Caracterização estrutural e funcional de um homólogo à exotoxina esfoliativa D de Staphylococcus aureus envolvido na mastite subclínica de pequenos ruminantes

Abstract

Mastitis is the most common and costly disease affecting dairy cattle, being S. aureus the most frequently isolated microorganism from clinical cases of this infection. This disease is characterized by inflammation of the mammary gland, with local symptoms that can result in systemic infection. A better understanding of the factors involved in pathogenicity, as well as the host response to infection by this microorganism, are key points for an efficient and satisfactory development of therapies to prevent and combat mastitis. Although considered an opportunistic pathogen, certain strains of S. aureus are more prone to cause pathologies than others, due to the variety of virulence factors associated to them. Through serological proteome analysis (SERPA) that enables the identification of differentially expressed immunoreactive proteins, the pathogenic profiles of S. aureus strains O46 - characteristic of subclinical mastitis - and O11 - characteristic of gangrenous mastitis - were evaluated. Based on these data, the aim of this study was to characterize a protein, encoded by the ORF O46_2740, immunoreactive only in S. aureus O46 and homologous to exfoliative toxin D. This protein is considered epidermolytic toxin and was associated to bullous impetigo and staphylococcal scalded skin syndrome (SSSS). A three-dimensional homology model was designed in silico using an ETB mold deposited in databases. For in vivo characterization we obtained recombinant strains of L. lactis containing the ORF O46_2740 coupled with the XIES expression system, but Western blotting did not confirm the production of ETD-like protein. Alternatively, the ORF was introduced in E. coli under control of T5 promoter, and the expression of ETD-like was successfully confirmed by Western blotting. ETD-like was purified using anti-His monoclonal antibody and applied on the back of newborn Balb/c mice to evaluate its effects on the skin. Preliminary results of in vivo assessment indicated the formation of bullous reactions also caused by the epidermolytic activity of S. aureus ETD; however, no exfoliative activity was observed.