Figure 3. Effect of codY mutation on phagocytosis and killing of pathogens by macrophages and the whole blood.

(a,b) Attenuated phagocytosis but enhanced killing of the ΔcodY strain by RAW264.7 macrophages. The wild type (WT) and codY mutated (ΔcodY) strain were incubated with RAW264.7 macrophages (MOI = 100:1) for 1 h followed by washing with PBS and then incubated in DMEM media containing ampicillin (100 μg/ml), when the time point is 0 h, to kill the bacteria cells outside of macrophages. After 1 or 2 hour of incubation, when the time point is 1 h or 2 h, the macrophages were washed, lysed with water followed by plating the lysate dilutions on TSB agar and determining the formed colonies associated to 1 h and 2 h time point respectively. The percentage of the CFU was normalized to WT group at 1 h designed as 100%. The data was showed as Means ± S.E.M; The statistic was conducted using Student’s t test (unpaired t test, two tailed); **P < 0.01 (a); The rates of the survival for WT strains and ΔcodY ones were compared. The survival rate was calculated as CFU at 2 h/ CFU at 1 h × 100%. Each assay was performed in triplicate (b,c) Growth factors of the WT and ΔcodY strains in pig blood. Approximate 10⁶ CFU of the WT and ΔcodY strains were incubated in heparinized pig blood and incubated for 2 h at 37 °C with end-to-end rotation. Growth factor was defined as the ratio of CFU in each sample after 2 h incubation over the CFU in the corresponding inoculum. Each assay was performed in triplicate. The data are expressed as the means ± S.D. of three independent experiment; ***P < 0.001; **P < 0.01; *P < 0.05.