Figure 2: EspJ inhibits FcγRIIa tyrosine phosphorylation and downstream actin polymerization.

(a–c) Macrophages were infected with GFP-EPEC, EPECΔespJ or EPECΔespJ expressing plasmid-encoded EspJ (pespJ), EspJ-R, EspJ-D or EspJ-R/D and then were challenged with IgG-opsonized RBC. Internalized RBCs (a) (total RBC are shown in red, external RBC in both red and cyan and actin in blue), tyrosine phosphorylation (b) and actin polymerization (c) (arrowheads) were visualized. Scale bars, 10 μm. Only EPEC and EPECΔespJ expressing EspJ reduced RBC phagocytosis, tyrosine phosphorylation and actin polymerization. (d) Cos-7 cells co-expressing FcγRIIa and myc-tagged (blue) EspJ, or EspJ-R/D were challenged with IgG-opsonized RBC and the percentage of internalized RBC quantified (arrowheads). Scale bars, 10 μm. Again, only EspJ could inhibit RBC phagocytosis. Results are the mean±s.d. of three independent experiments in which 100 (a) or 50 (b–d) cells were analysed. Data sets were analysed using one-way analysis of variance (GraphPad Prism v6.0). A significant result is defined as P<0.05 (shown as * and P<0.01 shown as **) as compared with uninfected or untransfected controls. (e) Cos-7 cells co-expressing GFP-tagged FcγRIIa or FcγRIIa Y282F/Y298F with EspJ, EspJ-R/D or an empty vector were treated with anti-FcγR IV.3 antibody with or without secondary antibody crosslinking. The FcγRIIa was immunoprecipitated and analysed by immunoblotting with anti-pTyr and anti-GFP antibodies, which shows phosphorylation of wild-type FcγRIIa in the control and EspJ-R/D-expressing cells (arrow), but not in cells expressing EspJ or FcγRIIa Y282F/Y298F. Similar results were obtained in three independent experiments. Representative immunoblots are shown. Full immunoblots are shown in Supplementary Fig. 3.