The deposition of C3b on the surface of aEPEC O55:H7 and STEC O55:H7 was determined by the dot blot technique. Briefly, nitrocellulose membranes (0.42 µM) were coated with 2 µL of bacterial samples (1 × 1013 CFU/mL). Subsequently, the membranes were incubated with 3% BSA in PBS overnight at room temperature. After incubation, the membranes were washed 3 times with PBS containing 0.05% Tween 20 (washing buffer) and incubated for 1 h at 37 °C with a solution of 10% normal human serum (NHS) in incubation buffer (1% BSA in PBS). NHS was used as a source of C3b. In sequence, the membranes were washed and incubated for 1 h with goat anti-C3b antibodies (Complement Technology, Tyler, TX, USA) diluted 1:5000 in incubation buffer. After incubation, the membranes were washed and incubated for 1 h with anti-goat IgG peroxidase conjugate (Sigma-Aldrich) diluted 1:10,000 in incubation buffer. The membranes were then washed, and the deposition of C3b on the bacterial surface was determined by chemiluminescence analyses utilizing SuperSignal West Pico Enhanced Chemiluminescent Substrate (Pierce Biotechnology, Inc.—Thermo Fisher Scientific, Waltham, MA, USA). The intensity of the signals was determined in pixels using Image J software. The results were plotted as “Mean Gray Values” (average intensity units in selection).
Supersignal west pico enhanced chemiluminescent substrate
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SuperSignal® West Pico Enhanced Chemiluminescent Substrate is a laboratory reagent used to detect and quantify proteins in Western blot analysis. It is designed to generate a chemiluminescent signal that can be measured using a compatible imaging system.
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5 protocols using supersignal west pico enhanced chemiluminescent substrate
1
Quantifying C3b Deposition on E. coli Strains
2
Quantifying C1q Deposition on EPEC
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The deposition of C1q on the surface of EPEC O26:H11 was also assessed using the dot blot technique. Nitrocellulose membranes (0.42 µM) were coated with 2 µL of EPEC O26:H11 culture (1 × 1013 CFU/mL) previously incubated for 1 h at 37 °C in the presence or absence of anti-O26 polysaccharide antibodies diluted 1/10 in PBS. As a positive control, 2 µL of C1q (125 ng/µL) were directly coated onto the membrane.
The membrane was blocked and washed as described above, and subsequently, incubated for 1 h with goat IgG anti-C1q (Complement Technology, Tyler, TX, USA) diluted 1:5000 in incubation buffer. After washing, the membranes were incubated for 1 h at room temperature with rabbit anti-goat IgG conjugated with peroxidase (Sigma-Aldrich) diluted 1:10,000 in incubation buffer. Following another round of washing, the deposition of C1q on the bacterial surface was detected by chemiluminescence using SuperSignal West Pico Enhanced Chemiluminescent Substrate (Pierce Biotechnology, Inc.—Thermo Fisher Scientific, Waltham, MA, USA). The intensity of the signals was determined in pixels using Image J software, Version 1.5, developed at the National Institutes of Health and the Laboratory for Optical and Computational Institutes (LOCI, University of Wisconsin, Madison, WI, USA), and the results were presented as ‘Mean gray values’ (average intensity units in pixels).
The membrane was blocked and washed as described above, and subsequently, incubated for 1 h with goat IgG anti-C1q (Complement Technology, Tyler, TX, USA) diluted 1:5000 in incubation buffer. After washing, the membranes were incubated for 1 h at room temperature with rabbit anti-goat IgG conjugated with peroxidase (Sigma-Aldrich) diluted 1:10,000 in incubation buffer. Following another round of washing, the deposition of C1q on the bacterial surface was detected by chemiluminescence using SuperSignal West Pico Enhanced Chemiluminescent Substrate (Pierce Biotechnology, Inc.—Thermo Fisher Scientific, Waltham, MA, USA). The intensity of the signals was determined in pixels using Image J software, Version 1.5, developed at the National Institutes of Health and the Laboratory for Optical and Computational Institutes (LOCI, University of Wisconsin, Madison, WI, USA), and the results were presented as ‘Mean gray values’ (average intensity units in pixels).
3
Extracellular Sat Protein Detection
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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).
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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).
4
Evaluating Sat Proteolytic Activity on Complement Proteins
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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.
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.
5
Western Blot Analysis of Nodule and Extracellular Proteins
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Nodule proteins and extracellular proteins were first resolved on 15% SDS-PAGE gels as described earlier. The separated proteins were electrophoretically transferred onto nitrocellulose membranes for 1 h. Following the transfer, the membranes were stained with Ponceau S to visually confirm the transfer. Nitrocellulose membrane were then incubated with 3% dry milk powder and dissolved in Tris-buffered saline (TBS; pH 7.4) for 1 h at room temperature with gentle shaking. After that, the nitrocellulose membrane was incubated with leghemoglobin or nitrogenase antibody that was diluted 1:20,000 in TBS containing 3% dry milk powder. For the detection of Nops, a cocktail of antibodies raised individual Nops were used at a final dilution of 1:10,000. Nonspecific binding was eliminated by washing the membrane three times (10 min for each wash) with TBS containing 0.05% Tween-20 (TBST). Bound antibodies were detected by incubating the nitrocellulose membrane with 1:20,000 of goat anti-rabbit IgG−horseradish peroxidase conjugate chemiluminescent antibody (Bio-Rad, Hercules, CA, USA) for 1 h. Following the incubation, the membrane was washed three times with TBST as mentioned above. Immunoreactive polypeptides were visualized by incubation of the membrane with SuperSignal West Pico enhanced chemiluminescent substrate (ThermoFisher, St. Peters, MO, USA).
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