Figure 3: EspJ inhibits EPEC- and Vaccinia virus-induced actin polymerization and recruitment of active Src to sites of bacterial attachment.
From: The Escherichia coli effector EspJ blocks Src kinase activity via amidation and ADP ribosylation
Cultured cells expressing Flag-tagged GFP, EspJ or, EspJ-R/D were infected with EPEC (a) or Vaccinia virus (b) and were quantified for actin polymerization associated with attached EPEC (DAPI stained) or Vaccinia virions (RFP-tagged A3 viral core protein). EspJ, but not EspJ-R/D, inhibited actin polymerization under both EPEC and Vaccinia virions. (c) Cells expressing Flag-tagged GFP, EspJ, or EspJ-R/D, were infected with EPEC and stained with anti-pY416, to detect active SFKs, and 4',6-diamidino-2-phenylindole (DAPI). EspJ, but not EspJ-R/D, inhibited accumulation of pY416 under adherent EPEC. Fifty cells were analysed in each of three independent experiments. Data sets were analysed using one-way analysis of variance (GraphPad Prism v6.0). A significant result is defined as P<0.05 (P<0.01 shown as ** and P<0.0001 shown as ****) as compared with control infections. Scale bars, 10 μm (a,c) or 20 μm (b). (d) Swiss 3T3 cells co-expressing GFP, Src-GFP or Src Y527F-GFP with Flag-tagged EspJ, EspJ-R/D or an empty vector were analysed by anti-pTyr immunoblot. EspJ inhibited general protein tyrosine phosphorylation. Similar results were obtained in three independent experiments. Representative immunoblots are shown. Full immunoblots are shown in Supplementary Fig. 7.
