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

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.

The IbeA-human BMEC (HBMEC) surface protein interaction and subsequently induced signal transduction were shown to be essential for bacterial invasion [6, 20,21]. Our previous studies have demonstrated that the specific interaction between IbeA and its primary receptor vimentin is the upstream signaling event that is required for the caveolae/lipid raft (LR)-dependent entry of E. coli K1 into HBMECs [6,21]. Subsequently, the co-receptor PSF and related signaling molecules (e.g., ERK, caveolin-1, α7 nAChR) are recruited to the LR microdomains [6, 20,22]. Bacterial invasion was positively correlated with phosphorylation of Vim at serine 82 by CaM kinase II (CaMKII), IbeA + E. coli-induced phosphorylation of Akt [5], and Erk [6]. IbeA + E. coli and IbeA-coated beads induced the clustering of Vim that was correlated with increased entry of bacteria and beads. These studies suggest that Vim-mediated signaling is essential for IbeA + E. coli K1 invasion. Vim has emerged as an important organizer of a number of critical proteins that contribute to attachment, migration, LR-mediated cell signaling [23][24]. It is also involved with bacterial infections caused by Group A streptococci, Salmonella and P. multocida [21,25]. It has been shown that caveolae/LRs are signaling platforms for IbeA-induced pathogenicities [6]. Caveolae/LRs are becoming increasingly recognized as significant entry portals for the endocytosis of a wide variety of microbial pathogens and toxins [26]. Recently, we have shown that caveolae/LRs are essential for C. neoformans penetration across the BBB [27]. OmpA (outer membrane protein A) + E. coli K1 entry into HBMEC was shown to be clathrin-independent but caveolae-dependent [28]. Caveolae, however, are not specifically associated with specific virulence factors in those studies, as OmpA is a major outer membrane protein present in both pathogenic E. coli K1 and nonpathogenic E. coli K12 [29]. Our studies show that the specific interaction between IbeA and its receptor, Vim, is the upstream signaling event, which is required for the caveolae/LR-dependent entry of E. coli K1 into HBMEC [6,8].
The NF-κB signal transduction pathway, which is an important regulator of the innate immunity, is becoming increasingly recognized for its signaling paradigm as a good therapeutic target for molecular biomedicine including microbial infections [30]. NF-κB activation has been shown to contribute to N. meningitidis invasion of epithelium and H. pylori-induced inflammation of gastric cells. NF-κB in the CNS is activated during bacterial meningitis [8]. Recent studies show that activation of NF-κB is required for E. coli-mediated expression of ICAM-1 [31]. Our recent studies demonstrated that inhibitors of NF-κB (caffeic acid phenethyl ester), Vim (withaferin A) and LRs (filipin) were able to block IbeA + E. coli K1 invasion [6,22]. Vim can form a complex with IκB, NF-κB and tubulins in the resting cells. This complex is dissociated upon the prolonged stimulation with IbeA. Vim and PSF act in concert to regulate IbeA + E. coli K1-induced activation and translocation of NF-κB, and subsequently upregulate expression of Vim, α7 nAChR and proinflammatory factors, which play an essential role in modulation of bacterial infections and inflammation associated with Ca 2+ signaling [8,[32][33][34][35].
PMN transmigration across the BBB is a hallmark feature of bacterial meningitis [31]. Transmigration of PMNs across the BBB is not only crucial for host defense against pathogens, but it may also cause significant damage to the CNS tissues, which results in devastating neurologic sequelae [19]. The adhesive interactions between transmigrating leukocytes and endothelial cells are well understood. However, the detailed mechanisms underlying the actual migration of leukocytes across the endothelium remain elusive. Recent studies have shown that blood lymphocytes and neutrophils preferentially can transmigrate across brain endothelial cells via a transcellular route [19]. Vim on both leukocytes and endothelial cells is able to form a highly dynamic anchoring structure at the site of contact between these two cell types [20]. Our previous studies showed that IbeA could play an important role in PMN transcellular migration across the BBB, which is mediated by Vim on both BMEC and leukocyte surfaces [31].
Over the past decades, the studies of neonatal bacterial meningitis caused by E. coli K1 revealed the importance and significance of IbeA contributing to bacterial adhesion/invasion, NF-κB activation and leukocyte transmigration, which are the three hallmarks (pathogenic triad) of this disease [8,19]. This occurs through interactions with host factors (e.g., Vim). One of the most challenging issues is the lack of a comprehensive understanding of the molecular basis underlying all three of the interrelated hallmark features of bacterial meningitis. Our previous findings suggest that IbeA induces all three of the hallmark features of bacterial meningitis through its receptor Vim. The underlying genetic mechanisms for these pathogenicities remain to be elucidated. In this report, using gene knockdown (cell culture) and knockout (mice) technologies, we examined how Vim contributes to the modulation of pathogen invasion, NF-κB activation and PMN recruitment/neuronal inflammation induced by IbeA + E. coli K1, which is the most common gram-negative pathogen causing neonatal bacterial meningitis. The gene knockout mouse models allow us to further dissect the role of Vim in modulation of host responses that are characterized as the pathogenic triad.

Ethics statement
All research involving human subjects has been approved by the Institutional Review Board (IRB) of Children's Hospital Los Angeles (CHLA The animal study with mice was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Our protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of The Saban Research Institute of CHLA (Permit number: A3276-01; IACUC Protocol # 157-13). All animals were maintained in a conventional production facility and given free access to diets (Purina Picovac Irradiated Mouse Diet #5062) and water. The mice were housed in HEPA filtered ventilated racks in polycarbonate micro isolator cages equipped with an automated reverse osmosis (RO) water delivery system. All cages and bedding materials were sterilized by autoclaving before the animals were placed in the cages according to the institutional guidelines. Bedding in ventilated rodent cages is changed every 7 days or more often as necessary to keep the mice clean and dry. All mice are maintained in static micro isolator rodent cages containing 3 or more adult mice, or a female mouse with a litter, or more often as necessary to keep the animals clean and dry. The health of the animals in the conventional housing facility was ensured by a sentinel monitoring program with quarterly screens. All mouse sentinels in the housing rooms were tested to be negative throughout the course of the study. Sanitization of cages containing mice was performed according to recommendations from the Guide or inhouse standards. Inhouse standards include a complete removal of bedding weekly with a complete cage sanitization every 2 wk. The evaluation of cage density was weekly done on the basis of individual mouse weight. Animals were housed in cages that provide ample space for opportunities for normal physiologic and behavioral needs. Mice were housed on contact bedding to provide opportunity for nest building and thermal regulation. At a minimum, all animals were provided with the opportunity for visual, auditory and olfactory communication with conspecifics when possible. In addition, singly housed animals may be provided with additional environmental enrichment. Mice were provided with Nestlets (or autoclaved Kimwipes for nude mice) as nest building materials. Mice were monitored every 24 hours by lab animal care technicians. Mouse well-being and health was assessed by observing the general appearance of animals as well as their activity prior, during, and after handling. No animals died before or during the experimental endpoint. The mice were anesthetized with Ketamine HCL (100 mg/kg) and xylazine (10 mg/kg) prior to blood collection. All efforts were made to minimize suffering. All animals received continual veterinary monitoring, and eventual euthanasia by pentabarbitol (100mg/kg) injection.
Bacterial strains, culture conditions, plasmids and media E44 is a rifampin-resistant strain derived from RS218 (018:K1: H7), which is a clinical isolate from the cerebrospinal fluid (CSF) of a neonate with meningitis [4]. Both RS218 and E44 have been characterized and have the same virulence phenotypes. ZD1 is an ibeA in-frame-deletion mutant of E44 [4]. Bacteria were cultured in brain-heart infusion (BHI) broth supplemented with ampicillin (100μg/ml), kanamycin (50 μg/ml) and/or rifampin (100 μg/ml) was used for bacterial culture if required.

Mice
Vim KO mice, previously obtained from Dr. Albee Messing at the University of Wisconsin, were originally provided by Professor Charles Babinet of the Institute Pasteur [46]. The absence of Vim has no apparent phenotypic changes in mouse reproduction and development [46]. Control mice were generated by back-breeding the KO mice with the WT strain (129/ SVEV) from which they were derived for at least ten generations [47]. Mice were housed in specific pathogen-free conditions at the Animal Maintenance Facility of Children's Hospital Los Angeles. The animals were used in transgenic breeding at 8 weeks of age for optimum reproductive performance. Male heterozygous (+/-) and female homozygous (-/-) were used in breeding. All experiments were approved by the Animal Care and Use Committee of Children's Hospital Los Angeles.

Hippocampal organotypic cultures (OTC)
Slices of the hippocampal formation of newborn (5-to 7-day-old) WT and Vim -/-mice were cultured as described previously [48]. Briefly, the hippocampal formations were prepared under sterile conditions, and cut transversely with a tissue chopper into slices of 400-μm thickness. After culturing 14 days, the OHCs were challenged by purified His-IbeA protein (0.1μg/ ml) in culture medium for live PI staining according to the protocol (BD Biosciences) for 3 and 6 hours, and fixed by 4% PFA. The images of hippocampus with DIC and PI were acquired with immuneflorescence microscope (200 x) and analyzed by image J5.0.

Mouse model of E. coli meningitis
Neonatal E. coli meningitis was induced as described in detail previously [6][7][8]. Mouse pups were randomly divided into different groups (5-6 pups/group). At 10 days of age, all animals were inoculated by i.p. injection of E. coli K1 strain E44 or ZD1 (10 5 colony-forming units, CFU). The inoculated animals were anaesthetized with ketamine and lidocaine at 18 h postinfection. Sectioning of brain tissues and sampling of blood and CSF were carried out as described previously [23,31]. To determine the recruitment of PMN into the CNS, CSF samples were stained with a FITC-conjugated rat anti-mouse Ly-6G (Gr-1) antibody and counted under the fluorescence microscope. The CSF concentration of albumin was measured using an ELISA kit from Bethyl laboratories (Montgomery, TX) according to the manufacturer's instructions. To measure soluble NF-κB, CD44 or ICAM1, five μl of the CSF samples were taken and subjected to ELISA using rabbit polyclonal antibodies against NF-κB (p65), CD44 or ICAM1 from Santa Cruz Biotechnology according to the manufacturer's instructions.

Histology immunostaining
Eighteen hours after E. coli inoculation, mouse brains were harvested, fixed in 10% buffered formalin for 24 h, and embedded in paraffin. Sections with 5 mm thickness were prepared, deparaffinized with xylene and then rehydrated with graded ethanol and distilled water. Heat treatment in a microwave, blockage of endogenous peroxidase activity with 3% H 2 O 2 and incubation with 10% goat serum were carried out as described in detail previously [7]. Anti-phospho-TAK1, anti-phospho-ERK, anti-NF-κB, or anti-phospho-CamKII (1:200 dilution) antibody was incubated with the sections overnight at 4°C. The following day, after washing, the sections were incubated with a HRP-conjugated goat anti-rabbit antibody at room temperature for 1 hour. Finally, all of the sections were stained with 3, 3-diaminobenzidine tetrahydrochloride (DAB) and then lightly counterstained with hematoxylin. Normal rabbit serum was used as a blocking agent and negative controls.

Measurements of intracellular (Ca2+]
To examine the role of vimentin in E. coli induced calcium signaling HBMEC, determination of intracellular calcium flux was carried out as described previously [7,[49][50][51]. Briefly, HBMEC were grown on culture dishes (MatTek, Ashland, MA) to about 40-50% confluence and then transfected with scramble, vimentin or α7nAChR siRNAs (Santa Cruz Biotechnonly) according to the manufacturer's protocol. Transfected cells were allowed to continue growth for 48 h until 80-90% confluence. After washing with phenol-red-free HBSS and then incubation for 60 min with 4 mM Fura-2 AM and 0.04% Pluronic-127, HBMEC were then washed with phenolred free HBSS 2 times and incubated in this buffer for an additional 20 min. Then, the intensities of fluorescence at 340 nm and 380 nm were monitored for 10 min at 4 seconds intervals. E44 or ZD1 (1×10 8 CFU) was added at 2 min to stimulate the cells, and changes in intensities at 340 nm and 380 nm were measured. These measurements were made using a Nikon inverted microscope (Melville, NY) equipped with a Nikon Fluor 406/1.3 NA Ph4DL oil immersion objective lens as described previously [7]. Additional pieces of equipment needed for the measurements included a Hamamatsu Corp. (Bridgewater, NJ) ORCA-100 (C4742-95-12NR) 12-bit digital camera (4×4 binning mode), a Ludl Electronics Products Ltd. (Hawthorne, NY) Mac2000 XYZ stage and a focus controller. MetaMorph 4.5 (Universal Imaging Corp., Downingtown, PA) was used for the control of the imaging rig. Changes in (Ca2+] were expressed as the F340:F380 ratio, where F340 and F380 were Fura-2 fluorescence intensities obtained at 340 nm and 380 nm excitation wavelengths, respectively.

Preparation of lipid rafts from BMEC
Lipid rafts that contained the protein caveolin were isolated using the Caveolae/Rafts Isolation kit from Sigma-Aldrich. HMBEC were transfected with scramble, vimentin or α7nAChR siR-NAs (Santa Cruz Biotechnology) as described above (in the section of SiRNA transfection). HBMEC were triggered by medium (control), E44, ZD1 (5×10 7 /plate) for 2 h. After incubation, membrane lipid raft fractions were prepared as described in detail previously [6,52]. The cell lysates were prepared by passage through a 22 gauge needle 20 times, and then centrifuged at 1000 g for 10 min. The post-nuclear supernatant was adjusted to 35% of OptiPrep with 60% original stock, transferred into SW40 centrifuge tubes and overlaid with 2 ml each of OptiPrep (30, 25 and 20%) and 1 ml of lysis buffer on the top. Nine fractions (1 ml for each fraction) were collected after centrifugation at 21000 rev./min for 3 h using an SW-41 rotor. These fractions were analyzed by Western blotting with the antibodies against caveolin-1, vimentin (V9) and PSF.

Statistical analysis
All results shown are the means+2S.D. of triplicate determinations in invasion and leukocyte transmigration assays. Microsoft Excel and the statistical package were used for the storage and analysis of raw data. Data from the animal experiments were analyzed using Software Graph Pad Prsim 5.0. ANOVA and covariates were followed by a multiple comparison test. P<0.05 was considered to be statistically significant.

Vimentin deficiency protects neonatal mice from E. coli K1-induced bacterial meningitis
In the neonatal murine model of E. coli meningitis, vimentin -/-and IbeA deletion in E. coli K1 (ZD1) did not change the bacteremia level (Fig 1A). However, the bacterial entry into brain, PMN transmigration across the BBB, and BBB permeability indicated by albumin concentration in CSF were all significantly reduced in vimentin knockout and with IbeA deletion (ZD1) as compared to wild-type mice with E44 infection (Fig 1B, 1C and 1D). Histological examination of brain sections with hematoxylin-eosin staining indicated that the blood infiltration into brain cortex (Fig 1E) and neuronal injury in the dentate gyrus of the hippocampus (Fig 1F and  1G) induced by E44 infection were both decreased in vimentin knockout by IbeA deletion (ZD1). Bacterial meningitis causes neuronal damage that predominates in the hippocampal dentate gyrus [53]. To verify the role of vimentin and IbeA in neuronal injury, the hippocampus from neonatal wildtype and vimentin knockout mice were isolated, cultured, and challenged with purified IbeA protein. Immunofluorescence examination showed the neuronal apoptosis indicated by live PI staining accumulated with IbeA stimulation in wildtype hippocampus, but not in vimentin knockout hippocampus after 3h (S1A and S1B Fig) and 6h (S1C and S1D Fig). These data demonstrated that the deficiency of vimentin protect neonatal mice from E. coli K1-induced bacterial meningitis.

Vimentin deficiency inhibits the CNS inflammatory response
The inflammatory response induced by E. coli K1 was also eliminated by both vimentin deficiency and IbeA deletion in this neonatal murine model of E. coli meningitis. Immunehistochemical staining showed that the expression of P65 increased by E44 infection in wildtype mice was decreased in both vimenin -/-mice and ZD1 infection in the brain cortex (Fig 2A), especially the BBB indicated by endothelial cells, which form the microvascular blood vessels (Fig 2B), as well as hippocampal CA1 (S2A Fig) and dentate gyrus (S2B Fig). The release of P65 and the adhesion molecules ICAM-1 and CD44 into CSF were all augmented by E44 infection and suppressed by both vimentin deficiency and IbeA deletion (Fig 2C, 2D and 2E), which confirmed IbeA-vimentin interaction could aggravate the inflammatory response in E. coli meningitis.

Vimentin-regulated α7 nAChR expression in mouse brain
Our previous findings indicated that vimentin-mediated initial activation of NF-κB in BMEC under E. coli K1 stimulation [8]. However, the underlying signaling pathway from vimetnin-IbeA interaction to NF-κB activation remains unclear. Since vimentin-mediated E. coli invasion is involved with calcium signaling [6], and α7 nAChR-mediated calcium signaling contributes to E. coli meningitis [7], we hypothesized that vimentin contributes to E. coli invasion and NF-κB activation via α7 nAChR-mediated calcium signaling. Immunehistochemical staining showed that vimentin expression was increased by E44 infection but not by ZD1 (the IbeA deletion mutant) in the brain cortex of wildtype mice, while there was no vimentin staining seen in vimentin knockout mice (Fig 3A). The α7 nAChR expression in the brain cortex was similarly increased by E44 infection and reduced by ZD1infection. The α7 nAChR level was largely suppressed in vimentin knockout mice under different treatments in contrast to wildtype mice (Fig 3B). Western blotting with the total brain tissues confirmed that vimentin deficiency suppressed α7 nAChR expression, and both were increased by IbeA + E. coli K1 (Fig 3C). Furthermore, the level of P65 expression was enhanced by IbeA + E. coli K1, and reduced in vimentin knockout (Fig 3C, lower panel).

E. coli K1-induced intracellular calcium flux is mediated by vimentin via α7 nAChR
Calcium ions are important for cellular signaling that is involved in the pathogenesis of bacterial infection [34]. Alpha7 nAChR has been found to play a regulatory role in the CNS inflammatory response to bacteria via its ion channel function for calcium signaling [7]. Since α7 nAChR expression was regulated by vimentin, we evaluated intracellular transient calcium flux induced by E. coli K1 using HBMEC with vimentin knockdown by siRNA. The strength of calcium flux indicated by the ratio of 340nm/380nm of Fura-2 AM was measured and calculated, showing that E44 stimulation resulted intracellular transient calcium flux immediately with a range of 0.8 fold increase on the HBMEC transfected with scrambled siRNA, while IbeA deletion (ZD1) abolished the calcium flux in these cells (Fig 4A and 4D). However, vimentin or α7 nAChR knockdown by siRNA both abolished the intracellular transient calcium flux induced by E44 and ZD1 stimulation (Fig 4B, 4C and 4D), which suggested that IbeA-vimentin meditated E. coli K1-induced intracellular calcium flux is α7 nAChR dependant. CaMKII activation is critical for E. coli K1-induced NF-κB activation CaMKII has been found to relate to E. coli K1 invasion [32] and to serve as the downstream target of calcium signal through α7 nAChR ion channel. CaMKII activation could result in TAK1 and Erk1/2 phosphorylation, thus increasing IKKα/β phosphorylation and NF-κB activation. In the neonatal murine model of E. coli meningitis, immunehistochemical staining showed that E44 but not ZD1 infection could significantly enhance the phosphorylation level of CaMKII as compared to control mice in brain cortex (Fig 5A). It is especially true in the BBB indicated by endothelial cells, which form the microvascular blood vessels (Fig 5B), as well as in the hippocampal CA1 (S3 Fig) and dentate gyrus (Fig 5C) in wildtpye mice. However, vimentin knockout abolished most of the CaMKII activation in both E44 and ZD1 infected mice, which suggested vimentin mediated E. coli K1 induced CaMKII activation through calcium signaling via α7 nAChR. Furthermore, the phosphorylation levels of TAK1(S4A and S4B in brain cortex, as well as endothelial cells, which form the microvascular blood vessels. These results were also confirmed by in vitro studies with HBMEC, which showed that the phosphorylation levels of CaM-KII, TAK1, ERK1/2, and IKK α/β and IκB degradation induced by E44 were all suppressed by ZD1 in a time course stimulation (S6 Fig). Furthermore, α7 nAChR siRNA knockdown could also abolish the phosphorylation of CaMKII, TAK1, ERK1/2, and IKK α/β and IκB degradation induced by E44. Taken together, these results suggest α7 nAChR-CaMKII play a critical role in the E. coli K1-induced NF-κB activation via a vimentin mediated calcium signaling pathway.

Vimentin-mediated calcium signaling is lipid raft dependent
In our previous studies, it has been shown that a complex containing vimentin, α7 nAChR and caveolin-1 is formed, and then recruited to caveolae upon E. coli K1 stimulation [6,7]. It suggests that caveolin-1-enriched lipid rafts may be the functionally important signaling platform in the vimentin-α7 nAChR-NF-κB pathway. However, the underlying mechanism for this signaling cascade was still unclear. Here, we performed lipid raft fractionation analysis using HMBEC transfected with vimentin or scrambled siRNA, to dissect the relationship between the vimentin-α7 nAChR signaling cascade and lipid rafts. As shown in S7B and S7C Fig,  vimentin knockdown abolished expression and recruitment of Vim and PSF to lipid raft fractions upon E44 infection, but did not change the pattern of caveolin-1 (S7A Fig). For the signaling cascade, CaMKII, Erk1/2 and TAK1 were all recruited by E44 stimulation in HBMEC transfected scramble siRNA, while ZD1 and vimentin siRNA knockdown both abolish the signaling molecules recruitment to lipid rafts (Fig 6A, 6B and 6C). The phosphorylated IKK α/β showed a trend to recruit into lipid rafts induced by E44 stimulation, while ZD1 and vimentin siRNA knockdown reduced both this trend and related phosphorylation levels (Fig 6D). These results indicate that lipid rafts play a role in vimentin-mediated NF-κB activation.

Discussion
Bacterial invasion and translocation across of BBB are critical steps during the development of neonatal E. coli meningitis. The IbeA protein has been identified as an important virulence factor for E. coli K1 invading into BMEC, which is the major component of BBB. The specific binding of IbeA to its primary receptor vimentin is the initial signaling event, which is required for the caveolae/LRs-dependent entry of E. coli K1 into BMECs [8,21]. Subsequently, the coreceptor PSF and related signaling molecules (e.g., ERK, caveolin-1, α7 nAChR) are recruited to the LR microdomains [6][7][8]20]. Vimentin is expressed in abundance in mesenchymal cells such as endothelial cells [54]. It is not only involved in the invasive epithelial-mesenchymal transition phenotype of tumor cells, but also contributes to the adhesive or invasive phenotype of microbial pathogens [25,55,56]. Our previous studies indicate that vimentin is an IbeAbinding protein on the surface of HBMECs. The middle region of IbeA (271-370 residues) and the head domain of vimentin are the binding sites of IbeA-vimentin interaction [57]. Vimentin serves as the primary receptor for IbeA-induced signaling activation and E. coli K1 invasion of HBMECs [6]. Our recent findings reveal that vimentin plays an important role in NF-κB activation and translocation [12]. Using the vimentin knockout (KO) mice in this study, we find that, vimentin is the key regulator of the pathogenic triad of bacterial meningitis, IbeA-induced E. coli K1 invasion, NF-κB activation and PMN transmigration across the BBB. All three of the interrelated host responses were significantly reduced in vimentin KO (Vim-/-) mice. It has been shown that endothelial surface expression of ICAM-1 and CD44 is significantly elevated upon E. coli infection but expression of ICAM-2, VCAM-1 or E-selectin in pulmonary endothelial cells is not affected [58,59]. We also found that IbeA mediated secretion of ICAM-1, CD44 and p65 in the CSF decreased significantly in vimentin deficient mice. These data suggest that vimentin plays important role in the IbeA-induced pathogenic triad during neonatal E. coli meningitis.
Calcium-mediated cell signaling has been shown to be important for the pathogenesis of bacterial infection [34] and the biological functions of α7 nAChR [35]. Elevated intracellular transient calcium flux in HBMEC can be induced by E. coli K1 virulence factors [7]. We have previously shown that α7 nAChR participates in the modulation of meningitic E. coli K1 invasion, PMN recruitment and neuronal inflammation, and that these events are related to the intracellular transient calcium flux regulated by α7 nAChR [7]. It was unclear, however, whether there is crosstalk between vimentin and α7 nAChR in IbeA-mediated pathogenesis. In this study, we found that the expression level of α7 nAChR was significantly reduced in the brain of vimentin KO mice. The increased expression of α7 nAChR in mouse brain cortex with E. coli K1 E44 infection was also abolished by vimentin KO. Additionally, we found that the intracellular transient calcium flux in HBMEC induced by E44 stimulation was blocked by knockdown (KD) of vimentin or α7 nAChR.
NF-κB in the CNS is activated during bacterial meningitis [60]. It has shown that blockage of NF-κB signaling can suppress CNS inflammation and protect rat brains from inflammatory injury resulting from transient focal cerebral ischemia [61] and bacterial meningitis [62]. In our previous studies, it has been shown that CaMKII-induced phosphorylation of vimentin at Ser 82 and the vimentin-binding domain of ERK are important for IbeA + E. coli K1 invasion of BMEC [6]. In this study, we shown that vimentin KO abolished most of the CaMKII activation in E44 infected mice. The phosphorylation levels of TAK1, ERK1/2, and IKK α/β and IκB degradation representing the activation of NF-κB induced by E44 were all suppressed by CaMKII siRNA knockdown. The phosphorylation levels of CaMKII, TAK1, ERK1/2, and IKK α/β and IκB degradation induced by E44 could be inhibited by vimentin siRNA knockdown, as well as the reduced expression of α7 nAChR. Alpha7 nAChR is involved in regulation of Ca 2+ /CaM-KII and /ERK1/2. So these results suggest that vimentin is critical for IbeA-mediated activation of NF-κB in HBMEC with E. coli K1infection through the vimentin-α7 nAChR singling pathway [63]. A proteomics study shows that vimentin is a raft protein present in lipid rafts of endothelial cells [64], suggesting that vimentin is required for lipid-raft-dependent cellular signaling. In our previous studies, it has been shown that E. coli K1 stimulation can induce formation of a complex containing vimentin, α7 nAChR and caveolin-1, and then recruitment of them into caveolae [6,7]. Caveolin-1-enriched lipid rafts may be the functionally important signaling platform in the vimentin-α7 nAChR-NF-κB pathway. In this report, we further explored the underlying mechanism for this signaling cascade, and found that CaMKII, Erk1/2 and TAK1 were all recruited by E44 stimulation in HBMEC, while the ibeA deletion mutant ZD1 and vimentin knockdown both significantly decreased the recruitment of signaling molecules into lipid rafts. The phosphorylated IKK α/β showed a trend to recruit into lipid rafts induced by E44 stimulation, while ZD1 and vimentin knockdown reduced both the trend and its phosphorylation levels. Gene knockdown of either Vim or α7 nAChR resulted in reduced calcium signaling, suggesting that the calcium ion may serve as a molecular bridge between these two proteins through lipid rafts [65]. These results indicated that lipid raft formation is required for the vimentin-α7 nAChR-NF-κB activation pathway (Fig 7).
Taken together, all the findings in this study indicate that the triad pathogenic features of E. coli meningitis are driven by the specific interactions between the meningitic virulence factor IbeA and its primary receptor vimentin. Genetic downmodulation of both vimentin and α7 nAChR significantly blocks IbeA + E. coli K1 invasion, NF-κB activation and leukocyte transmigration that occur at the BBB, which are the triad hallmark features of this disease. In this report, TAK1 has been identified as the upstream signaling molecule (kinase) to regulate NF-κB in the vimentin-α7 nAChR pathway during E. coli meningitis. The molecular and cellular mechanisms underlying crosstalk between vimentin and α7 nAChR, however, remain to be defined. Further dissecting the vimentin-α7 nAChR axis may lead to the discovery and development of novel therapeutic interventions against the triad features of bacterial meningitis.