC-binding peptides. Subsequently, high-precision computational methods will map, select and evaluate protein-peptide interaction for screening of exosites, allosteric sites and identification of potential inhibitors. The identity of the peptide sequences with fragments of Dsgs-1 and other mammalian cell proteins will also be investigated by in silico analysis. Finally, the low, medium or high affinity of interaction between ExhC-peptides will be confirmed by the Surface Plasmon Resonance technique, providing a better direction for the assays of inhibition of the necrotic and/or epidermiolytic activity of ExhC. (AU)