Fig 1. Meningitic E. coli exploits EGFR for its penetration of the BBB in vitro and in vivo.

(A) The EGFR-selective inhibitor gefitinib inhibits meningitic E. coli RS218 invasion of HBMEC in a dose-dependent manner, but does not affect its adhesion. * p<0.05, ** p<0.01. (B) Bacterial growth was not affected by the treatment with gefitinib. Overnight bacterial culture was 1:100 transferred into fresh medium with or without gefitinib at indicated concentrations, and further incubated for 2 h. Viable bacterial counts were determined by series dilution and plating at 30 min interval. (C) Gefitinib did not lead to an inhibition of cell proliferation when used at the indicated concentrations. (D) Meningitic E. coli invasion of the EGFR knock-out HBMEC (KO#35) was significantly decreased compared to the invasion of the control cells. * p<0.05. (E) A time-dependent activation of EGFR occurs in response to E. coli RS218 in HBMEC. The ratio of p-EGFR and EGFR was calculated based on densitometry analysis. ** indicates the difference was significant compared to time 0 (p<0.01). (F) EGFR mRNA transcription levels did not change in response to E. coli RS218 in HBMEC, as assessed by real-time PCR analysis. GAPDH was used as the endogenous reference. Representative results from three individual experiments are shown. (G) EGFR protein expression levels were not affected in response to E. coli RS218 in HBMEC. Actin was probed in the same lysate and used as a loading control. (H) RS218 invasion was significantly reduced in HBMEC transfected with the dominant-negative EGFR construct pcDNA-EGFR-GGS compared with pcDNA3.1 control vector-transfected HBMEC. ** p<0.01. (I) E. coli RS218 penetration into the brain was significantly inhibited by administration of gefitinib (10 mg/kg) compared with vehicle treatment. In contrast, the magnitudes of bacteremia did not differ between the recipients of gefitinib and vehicle control. * p <0.05. (J) Co-localization of E. coli strain RS218 and EGFR is demonstrated in HBMEC. Scale bar = 10 μm.