GBS pigment was purified as previously described (Rosa-Fraile et al., 2006 (link)) with some modifications. In brief, WT GBS were grown at 37°C in New Granada Media (de la Rosa et al., 1992 (link)) until the broth turned red (48–72 h). Bacterial cells were pelleted, washed three times with distilled water and twice with DMSO. The cell pellet was then resuspended in DMSO:0.1% TFA overnight to extract the pigment, cell debris was pelleted, and the supernatant containing the pigment was saved. The above process was repeated until the supernatant obtained from GBS cells was clear. Pigment was then precipitated by addition of 25% NH4OH to a final concentration of 0.25% as previously described (Vanberg et al., 2007 (link)). Precipitated pigment was washed three times with HPLC grade water and twice in DMSO, redissolved in DMSO:0.1%TFA, and purified using a Sephadex LH-20 (GE Healthcare) column as previously described (Rosa-Fraile et al., 2006 (link); Vanberg et al., 2007 (link)). Fractions containing purified pigment were pooled and precipitated with NH4OH (Scientific Products) as described above, washed three times with HPLC grade water, twice with DMSO, and lyophilized. As a control, GBSΔcylE was also grown in New Granada Media and pigment extraction protocol was followed as described above. For NMR analysis, purified pigment or control ΔcylE samples were resuspended in DMSO-d6:0.1% d-TFA (Sigma-Aldrich). 1H,13C, 1H-COSY NMR experiments were performed at 298K on a Bruker AV-500 NMR Spectrometer. Residual DMSO-d5 was used to calibrate chemical shifts. For MS experiments, lyophilized pigment or control ΔcylE samples were dissolved in DMSO:0.1%TFA and analyzed by Fourier Transform Ion Cyclotron Resonance mass spectrometry on a Bruker AutoFlex APEX Qe 47e instrument. For hemolytic and cytotoxic assays, lyophilized pigment or control ΔcylE extract was dissolved in DTS to a final concentration of 1mM. The samples were incubated overnight at room temperature in the dark before use.
Hemolytic titer assays was performed using methods described with some modifications (Nizet et al., 1996 (link)). In brief, twofold serial dilutions of purified pigment or control ΔcylE extract in DTS was performed in PBS + 0.2% glucose in a final volume of 100 µl. These samples were then incubated with 100 µl of heparin-treated hRBCs (1%) in 96-well plates at 37°C for 1 h, after which the plates were spun for 4 min at 3,000 g to pellet unlysed hRBC. The supernatants were transferred to a replica 96-well plate and hemoglobin release was measured by recording the absorbance at 420 nm. Positive and negative controls included wells that contained hRBC with 0.1% SDS or PBS, respectively. Solvent control for each pigment concentration was included in the analysis. The effective concentration 50 is the concentration of pigment that produces 50% hemoglobin release compared with the SDS control and was determined using non linear regression. The experiment was performed in triplicate using three independent preparations of purified pigment.
For proteinase K treatment of the pigment before hemolytic assays, pigment and control ΔcylE samples in DTS was lyophilized and dissolved in proteinase K buffer (20 mM Tris, pH 8.0, and 1 mM CaCl2). Each sample was divided into two and proteinase K was added at a final concentration of 0.25 mg/ml, as previously described (Vanberg et al., 2007 (link)), to one of the aliquots and all samples were incubated at 37°C for 1 h. Hemolytic titer assays were then performed on pigment and control samples that were treated with and without proteinase K. Buffer controls were also included. The activity of proteinase K used in these experiments was confirmed by digesting 100 µg BSA with 0.25 mg/ml proteinase K at 37°C for 1 h followed by 12% SDS-PAGE and SYPRO Ruby staining.
Hemolytic titer assays was performed using methods described with some modifications (Nizet et al., 1996 (link)). In brief, twofold serial dilutions of purified pigment or control ΔcylE extract in DTS was performed in PBS + 0.2% glucose in a final volume of 100 µl. These samples were then incubated with 100 µl of heparin-treated hRBCs (1%) in 96-well plates at 37°C for 1 h, after which the plates were spun for 4 min at 3,000 g to pellet unlysed hRBC. The supernatants were transferred to a replica 96-well plate and hemoglobin release was measured by recording the absorbance at 420 nm. Positive and negative controls included wells that contained hRBC with 0.1% SDS or PBS, respectively. Solvent control for each pigment concentration was included in the analysis. The effective concentration 50 is the concentration of pigment that produces 50% hemoglobin release compared with the SDS control and was determined using non linear regression. The experiment was performed in triplicate using three independent preparations of purified pigment.
For proteinase K treatment of the pigment before hemolytic assays, pigment and control ΔcylE samples in DTS was lyophilized and dissolved in proteinase K buffer (20 mM Tris, pH 8.0, and 1 mM CaCl2). Each sample was divided into two and proteinase K was added at a final concentration of 0.25 mg/ml, as previously described (Vanberg et al., 2007 (link)), to one of the aliquots and all samples were incubated at 37°C for 1 h. Hemolytic titer assays were then performed on pigment and control samples that were treated with and without proteinase K. Buffer controls were also included. The activity of proteinase K used in these experiments was confirmed by digesting 100 µg BSA with 0.25 mg/ml proteinase K at 37°C for 1 h followed by 12% SDS-PAGE and SYPRO Ruby staining.
