Alliance image system

E. coli EC071 was grown for 18 h in 5 mL of LB broth at 37°C under constant shaking (250 rpm). The culture was harvested at 2.000 x g for 15 min at 4°C and 1 mL aliquots of the supernatant were precipitated with 10% trichloroacetic acid (TCA) (Sigma-Aldrich, USA), as described elsewhere (48 (link)). Culture supernatants of enteroaggregative E. coli (EAEC) EC233/93 and diffusely-adherent E. coli (DAEC) FBC 114 were prepared as described above and used as Sat-producing strains (positive controls). Shigella flexneri M90T culture supernatant, similarly prepared, was used as a negative control (41 (link), 44 (link), 49 (link)).
The resulting precipitated supernatants were denatured with β-mercaptoethanol at 96°C for 5 min for further analysis by 10% SDS-PAGE (2 independent gels) (50 (link)). The first gel was stained by silver nitrate (51 (link)) and the second one was used for immunoblotting assays, employing polyclonal anti-Sat serum (44 (link)) and peroxidase-conjugated anti-rabbit IgG as secondary antibody (Sigma-Aldrich). Signal detection was performed using SuperSignal^® West Pico Enhanced Chemiluminescent Substrate (ThermoFisher Scientific) and the Alliance Image System (UVITEC, UK).

Initially, the proteolytic activity of purified Sat was tested against the following purified complement proteins (Complement Technology, USA): C1q, C2, C3 and C3b, C4 and C4b, C5, C6, C7, C8 and C9. To identify possible Sat substrates among these complement proteins, 5 µg of Sat were incubated with 0.5-1.0 µg of each complement molecule in the presence of MOPS buffer (0,1 M MOPS, 0,2 M NaCl and 0,01 mM ZnSO4, pH 7,3) (40 (link)) at 37°C for 5 or 24 h. As a control for spontaneous cleavage, complement molecules diluted in MOPS buffer were incubated under the same conditions. Incubation products were analyzed by immunoblotting using specific antibodies to each complement protein (Complement Technology, USA), and peroxidase-conjugated anti-goat IgG as the secondary antibody (Sigma-Aldrich). Signal detection was performed using the SuperSignal^® West Pico Enhanced Chemiluminescent Substrate (ThermoFisher Scientific) and the Alliance Image System (UVITEC, UK).
Dose dependency of Sat-induced cleavage of the substrates was evaluated using lower concentration of purified Sat (0.5 or 1.0 µg). Also, inhibition of Sat proteolytic activity was assessed by incubating purified Sat (0.5 or 1.0 µg) with 1.0 mM phenylmethylsulfonyl fluoride (PMSF) for 30 min at room temperature before the addition of complement proteins. Incubation products were analyzed as described above.