Acetate assimilation system encoded by acs-yjcH-actP operon contributed to APEC intracellular replication within macrophages. A Deletion of acs-yjcH-actP operon impaired FY26 intracellular replication within HD11 macrophages. The survival rates of wild-type FY26, four mutants (FY26Δacs, FY26ΔactP, FY26Δacs-yjcH-actP, and FY26ΔsatP), and the complemented strain FY26Cacs-yjcH-actP were measured to investigate the contribution of the acetate assimilation system on APEC proliferation. B To determine the contribution of acetate assimilation system on APEC replication within RAW264.7 macrophages. Macrophages were infected at a MOI of 10. Data acquired from at least four individual experiments, and each assay was performed by three biological repetitions. The significant differences were identified using two-way ANOVA analysis (*P < 0.01), and the mean values ± SEs were shown in each plotting. C, D Determine the effect of acetate assimilation system on APEC replication within macrophages by immunofluorescence assays. HD11 cells were infected at a MOI of 5. C Quantification of wild-type FY26, FY26Δacs, FY26Δacs-yjcH-actP, and FY26Cacs-yjcH-actP intracellular proliferation in macrophages was indicated. The percentage of infected HD11 cells containing ≤ 2, 3 to 5, 6 to 10, and ≥ 11 bacteria per cell at 8 hpi was counted by fluorescence microscopy. Data for quantification of bacteria proliferation was acquired from more than 100 infected cells for three independent experiments. The significant differences were identified using one-way ANOVA analysis (*P < 0.01), and the mean values ± SEs were shown. D Representative confocal microscopy images of wild-type FY26, FY26Δacs, FY26Δacs-yjcH-actP, and FY26Cacs-yjcH-actP intracellular proliferation in macrophages at 8 hpi were shown. The infected HD11 cells were fixed and labeled with DAPI and Phalloidin (actin staining; TRITC conjugated). Bacteria were incubated with an anti-APEC antibody and immediately labeled with a fluorescent secondary antibody (Alexa 488, green). Scale bar = 10 μm.