Ca2+/calmodulin-dependent invasion of microvascular endothelial cells of human brain by Escherichia coli K1

Escherichia coli K1 invasion of microvascular endothelial cells of human brain (HBMEC) is required for E. coli penetration into the central nervous system, but the microbial-host interactions that are involved in this invasion of HBMEC remain incompletely understood. We have previously shown that FimH, one of the E. coli determinants contributing to the binding to and invasion of HBMEC, induces Ca²⁺ changes in HBMEC. In the present study, we have investigated in detail the role of cellular calcium signaling in the E. coli K1 invasion of HBMEC, the main constituents of the blood-brain barrier. Addition of the meningitis-causing E. coli K1 strain RS218 (O18:K1) to HBMEC results in transient increases of intracellular free Ca²⁺. Inhibition of phospholipase C with U-73122 and the chelating of intracellular Ca²⁺ by BAPTA/AM reduces bacterial invasion of HBMEC by approximately 50%. Blocking of transmembrane Ca²⁺ fluxes by extracellular lanthanum ions also inhibits the E. coli invasion of HBMEC by approximately 50%. In addition, E. coli K1 invasion is significantly inhibited when HBMEC are pretreated by the calmodulin antagonists, trifluoperazine or calmidazolium, or by ML-7, a specific inhibitor of Ca²⁺/calmodulin-dependent myosin light-chain kinase. These findings indicate that host intracellular Ca²⁺ signaling contributes in part to E. coli K1 invasion of HBMEC. This work was supported by the American Heart Association (grant SDG 0435177N to Y.K.) and by NIH grants (to K.S.K.).     

Regulation of protein kinase C in Escherichia coli K1 invasion of human brain microvascular endothelial cells.

Escherichia coli is one of the most important pathogens involved in the development of neonatal meningitis in many parts of the world. Traversal of E. coli across the blood-brain barrier is a crucial event in the pathogenesis of E. coli meningitis. Our previous studies have shown that outer membrane protein A (OmpA) expression is necessary in E. coli for a mechanism involving actin filaments in its passage through the endothelial cells. Focal adhesion kinase (FAK) and phosphatidylinositol 3-kinase (PI3K) have also been activated in host cells during the process of invasion. In an attempt to elucidate the mechanisms leading to actin filament condensation, we have focused our attention on protein kinase C (PKC), an enzyme central to many signaling events, including actin rearrangement. In the current study, specific PKC inhibitors, bisindolmaleimide and a PKC-inhibitory peptide, inhibited E. coli invasion of human brain microvascular endothelial cells (HBMEC) by more than 75% in a dose-dependent manner, indicating a significant role played by this enzyme in the invasion process. Our results further showed that OmpA+ E. coli induces significant activation of PKC in HBMEC as measured by the PepTag nonradioactive assay. In addition, we identified that the PKC isoform activated in E. coli invasion is a member of the conventional family of PKC, PKC-alpha, which requires calcium for activation. Immunocytochemical studies have indicated that the activated PKC-alpha is associated with actin condensation beneath the bacterial entry site. Overexpression of a dominant negative mutant of PKC-alpha in HBMEC abolished the E. coli invasion without significant changes in FAK phosphorylation or PI3K activity patterns. In contrast, in HBMEC overexpressing the mutant forms of either FAK or PI3K, E. coli-induced PKC activation was significantly blocked. Furthermore, our studies showed that activation of PKC-alpha induces the translocation of myristoylated alanine-rich protein kinase C substrate, an actin cross-linking protein and a substrate for PKC-alpha, from the membrane to cytosol. This is the first report of FAK- and PI3K-dependent PKC-alpha activation in bacterial invasion related to cytoskeletal reorganization.     

Escherichia coli K1 induces IL-8 expression in human brain microvascular endothelial cells

Microbial penetration of the blood-brain barrier (BBB) into the central nervous system is essential for the development of meningitis. Considerable progress has been achieved in understanding the pathophysiology of meningitis, however, relatively little is known about the early inflammatory events occurring at the time of bacterial crossing of the BBB. We investigated, using real-time quantitative PCR, the expression of the neutrophil chemoattractants alpha-chemokines CXCL1 (Groalpha) and CXCL8 (IL-8), and of the monocyte chemoattractant beta-chemokine CCL2 (MCP-1) by human brain microvascular endothelial cells (HBMEC) in response to the meningitis-causing E. coli K1 strain RS218 or its isogenic mutants lacking the ability to bind to and invade HBMEC. A nonpathogenic, laboratory E. coli strain HB101 was used as a negative control. CXCL8 was shown to be significantly expressed in HBMEC 4 hours after infection with E. coli K1, while no significant alterations were noted for CXCL1 and CCL2 expression. This upregulation of CXCL8 was induced by E. coli K1 strain RS218 and its derivatives lacking the ability to bind and invade HBMEC, but was not induced by the laboratory strain HB101. In contrast, no upregulation of CXCL8 was observed in human umbilical vein endothelial cells (HUVEC) after stimulation with E. coli RS218. These findings indicate that the CXCL8 expression is the result of the specific response of HBMEC to meningitis-causing E. coli K1.     

Effect of rpoS mutations on stress-resistance and invasion of brain microvascular endothelial cells in Escherichia coli K1

Escherichia coli K1 strains are predominant in causing neonatal meningitis. We have shown that invasion of brain microvascular endothelial cells (BMEC) is a prerequisite for E. coli K1 crossing of the blood-brain barrier. BMEC invasion by E. coli K1 strain RS218, however, has been shown to be significantly greater with stationary-phase cultures than with exponential-phase cultures. Since RpoS participates in regulating stationary-phase gene expression, the present study examined a possible involvement of RpoS in E. coli K1 invasion of BMEC. We found that the cerebrospinal fluid isolates of E. coli K1 strains RS218 and IHE3034 have a nonsense mutation in their rpoS gene. Complementation with the E. coli K12 rpoS gene significantly increased the BMEC invasion of E. coli K1 strain IHE3034, but failed to significantly increase the invasion of another E. coli K1 strain RS218. Of interest, the recovery of E. coli K1 strains following environmental insults was 10-100-fold greater on Columbia blood agar than on LB agar, indicating that growing medium is important for viability of rpoS mutants after environmental insults. Taken together, our data suggest that the growth-phase-dependent E. coli K1 invasion of BMEC is affected by RpoS and other growth-phase-dependent regulatory mechanisms.     

Phosphatidylinositol 3-kinase activation and interaction with focal adhesion kinase in Escherichia coli K1 invasion of human brain microvascular endothelial cells

Invasion of brain microvascular endothelial cells (BMEC) is a prerequisite for successful crossing of the blood-brain barrier by Escherichia coli K1. We have previously demonstrated the requirement of cytoskeletal rearrangements and activation of focal adhesion kinase (FAK) in E. coli K1 invasion of human BMEC (HBMEC). The current study investigated the role of phosphatidylinositol 3-kinase (PI3K) activation and PI3K interaction with FAK in E. coli invasion of HBMEC. PI3K inhibitor LY294002 blocked E. coli K1 invasion of HBMEC in a dose-dependent manner, whereas an inactive analogue LY303511 had no such effect. In HBMEC, E. coli K1 increased phosphorylation of Akt, a downstream effector of PI3K, which was completely blocked by LY294002. In contrast, non-invasive E. coli failed to activate PI3K. Overexpression of PI3K mutants Deltap85 and catalytically inactive p110 in HBMEC significantly inhibited both PI3K/Akt activation and E. coli K1 invasion of HBMEC. Stimulation of HBMEC with E. coli K1 increased PI3K association with FAK. Furthermore, PI3K/Akt activation was blocked in HBMEC-overexpressing FAK dominant-negative mutants (FRNK and Phe397FAK). These results demonstrated the involvement of PI3K signaling in E. coli K1 invasion of HBMEC and identified a novel role for PI3K interaction with FAK in the pathogenesis of E. coli meningitis.     

Enhanced Escherichia coli invasion of human brain microvascular endothelial cells is associated with alternations in cytoskeleton induced by nicotine

Although epidemiological studies have shown that exposure to tobacco smoking significantly increases the risk of bacterial meningitis, heretofore the pathogenic effects of smoking on this disease have been poorly understood. In order to dissect this issue, we have investigated the effects of nicotine, the major component of tobacco, on E. coli invasion of human brain microvascular endothelial cells (HBMEC). Our studies showed that E. coli invasion of HBMEC was significantly enhanced by nicotine in a dose-dependent manner. The nicotine-mediated enhancement was associated with actin cytoskeleton rearrangement and morphological changes in the eukaryotic host cell that are essential for bacterial entry. The recombinant IbeA protein and alpha-bungarotoxin (a nicotinic acetylcholine receptor antagonist) were able to efficiently block the nicotine-mediated cellular effects, suggesting the involvement of the IbeA and nicotinic receptors. Blocking of phosphatidylinositol 3-kinase (PI3K) by LY294002 abolished the entry of E. coli in HBMECs treated with nicotine in a dose-dependent manner. Inhibition of PI3K was associated with decreased phosphorylation of Akt and actin cytoskeletal rearrangement. In contrast to PI3K, blockage of Rho kinase (ROCK) by Y27632 upregulated both nicotine- and E. coli-mediated cellular responses. Thus, this study provides experimental evidence for the first time that the major component of tobacco, nicotine, enhances meningitic E. coli invasion of HBMEC through modulation of cytoskeleton.     

Transforming growth factor-beta1 inhibits thrombin activation of endothelial cells

Transforming growth factor-beta1 (TGF-beta1) is reported to exert both pro- and anti-inflammatory effects on the chronic activation of endothelial cells (ECs) in vitro by cytokines such as tumour necrosis factor-alpha (TNF-alpha). However, the effects of TGF-beta1 on acute inflammatory responses of ECs in vitro (e.g. to thrombin) have not been characterised. Pretreatment with TGF-beta1 (10 ng/mL) effectively inhibited all the thrombin-stimulated responses in rat aortic endothelial cells (RAECs) examined: adhesion and migration of polymorphonuclear leukocytes, adhesion of platelets and lymphocytes. Substantial inhibition of thrombin stimulation occurred after 30 min of pretreatment with TGF-beta1 and maximal inhibition was obtained after 1-20 h of pretreatment. Inhibition by TGF-beta1 pretreatment for 30 min was not affected by cycloheximide and was therefore independent of protein synthesis. Treatment with TGF-beta1 for 20 h did not affect the total levels of P-selectin and von Willebrand factor (vWF) in RAECs, but reduced thrombin-stimulated recruitment of P-selectin and vWF to the cell surface. The data demonstrate that TGF-beta1 exerts a potent anti-thrombin effect on ECs, effective after long and short pretreatment times.     

A novel genetic island of meningitic Escherichia coli K1 containing the ibeA invasion gene (GimA): functional annotation and carbon-source-regulated invasion of human brain microvascular endothelial cells

The IbeA (ibe10) gene is an invasion determinant contributing to E. coli K1 invasion of the blood-brain barrier. This gene has been cloned and characterized from the chromosome of an invasive cerebrospinal fluid isolate of E. coli K1, strain RS218 (018:K1: H7). In the present study, a genetic island of meningitic E. coli containing ibeA (GimA) has been identified. A 20.3-kb genomic DNA island unique to E. coli K1 strains has been cloned and sequenced from an RS218 E. coli K1 genomic DNA library. Fourteen new genes have been identified in addition to the ibeA. The DNA sequence analysis indicated that the ibeA gene cluster was localized to the 98 min region and consisted of four operons, ptnIPKC, cglDTEC, gcxKRCI and ibeRAT. The G+C content (46.2%) of unique regions of the island is substantially different from that (50.8%) of the rest of the E. coli chromosome. By computer-assisted analysis of the sequences with DNA and protein databases (GenBank and PROSITE databases), the functions of the gene products could be anticipated, and were assigned to the functional categories of proteins relating to carbon source metabolism and substrate transportation. Glucose was shown to enhance E. coli penetration of human brain microvascular endothelial cells and exogenous cAMP was able to block the stimulating effect of glucose, suggesting that catabolic regulation may play a role in control of E. coli K1 invasion gene expression. Our data suggest that this genetic island may contribute to E. coli invasion of the blood-brain barrier through a carbon-source-regulated process. Electronic Publication     

Escherichia coli outer membrane protein A adheres to human brain microvascular endothelial cells

Escherichia coli K1 is the most common gram-negative bacterium causing neonatal meningitis. The outer membrane protein A (OmpA) assembles a beta-barrel structure having four surface-exposed loops in E. coli outer membrane. OmpA of meningitis-causing E. coli K1 is shown to contribute to invasion of the human brain microvascular endothelial cells (HBMEC), the main cellular component of the blood-brain barrier (BBB). However, the direct evidence of OmpA protein interacting with HBMEC is not clear. In this study, we showed that OmpA protein, solubilized from the outer membrane of E. coli, adhered to HBMEC surface. To verify OmpA interaction with the HBMEC, we purified N-terminal membrane-anchoring beta-barrel domain of OmpA and all surface-exposed loops deleted OmpA proteins, and showed that the surface-exposed loops of OmpA were responsible for adherence to HBMEC. These findings indicate that the OmpA is the adhesion molecule with HBMEC and the surface-exposed loops of OmpA are the determinant of this interaction.     

Transforming growth factor-beta 1 increases the adhesion of MDA-MB-231 mammary adenocarcinoma cells to the microvascular subendothelium

The increase of tumor cell adhesion to the subendothelium in the presence of TGF-beta 1 is thought to be mediated by two major events: an enrichment of extracellular matrix proteins secreted by endothelial cells and an increase of the integrins on the surface of tumor cells. In this study, we analyzed the effect of TGF-beta 1 on the adhesion of a mammary adenocarcinoma cell line (MDA-MB-231) to the matrix of human microvascular endothelial cells (HMEC-1). The adhesion of TGF-beta 1-treated tumor cells to a non-treated matrix or to purified matrix proteins was enhanced, while no increase was observed when non-treated tumor cells were let to adhere to a matrix secreted by HMEC-1 in the presence of the cytokine. Thus, the increase of cell adhesion was due to the effect of TGF-beta 1 on tumor cells and not to the matrix enrichment induced by this cytokine. The hyper-adhesion was inhibited by the RGD peptide and EDTA indicating that integrins were involved. Integrin subunits concentrations (alpha 5, alpha v and beta 1) on the surface of TGF-beta 1-treated tumor cells were not modified, while confocal microscopy showed a reorganization of beta 1 integrin subunits on the cell surface and in the cytoplasm resulting in actin fibers reorganization in the cytoskeleton. This indicates that the enhanced adhesion of TGF-beta 1-treated MDA-MB-231 cells to the subendothelium is due to a qualitative change of integrins.     

The type III secretion system is involved in the invasion and intracellular survival of Escherichia coli K1 in human brain microvascular endothelial cells

Two thermostable phytases were identified from Thai isolates of Aspergillus japonicus BCC18313 (TR86) and Aspergillus niger BCC18081 (TR170). Both genes of 1404 bp length, coding for putative phytases of 468 amino acid residues, were cloned and transferred into Pichia pastoris . The recombinant phytases, r-PhyA86 and r-PhyA170, were expressed as active extracellular, glycosylated proteins with activities of 140 and 100 U mL⁻¹, respectively. Both recombinant phytases exhibited high affinity for phytate but not for p -nitrophenyl phosphate. Optimal phytase activity was observed at 50 °C and pH 5.5. High thermostability, which is partly dependent on glycosylation, was demonstrated for both enzymes, as >50% activity was retained after heating at 100 °C for 10 min. The recombinant phytases also exhibited broad pH stability from 2.0 to 8.0 and are resistant to pepsin. In vitro digestibility tests suggested that r-PhyA86 and r-PhyA170 are at least as efficient as commercial phytase for hydrolyzing phytate in corn-based animal feed and are therefore suitable sources of phytase supplement.     

Identification of a surface protein on human brain microvascular endothelial cells as vimentin interacting with Escherichia coli invasion protein IbeA

Escherichia coli K1 is the most common gram-negative bacteria that cause meningitis during the neonatal period. The ibeA gene product in E. coli K1 has been characterized as a virulence factor that contributes to the binding to and invasion of brain microvascular endothelial cells (BMEC). Here, we identified a surface protein on human BMEC, vimentin, that interacts with the E. coli invasion protein IbeA. The binding sites of the IbeA-vimentin interaction are located in the 271-370 residue region of IbeA and the vimentin head domain. The regulatory protease factor Xa is able to cleave IbeA between R297 and K298 residues, and this cleavage abolishes the IbeA-vimentin interaction.     

Responses of brain and non-brain endothelial cells to meningitis-causing Escherichia coli K1

Bacterial interaction with specific host tissue may contribute to its propensity to cause an infection in a particular site. In this study, we examined whether meningitis-causing Escherichia coli K1 interaction with human brain microvascular endothelial cells, which constitute the blood-brain barrier, differed from its interaction with non-brain endothelial cells derived from skin and umbilical cord. We showed that E. coli K1 association was significantly greater with human brain microvascular endothelial cells than with non-brain endothelial cells. In addition, human brain microvascular endothelial cells maintained their morphology and intercellular junctional resistance in response to E. coli K1. In contrast, non-brain endothelial cells exhibited decreased transendothelial electrical resistance and detachment from the matrix upon exposure to E. coli K1. These different responses of brain and non-brain endothelial cells to E. coli K1 may form the basis of E. coli K1's propensity to cause meningitis.     

Transforming growth factor-beta1 enhanced vascular endothelial growth factor synthesis in mesenchymal stem cells

Angiogenesis is essential for transplantation of mesenchymal stem cells (MSCs). Vascular endothelial growth factor (VEGF) is one of the most potent angiogenic factors identified to date. Elevated VEGF levels in MSCs correlate with the potential of MSCs transplantation. As an indirect angiogenic agent, transforming growth factor-β1 (TGF-β1) plays a pivotal role in the regulation of vasculogenesis and angiogenesis. However, the effect of TGF-β1 on VEGF synthesis in MSCs is still unknown. Besides, the intracellular signaling mechanism by which TGF-β1 stimulates this process remains poorly understood. In this article, we demonstrated that exposure of MSCs to TGF-β1 stimulated the synthesis of VEGF. Meanwhile, TGF-β1 stimulated the phosphorylation of Akt and extracellular signal-regulated kinase 1/2 (ERK1/2). Moreover, Ly 294002, a specific inhibitor of phosphatidylinositol-3-kinase (PI3K)/Akt significantly attenuated the VEGF synthesis stimulated by TGF-β1. Additionally, U0126, a specific inhibitor of ERK1/2, also significantly attenuated the TGF-β1-stimulated VEGF synthesis. These results indicated that TGF-β1 enhanced VEGF synthesis in MSCs, and the Akt and ERK1/2 activation were involved in this process.     

Group B streptococcal pilus proteins contribute to adherence to and invasion of brain microvascular endothelial cells

Surface filamentous structures known as pili have been discovered recently in the gram-positive streptococcal pathogens that cause invasive disease in humans, including group B Streptococcus (GBS). We show that two GBS proteins involved in pilus formation, encoded by pilA and pilB, also facilitate the interaction of this important agent of central nervous system infection with endothelial cells of the human blood-brain barrier.     

HIV-1 Tat-mediated apoptosis in human brain microvascular endothelial cells

The integrity of the blood-brain barrier (BBB) is critical for normal brain function. Neuropathological abnormalities in AIDS patients have been associated with perivascular HIV-infected macrophages, gliosis, and abnormalities in the permeability of the BBB. The processes by which HIV causes these pathological conditions are not well understood. To characterize the mechanism by which HIV-1 Tat protein modulates human brain microvascular endothelial cell (HBMEC) functions, we studied the effects of HIV-1 Tat in modulating HBMEC apoptosis and permeability. Treatment of HBMEC with HIV-1 Tat led to Flk-1/KDR and Flt-4 receptor activation and the release of NO. The protein levels of endothelial NO synthase (NOS) and inducible NOS were increased by HIV-1 Tat stimulation. Importantly, HIV-1 Tat caused apoptosis of HBMEC, as evidenced by changes in the cleavage of poly(A)DP-ribose polymerase, DNA laddering, and incorporation of fluorescein into the nicked chromosomal DNA (TUNEL assay). HIV-1 Tat-mediated apoptosis in HBMEC was significantly inhibited in the presence of N-nitro-L-arginine methyl ester (an inhibitor of NOS) and wortmannin (a phosphoinositol 3-kinase inhibitor). Furthermore, HIV-1 Tat treatment significantly increased HBMEC permeability, and pretreatment with both N-nitro-L-arginine methyl ester and wortmannin inhibited the Tat-induced permeability. Taken together, these results indicate that dysregulated production of NO by HIV-1 Tat plays a pivotal role in brain endothelial injury, resulting in the irreversible loss of BBB integrity, which may lead to enhanced infiltration of virus-carrying cells across the BBB.     

Outer membrane protein A of Escherichia coli K1 selectively enhances the expression of intercellular adhesion molecule-1 in brain microvascular endothelial cells

Escherichia coli K1 meningitis is a serious central nervous system disease with unchanged mortality and morbidity rates for last few decades. Intercellular adhesion molecule 1 (ICAM-1) is a cell adhesion molecule involved in leukocyte trafficking toward inflammatory stimuli at the vascular endothelium; however, the effect of E. coli invasion of endothelial cells on the expression of ICAM-1 is not known. We demonstrate here that E. coli K1 invasion of human brain microvascular endothelial cells (HBMEC) selectively up-regulates the expression of ICAM-1, which occurs only in HBMEC invaded by the bacteria. The interaction of outer membrane protein A (OmpA) of E. coli with its receptor, Ecgp, on HBMEC was critical for the up-regulation of ICAM-1 and was depend on PKC-alpha and PI3-kinase signaling. Of note, the E. coli-induced up-regulation of ICAM-1 was not due to the cytokines secreted by HBMEC upon bacterial infection. Activation of NF-kappaB was required for E. coli mediated expression of ICAM-1, which was significantly inhibited by over-expressing the dominant negative forms of PKC-alpha and p85 subunit of PI3-kinase. The increased expression of ICAM-1 also enhanced the binding of THP-1 cells to HBMEC. Taken together, these data suggest that localized increase in ICAM-1 expression in HBMEC invaded by E. coli requires a novel interaction between OmpA and its receptor, Ecgp.     

Transforming growth factor-beta signalling in brain disorders

Transforming growth factor-beta (TGF-beta) has been characterized as an injury-related factor, based on the observation that it is strongly up-regulated in many acute or chronic central nervous system disorders. TGF-beta is generally thought to be neuroprotective and several mechanisms have been proposed to explain this beneficial action. For instance, TGF-beta protects neurons against the potentiating effect of tissue-type plasminogen activator on NMDA receptor-mediated excitotoxicity, by up-regulating type-1 plasminogen activator inhibitor expression in astrocytes. TGF-beta has also anti-apoptotic properties, through a recruitment of a mitogen-activated protein kinase pathway and a concomitant activation of anti-apoptotic members of the Bcl-2 family. These multiple mechanisms might reflect the pleiotropic nature of TGF-beta, reinforcing the potential therapeutic value of this cytokine in several central nervous system disorders.