IbeA and OmpA of Escherichia coli K1 exploit Rac1 activation for invasion of human brain microvascular endothelial cells

Abstract

Meningitis-causing Escherichia coli K1 internalization of the blood-brain barrier is required for penetration into the brain, but the host-microbial interactions involved in E. coli entry of the blood-brain barrier remain incompletely understood. We show here that a meningitis-causing E. coli K1 strain RS218 activates Rac1 (GTP-Rac1) of human brain microvascular endothelial cells (HBMEC) in a time-dependent manner. Both activation and bacterial invasion were significantly inhibited in the presence of a Rac1 inhibitor. We further showed that the guanine nucleotide exchange factor Vav2, not β-Pix, was involved in E. coli K1-mediated Rac1 activation. Since activated STAT3 is known to bind GTP-Rac1, the relationship between STAT3 and Rac1 was examined in E. coli K1 invasion of HBMEC. Downregulation of STAT3 resulted in significantly decreased E. coli invasion compared to control HBMEC, as well as a corresponding decrease in GTP-Rac1, suggesting that Rac1 activation in response to E. coli is under the control of STAT3. More importantly, two E. coli determinants contributing to HBMEC invasion, IbeA and OmpA, were shown to affect both Rac1 activation and their association with STAT3. These findings demonstrate for the first time that specific E. coli determinants regulate a novel mechanism of STAT3 cross talk with Rac1 in E. coli K1 invasion of HBMEC.

Fig 1

Examination of Rac1 activation in HBMEC incubated with E. coli K1 strain RS218. (A) To analyze the role of GTP-Rac1 in E. coli adhesion to and invasion of HBMEC, host cells were pretreated with either the Rac1 inhibitor (NSC23766) or vehicle control, incubated with the bacteria, and examined for bacterial adhesion and invasion. Values were graphically represented, with the results from control cells being taken as 100%. *, P < 0.05 compared to no inhibitor. (B) HBMEC treated with NSC23766 (100 μM) or vehicle control were incubated with E. coli for various time points and then lysed. Lysates were incubated with PBD-GST beads, boiled in SDS-PAGE buffer, and analyzed for activated Rac1 (GTP-Rac1) by Western blotting. Lysates were also examined for total Rac1. (C) Band intensities were estimated densitometrically by ImageJ software and expressed graphically as the ratio of GTP-Rac1 to total Rac1. The fold change was estimated with respect to time zero.

Fig 2

Role of GEFs on Rac1 activation in response to E. coli K1 invasion. To identify the role of guanine nucleotide exchange factors (GEFs) in E. coli-dependent Rac1 activation, HBMEC were transfected with downregulating (DN) plasmids of the Rac1 GEFs β-Pix (A) and Vav2 (B) and analyzed for expression of either FLAG (β-Pix) or c-myc (Vav2) present in the constructs. Arrows denote the expression of either the wild-type or downregulating molecules. Next, HBMEC transfected with either β-Pix (C) or Vav2 (D) were examined for E. coli adhesion and invasion in comparison to control cells. Bacterial adhesion and invasion were graphically represented as the relative frequency (%), with values from control cells taken as 100%. *, P < 0.05 compared to wild-type transfected HBMEC. Cells transfected with either with β-Pix (E) or Vav2 (F) were grown in monolayers and incubated with E. coli for various time periods. Lysates were analyzed for either activated Rac1 (GTP-Rac1) using GST-PBD beads or total Rac1 by Western blot assays.

Fig 3

STAT3 regulation of GTP-Rac1 in E. coli invasion of HBMEC. (A) To examine the effect of STAT3 on E. coli adhesion and invasion of HBMEC, the host cells were transfected with STAT3β or the control vector and examined for E. coli adhesion and invasion. *, P < 0.05 compared to vector control. (B) To analyze the effect of STAT3 downregulation on the activation of Rac1, HBMEC transfected with either STAT3β or control vector were incubated with E. coli for various time periods. Cell lysates were then examined for either pSTAT3 (i) or Rac1 activation (ii) by Western blot analysis.

Fig 4

Effect of 17-AAG on STAT and Rac1 activation, as well as E. coli invasion of HBMEC. (A) To examine the effect of 17-AAG (a geldanomycin and HSP90/STAT3 inhibitor) on E. coli adhesion to and invasion of HBMEC, HBMEC were treated with the inhibitor or vehicle control and then examined for E. coli adhesion and invasion. *, P < 0.05 compared to vehicle control. (B and C) HBMEC treated with 17-AAG or vehicle control were incubated with E. coli for various time points, and the lysates were analyzed for either p-STAT3 (Bi) or Rac1 (Bii) activation by Western blotting. Band intensities were estimated densitometrically and are expressed as the fold increase in the pSTAT3/STAT3 (Ci) or GTP-Rac1/total Rac1 (Cii) ratio.

Fig 5

Specific virulence factors of E. coli RS218 activate host cell Rac1. (A) To examine the association of STAT3 with Rac1 in response to RS218, HBMEC were incubated with the bacteria for 30 min, and lysates were examined for GTP-Rac1 using PBD-PAK1 beads. The beads were also examined for STAT3 in a Western blot assay. (B) To assess the contribution of the microbial factors to Rac1 activation, HBMEC were incubated with either wild-type RS218 or its mutants. Lysates were mixed with PBD-GST beads and examined for activated Rac1 (GTP-Rac1) and GTP-Rac1-associated STAT3. Total amounts of Rac1 in the lysates were also determined by Western blot analysis. The band intensities of Rac1, GTP-Rac1, and Rac1-bound STAT3 were measured by ImageJ software, and the results expressed as ratios of GTP-Rac1/total Rac1 or GTP-Rac1 bound STAT3/total Rac1 with the values of time zero as 1.

Fig 6

Diagrammatic representation of STAT3 regulation of Rac1 in E. coli K1 invasion of HBMEC. Whereas earlier studies showed that FimH and CNF1 activate RhoA for E. coli internalization of HBMEC (12, 13), we hypothesize, based on the information obtained in the present study, that OmpA and IbeA trigger the phosphorylation of STAT3, which aids in converting GDP-Rac1 to GTP-Rac1 (through an as-yet-unknown mechanism involving the GEF, Vav2). Inhibitors or downregulators of either STAT3 or Rac1 decrease E. coli invasion of HBMEC. This study shows for the first time a molecular mechanism of E. coli regulation of Rac1 activation via STAT3 for E. coli internalization into HBMEC.