Resumo

B. fragilis is a member of the commensal flora of the human intestine, but is also frequently found in severe intra-abdominal infections. Recently, a putative plasminogen-binding protein, Bfp60 located in the outer membrane (OM), was identified and characterized in B. fragilis by our group. This molecule was assayed and showed to recognize laminin-1 and convert plasminogen (Plg) into plasmin. Several pathogenic bacteria can release from their surface outer membrane vesicles (OMV). These OMV usually contain OM and periplasmic constituents, including proteins, lipoproteins, phospholipids and LPS. In addition some species can incorporate to their vesicle surface membrane proteins, toxins and signaling molecules. B. fragilis releases OMV and contain several enzymes in their cytoplasm and the polysaccharide, named PSA, in their surface that mediate immune regulation and disease. Hence, the main goal of this study was to investigate if B. fragilis OMV possess the plasminogen-binding protein Bfp60 in their surface. The OMV of the B. fragilis strain 630 were isolated after inoculating an overnight culture grown under anaerobic conditions at 37oC into 100mL of defined media previously described as OMV inducer in the species. The vesicles were separated by ultracentrifugation and observed in the transmission electron microscopy (MEV)for confirmation. An immunogold electron microscopy was performed using an anti-Bfp60 against the OMV and a protein extraction followed by a SDS-PAGE and Western blotting (WB). Our results showed that the defined medium was able to over produce the OMV in B. fragilis and the methodology used to isolate the vesicles was also efficient, as it was shown in the MET. The immunogold using the anti-Bfp60 demonstrated the presence of this protein in the OMV, which has also been visualized in the whole cell, and a WB is been performed to prove this result. After the confirmation with the WB, the protein will be sequenced and compared with the Bfp60 found in the surface of B. fragilis to be characterized. The presence of the plasminogen-binding protein in the OMV might help to understand if the Plg recognition and activation can contribute for the abscess formation in B. fragilis.