Human plasminogen

GAS cultures were grown overnight to stationary phase. The next day, 300 μl of culture was added to 3 ml of THB supplemented with 1 unit/ml of human plasminogen (Calbiochem) and 7 μm human fibrinogen (Calbiochem) to facilitate cell surface plasmin acquisition, or THB only as the negative control. Cultures were grown to A₆₀₀ = 0.4, divided into 3 × 1-ml aliquots in siliconized tubes, centrifuged for 5 min at 6,000 × g, and bacterial pellets were washed once with 1 ml of sterile PBS. Following resuspension in 200 μl of PBS, 10-μl aliquots were collected for cfu enumeration, prior to transferring 180 μl into V-bottom 96-well plates (Costar) and adding 20 μl of substrate S-2251 (Chromogenix). The plate was incubated in the dark for 1 h at 37 °C, centrifuged for 10 min at 500 × g, and 100 μl from each well was transferred into a flat bottom 96-well plate (Costar). The A₄₀₅ was measured using SpectraMax 250 (Molecular Devices). Cell surface plasmin activity was calculated as absorbance units/cfu. The following control wells were used: positive control, 1 unit/ml of human plasminogen + 1 μg of streptokinase from group C Streptococcus (Sigma); negative control, 1 unit/ml of human plasminogen only; substrate negative control, PBS only.

Human IgG, papain-treated fragments of Human IgG, human plasminogen and fibrinogen were purchased from Sigma. Polyclonal antibodies against Human IgG and plasminogen as well as HRP-conjugated anti-human antibodies were purchased from Dako. Antibodies directed against human C1q were from Calbiochem. Ethidium homodimer-1 and the secondary rabbit anti-mouse IgG Alexa Fluor 488 conjugated antibody (Cat#: A-11059; RRID: AB_2534106) were obtained from Life Technologies. 16% formaldehyde without methanol and CitiFluor™ CFM3 mounting medium were obtained from Electron Microscopy Science.

Briefly, a 96-well plate was coated with 1.0 μg/well of rNTHiENO diluted in 100 μL of PBS (phosphate-buffered saline) and incubated overnight at 4 °C. After that, three washes with PBS-Tween (PBST) 0.5% were performed, and the wells were blocked with 2% BSA for 2 h at 37 °C. The plate was washed three times again and incubated with 0.01–10 μg/well of human plasminogen (Sigma-Aldrich, USA) diluted in 100 μL of PBS or with BSA as a negative control at the same concentrations for 2 h at 37 °C. Binding protein was detected with polyclonal anti-Plg antibodies as first antibody and goat anti-mouse IgG-HRP-conjugated as the secondary antibody.
Inhibition experiments were performed by adding polyclonal anti-rNTHiENO antibodies (serial dilutions from 1/100 to 1/40,000) prior to the addition of Plg (1.0 μg/well). These antibodies were obtained in a previous work [20 (link)]. The interaction was visualized using 3,3’,5,5’-Tetramethylbenzidine (TMB; Sigma-Aldrich), and the reaction was stopped with the addition of 50 μL of 0.5 M sulfuric acid, and, finally, the OD was read at 450 nm. All experiments were repeated three times.