Figure 4.

B. miyamotoi CbiA-bound plasminogen is converted to active plasmin by uPA. Microtiter plates were coated with 5 μg/ml of plasminogen (Plg) (A), BSA (B), CbiA (C), CspA (D), or BBA70 (E). The latter four proteins were subsequently incubated with 10 μg/ml plasminogen. Following several wash steps, a reaction mixture containing the plasminogen activator uPA (final concentration of 0.1 μg/ml) and the chromogenic substrate D-Val-Leu-Lys-p-nitroanilide dihydrochloride (S-2251) was added (■). Control reactions included 50 mM of the lysine analog tranexamic acid (T) (♦) or omitted plasminogen (▾) or uPA (▴), respectively. Microtiter plates were incubated at room temperature for 24 h and absorbance at 405 nm was measured at 30 min intervals. At least three independent experiments were conducted, each in triplicate. Data shown are from a representative experiment. Evaluation of the statistical significance (F). The OD values of the final measuring point (24 h) of CbiA, BBA70, DbpA, and BSA were used for the calculation using GraphPad prism 7. ***p ≤ 0.002, ****p ≤ 0.0001 compared with BSA as negative control. Raw data were analyzed by one-way ANOVA with post-hoc Bonferroni multiple comparison test.