Interaction of OnCL-K1 with OnCD93

The Mϕs were preprepared and adjusted to a concentration of 1 × 10⁶ cells/ml using fresh L-15 medium containing 10% FBS and 1% penicillin/streptomycin. The treatment group was exposed to OnCL-K1 eukaryotic protein (5 μg/ml), anti–OnCL-K1 pAb (5 μg/ml), BSA (5 μg/ml), and PBS in the presence of formalin-inactivated S. agalactiae or A. hydrophila (1 × 10⁶ CFU/ml). All groups were maintained at 25°C, and cells were collected and lysed with TRIzol reagent for RNA extraction at 6, 12, 24, and 48 h postchallenge. The expression of OnCD93 in the Mϕs was performed using qRT-PCR. Furthermore, the impact of OnCL-K1 knockdown on OnCD93 expression in infected tilapia was assessed using qRT-PCR and Western blotting.

To confirm the interaction between OnCL-K1 and OnCD93, ELISA and IP assays were conducted. For the ELISA experiment, (r)OnCD93 and Trx were labeled with biotin hydrazide. The 96-well plates were coated with OnCL-K1 eukaryotic protein (2 μg/ml) and incubated at 4°C overnight, followed by blocking for 2 h at 37°C. After washing, different concentrations of (r)OnCD93 (0, 2, 5, 10, and 20 μg/ml) were added and incubated for 1 h. After washing three times, we detected (r)OnCD93 using streptavidin-HRP conjugate (1:2500, SouthernBiotech) for 1 h at 37°C. Then, the reaction was measured at OD₄₅₀ using a microplate reader. Additionally, the binding of OnCL-K1 and (r)OnCD93 was detected by IP using the same method as described above.

To investigate whether the binding sites of CL-K1 and CD93 were the same as those of MASP-1/3, we performed a competitive ELISA according to the previously described method (29). Briefly, fixed concentrations of (r)OnCD93 (2 μg/ml) and OnCL-K1 eukaryotic protein (2 μg/ml) were incubated with increasing concentrations (0, 2, 5, 10, 20, 40, 80, and 100 μg/ml) of (r)OnMASP-1/3 and Trx, respectively. The above mixtures were then incubated in mannan (10 μg/ml)-coated microtiter wells. After incubation and washing, the amounts of (r)OnMASP-1/3 binding to OnCL-K1 were measured by ELISA using streptavidin-HRP conjugate (1:2500). The color reaction and reading were performed as described above.

To investigate the impact of the interaction between OnCL-K1 and CD93 on complement-mediated cell lysis, the hemolysis assay of CRBCs was conducted as described above. Briefly, fresh and healthy tilapia serum was preincubated with mannan (1 μg/ml) for 1 h. Various concentrations of (r)OnCD93 (0, 5, 10, 20, and 50 μg/ml) were mixed with 100 μl of 2% CRBCs and the preincubated serum, bringing the total volume to 250 μl. The mixture was incubated at 28°C for 1 h, and the cell supernatant (100 μl) was detected at OD₄₀₅ nm by a microplate reader (Thermo Fisher Scientific, Waltham, MA). The BSA (50 μg/ml) group was used as a control. Additionally, the effect of the association between OnCL-K1 eukaryotic protein (5 μg/ml) and (r)OnCD93 (0, 5, 10, 20, and 50 μg/ml) on complement-mediated cell lysis was also assessed using the hemolysis assay as described above.