Search for peptide ligands for inhibition against exfoliative toxin c (exhc) from Mammaliicoccus sciuri via Phage Display

Mammaliicoccus sciuri is a pathogenic and commensal bacterium with clinical and veterinary importance. M. sciuri was described as the etiological agent of exudative epidermitis in swine and the main virulence factor involved was exfoliative toxin C (ExhC). M. sciuri ExhC also causes epidermal exfoliation in newborn mice, necrosis in renal fibroblasts in newborn hamsters, and inhibition of phagocytic action by RAW 264.7 macrophages. These last two properties are so far unknown for other exfoliative toxins (ETs) produced by bacteria of the genus Staphylococcus. The inhibition and death of RAW 264.7 macrophages decreased the phagocytosis of a M. sciuri strain by such cells. Cell death by necrosis generates an immune response in the host, involving the action of macrophages in the target tissue of necrosis. Recently, the production of recombinant ExhC fragments led to the conclusion that specific interactions in the domain containing certain amino acid residues are responsible for the necrotic activity in the BHK-21 cell line. The alignment of amino acids in the necrotic domain with the corresponding regions of the other ETs indicated the presence of amino acid residues that are fully conserved or have similar biochemical properties in most ETs, except for M. sciuri ExhC. However, there are still unknown fragments and/or residues of ExhC amino acids involved in the death of RAW 264.7 macrophages. The main objective is to find peptide ligands that can modulate the different activities of ExhC: epidermal exfoliation (1), cell necrosis (2) and inhibition of the host's innate immune response (3). Furthermore, these ligands will be in silico analyzed to assess whether the peptide sequences correspond to partner protein sequences involved in triggering different mechanisms of action (1, 2 and 3). These goals will be achieved through the formation of peptide-ExhC complexes, so that there is interaction with the active site of ExhC and/or secondary sites (exosites or allosteric sites). For this, we will express and purify ExhC from M. sciuri, and use it in Phage Display experiments (technology that allows the expression of exogenous (poly)peptides on the surface of bacteriophage capsids). Through this technology, it is possible to find peptides that bind to protein targets with high affinity and specificity. The peptides then identified will be tested in cell line experiments and enzymatic assays, according to the results obtained through in silico analyses.

This work is supported by FAPESP (2023/05158-3) and associated to FAPESP thematic project (2020/08615-8).