| Congresso Brasileiro de Microbiologia 2023 | Resumo: 858-1 | ||||
Resumo:The serine protease Pet is an autotransporter protein of the SPATEs family, important in the pathogenicity of Escherichia coli. The pet gene was initially found in the E. coli virulence plasmid, pAA2. Although this virulence factor was initially described in an intestinal pathotype, pet may also be present in other E. coli pathotypes, such as ExPEC. The complement system is an important defense mechanism of the immune system that can be activated by invading pathogens. Proteases produced by pathogenic bacteria, such as SPATEs, have proteolytic activity and can cleave components of the complement system, promoting bacterial resistance to human serum. Considering these factors, the proteolytic activity of Pet and its role in evading the complement system were investigated. Proteolytic assays were performed by incubating commercial components of the complement system with Pet and Pet S260I proteins, obtained from the supernatants of clone HB101 (pCEFN1) and clone HB101 (pCEFN2). Cleavage patterns were analyzed by Immunoblotting using specific antibodies for C3, C5 and C9. Furthermore, the inhibition of C9 polymer formation induced by the ZnCl2 catalyst was also analyzed in vitro. Finally, resistance of E. coli DH5a incubated with human serum pre-treated with Pet and with the negative control was evaluated. The obtained results showed that Pet can cleave C3, C5 and C9 components. Pet also inhibited the natural formation of C9 polymers in proteolytic assays. Furthermore, a dose-dependent inhibition of ZnCl2-induced C9 polymerization in vitro was observed. E. coli DH5a survived incubation with human serum pre-treated with the supernatant containing Pet. The polymerization of C9 monomers and the formation of the membrane attack complex, mediated by the complement system, are essential for cell lysis of invading pathogens. Therefore, Pet can potentially interfere with the classical, alternative and lectin pathways, and may even inhibit the formation of important by-products of the cascade, as well as inhibiting the formation of MAC in the bacterial outer membrane. Thus, theses results demonstrate that Pet is a virulence factor capable of cleaving components of the complement system and that it may contribute to the resistance of Escherichia coli to human serum. Palavras-chave: Bacterial toxins, Complement system, Escherichia coli, Immune system cells, Serine proteases Agência de fomento:FAPESP & Fundação Butantan | |||||