| Congresso Brasileiro de Microbiologia 2023 | Resumo: 41-1 | ||||
Resumo:INTRODUCTION: The main virulence phenotype of enteropathogenic Escherichia coli (EPEC) is the formation of the attaching and effacing lesion, which is characterized by the destruction of host cell microvilli through the recruitment of polymerized F-actin at the adherence site, leading to the formation of a pedestal-like structure. According to the presence or absence of the bfp operon, EPEC isolates can be classified as typical (tEPEC) or atypical (aEPEC), respectively. In previous studies, our group demonstrated that aEPEC is the main diarrheagenic E. coli pathotype isolated in Brazil, as well as the association of specific serotypes, including the serotype O2:H16, with diarrheal outbreaks. A comparative gene analysis of 106 sequenced aEPEC, including 7 aEPEC of serotype O2:H16, revealed a set of 31 genes exclusively detected in this serotype. Among these genes, we identified one responsible for encoding an uncharacterized autotransporter (AT) protein. Therefore, the objective of the present study was to evaluate the contribution of the novel AT protein in biofilm formation and its ability to bind to extracellular matrix components. MATERIAL AND METHODS: The gene encoding the novel AT protein was cloned into the vector pBAD/Myc-His-A, generating the recombinant plasmid pIC, which was transformed into the E. coli MS427 strain, an MG1655 derivative delete for the agn43 aggregation factor. This strain was used in a biofilm formation assay performed on a polystyrene surface with 24, 48, and 72 hours of incubation using lysogeny broth. Additionally, we cloned the passenger domain-encoding region (amino acids 22 to 471) into the pET-28a vector, which was transformed into a BL21(DE3). The recombinant protein carrying a His-Tag was expressed through IPTG induction, purified via immobilized metal affinity chromatography, and used for rabbit anti-serum production. Assays to measure binding to macromolecules of the extracellular matrix were performed using 0.1 µM of the purified passenger domain recombinant protein against 1 µg of collagens I, III, IV and V, cellular and plasma fibronectin, laminin, fibrinogen and vitronectin. RESULTS: Production of the novel AT protein in MS427(pIC) and the wild type BA92 was confirmed by immunoblotting and immunogold. Biofilm formation was significantly increased (p<0.0001) in MS427 harboring the pIC recombinant plasmid, compared to the MS427 carrying the empty pBAD/Myc-His-A vector. Furthermore, the purified recombinant passenger domain attached to plasma fibronectin. In conclusion, we demonstrated that the novel AT protein characterized in the present study may have a role on the pathogenicity of EPEC isolates by mediating biofilm formation and binding to plasma fibronectin. Palavras-chave: atypical EPEC, autotransporter proteins, virulence Agência de fomento:CAPES (88887.482477/2020-00) e FAPESP (2017/14821-7) | |||||