Figure 6. SzP/TRX interaction contributed to the FH recruitment and reduced the C3 deposition on the bacterial surface, beneficial for S. zooepidemicus to evade phagocytosis of the host immune system.

A: The binding of FH to SzP (lane 1), TRX (lane 2) and the SzP/TRX complex (Lane 3) was demonstrated by using polyclonal goat antiserum to FH in an immunoblot analysis. B: TRX inhibited C3 convertase activity in the fluid phase. Fluid phase alternative pathway C3 convertase was generated by the addition of purified C3, C3i, factor B, and 0.1 M MgCl₂. 10 µg of TRX or FH were added followed by the addition of factor D to a final volume of 125 µl. Purified components only (control+); purified complement components without factor D (control-). The inhibition of C3 convertase was determined by C3a generation after 30 min of incubation and measured by a C3a ELISA. The effect of dosage increase of FH and TRX on C3a generation (ng/ml) was shown here. Reduction in C3a generation was correlated with the decreased C3 convertase activity. (Symbol * indicates that a value significantly differs from the control+ group). C: Immunoblot showing C3 components eluted from the surface of the S. zooepidemicus wild strains and the SzP knockout strains following treatment of TRX, FH or TRX and FH in porcine plasma. The blot was developed with the affinity purified antiserum to C3. C3 components eluted from the S. zooepidemicus wild strains and the SzP knockout strains after incubation in porcine plasma were used as the negative control. D: Flow cytometry analysis of S. zooepidemicus surface-bound C3b, a total of 10,000 events were collected per sample and a single gate was used to exclude debris. E: Flow cytometry analysis of gradient concentration TRX pretreated S. zooepidemicus surface-bound C3b. The antiphagocytic effectiveness of the TRX was dose-dependent until saturation. These results were the mean±SD for n = 3, *p<0.05, **p<0.01, ***p<0.001.