Vimentin, a Novel NF-κB Regulator, Is Required for Meningitic Escherichia coli K1-Induced Pathogen Invasion and PMN Transmigration across the Blood-Brain Barrier

Abstract

Background: NF-κB activation, pathogen invasion, polymorphonuclear leukocytes (PMN) transmigration (PMNT) across the blood-brain barrier (BBB) are the pathogenic triad hallmark features of bacterial meningitis, but the mechanisms underlying these events remain largely unknown. Vimentin, which is a novel NF-κB regulator, is the primary receptor for the major Escherichia coli K1 virulence factor IbeA that contributes to the pathogenesis of neonatal bacterial sepsis and meningitis (NSM). We have previously shown that IbeA-induced NF-κB signaling through its primary receptor vimentin as well as its co-receptor PTB-associated splicing factor (PSF) is required for pathogen penetration and leukocyte transmigration across the BBB. This is the first in vivo study to demonstrate how vimentin and related factors contributed to the pathogenic triad of bacterial meningitis.

Methodology/principal findings: The role of vimentin in IbeA+ E. coli K1-induced NF-κB activation, pathogen invasion, leukocyte transmigration across the BBB has now been demonstrated by using vimentin knockout (KO) mice. In the in vivo studies presented here, IbeA-induced NF-κB activation, E. coli K1 invasion and polymorphonuclear neutrophil (PMN) transmigration across the BBB were significantly reduced in Vim-/- mice. Decreased neuronal injury in the hippocampal dentate gyrus was observed in Vim-/- mice with meningitis. The major inflammatory regulator α7 nAChR and several signaling molecules contributing to NF-κB activation (p65 and p-CamKII) were significantly reduced in the brain tissues of the Vim-/- mice with E. coli meningitis. Furthermore, Vim KO resulted in significant reduction in neuronal injury and in α7 nAChR-mediated calcium signaling.

Conclusion/significance: Vimentin, a novel NF-κB regulator, plays a detrimental role in the host defense against meningitic infection by modulating the NF-κB signaling pathway to increase pathogen invasion, PMN recruitment, BBB permeability and neuronal inflammation. Our findings provide the first evidence for Vim-dependent mechanisms underlying the pathogenic triad of bacterial meningitis.

Fig 1. Effects of vimentin deficiency on E. coli K1-induced bacterial meningitis.

A-B: Bacterial loads in blood (A) and brain (B) of WT and vimentin KO mice. C: Transmigration of PMN into the CSF of WT and KO mice. D: Albumin level in CSF of WT and KO mice. E-G: Histological examination of brain cortex (E), dentate gyrus (F) and hippocampus (G). Images (E and F) photographed at 200X magnification. Boxes in (F) show the relationship between F and G (G1–6). WT: wild-type. KO: knockout. NS: not statistically significant. Bar graphs show the means ± SD. In both invasion and PMN transmigration assays, significant differences between different groups (5–6 pups/group) are marked by asterisks (*P<0.05; **P<0.01).

Fig 2. Effects of vimentin deficiency on E. coli K1-induced CNS inflammatory response.

A-B: Immunehistochemical analysis of p65 expression in the brain cortex (A) and endothelial cells (B). Image (A) photographed at 200X magnification. Boxes in (A) show the relationship between A and B (B1–6). C-E: The protein level of soluble P65 (C), adhesion molecules ICAM-1 (D) and CD44 (E) in the CSF. WT and KO mice were divided into 4 groups (5–6 pups/group). Each experiment was performed three times. *P<0.05, **P<0.01.

Fig 3. Effects of vimentin deficiency on α7 nAChR expression and NF-κB activation in mouse brain.

A-B: Immunehistochemical analysis of vimentin (A) and α7 nAChR (B) expression in mouse brain cortex. Images (A and B) photographed at 200X magnification C: Western blotting analysis of vimentin, α7 nAChR, phospho-IKKα/β and P65 expression levels in mouse vascular endothelial cells.

Fig 4. Effects of vimentin deficiency on E. coli K1-induced intracellular calcium flux.

Elevation of intracellular calcium flux in HBMEC stimulated with E44 or ZD1 strains. HBMEC transfected with siRNAs of a scrambled sequence (CON), vimentin (Vim KD) and α7 nAChR (A7 KD) were loaded with Fura-2 AM as described in Methods and Materials. The monolayer was monitored for intracellular calcium flux for 10 minutes with 4 s intervals under an automated fluorescent microscope. Monolayer cells were stimulated with E44 or ZD (10⁸ CFU) at the 120 s time point. The intensity of fluorescence at 340 nm and 380 nm was measured. The ratios of intensity of fluorescence at 340 nm and 380 nm were calculated for each time interval and depicted as continuous lines in (A–C). (G) The y axis represents the ratio, and x axis represents time (s). The 340 nm/380 nm ratio changes in each treatment were calculated and subjected to statistical analysis. WT HBMEC without any pre-treatment served as a control and are defined as one-fold (1.0). (*P<0.05; **P<0.01).

Fig 5. Role of vimentin in E. coli K1-induced activation of CaMKII and NF-κB.

A-C: Immunehistochemical staining (DAB) was used to examine the phosphorylation level of CaMKII in the brain cortex (A), especially in the BBB indicated by endothelial cells (B), and dentate gyrus (C) of WT or vimentin KO mice infected with E. coli. The nucleus is stained as blue, and CaMKII is stained as brown. Images (A and C) photographed at 200X magnification. Boxes in (A) show the relationship between A and B (B1–6).

Fig 6. Activation of vimentin-α7 nAChR signaling cascades is lipid raft-dependent.

Wild-type BMEC or vimentin KO BMEC were triggered by medium (control), E44 or ZD1 (5×10⁷/plate) for 2 h. Lipid rafts were isolated with the Caveolae/Rafts Isolation kit, which was purchased from Sigma–Aldrich. (A-D) Western blotting was used to detect distribution of CaMKII, TAK1, ERK1/2 and phospho-IKKα/β in the lipid rafts of HBMECs. Fractions 1–3 marked with an asterisk (*) consisted of caveolin-1-enriched lipid rafts. Fractions 1–9 represent the gradients from top to bottom.

Fig 7. A schematic diagram of the Vim- signaling model.

E. coli K1 infection triggers IbeA-dependent activation of the vimentin signaling pathway at the host cell membrane. IbeA binds to its receptor Vim and co-receptor PSF that interact with α7n nAChR through lipid rafts. These communications trigger phosphorylation of signaling proteins (e.g., Vim, TAK1), which in turn activates the nuclear factor-kappaB (NF-κB) pathways via activation of the IκB kinase (IKK) complex. NF-κB activation resulted to the nuclear translocation of NF-κB, which induces the production of cytokines, chemokines, and others proinflammatory molecules in response to bacterial stimuli.