Involvement of Focal Adhesion Kinase inEscherichia coli Invasion of Human Brain Microvascular Endothelial Cells

ABSTRACT Escherichia coli K1 traversal across the blood-brain barrier is an essential step in the pathogenesis of neonatal meningitis. We have previously shown that invasive E. colipromotes the actin rearrangement of brain microvascular endothelial cells (BMEC), which constitute a lining of the blood-brain barrier, for invasion. However, signal transduction mechanisms involved in E. coli invasion are not defined. In this report we show that tyrosine kinases play a major role in E. coli invasion of human BMEC (HBMEC). E. coli induced tyrosine phosphorylation of HBMEC cytoskeletal proteins, focal adhesion kinase (FAK), and paxillin, with a concomitant increase in the association of paxillin with FAK. Overexpression of a dominant interfering form of the FAK C-terminal domain, FRNK (FAK-related nonkinase), significantly inhibited E. coli invasion of HBMEC. Furthermore, we found that FAK kinase activity and the autophosphorylation site (Tyr397) are important in E. coli invasion of HBMEC, whereas the Grb2 binding site (Tyr925) is not required. Immunocytochemical studies demonstrated that FAK is recruited to focal plaques at the site of bacterial entry. Consistent with the invasion results, overexpression of FRNK, a kinase-negative mutant (Arg454 FAK), and a Src binding mutant (Phe397 FAK) inhibited the accumulation of FAK at the bacterial entry site. The overexpression of FAK mutants in HBMEC also blocked theE. coli-induced tyrosine phosphorylation of FAK and its association with paxillin. These observations provide evidence that FAK tyrosine phosphorylation and its recruitment to the cytoskeleton play a key role in E. coli invasion of HBMEC.

Download Full-text

Identification and Characterization of a Novel Ibe10 Binding Protein That Contributes to Escherichia coliInvasion of Brain Microvascular Endothelial Cells

Infection and Immunity ◽

10.1128/iai.67.3.1131-1138.1999 ◽

1999 ◽

Vol 67 (3) ◽

pp. 1131-1138 ◽

Cited By ~ 55

Author(s):

Nemani V. Prasadarao ◽

Carol A. Wass ◽

Sheng-He Huang ◽

Kwang Sik Kim

Keyword(s):

Endothelial Cells ◽

Amino Acid ◽

Amino Acid Sequence ◽

Affinity Chromatography ◽

Blood Brain Barrier ◽

Brain Barrier ◽

Microvascular Endothelial Cells ◽

Brain Microvascular Endothelial Cells ◽

E Coli ◽

Blood Brain

ABSTRACT The molecular basis of Escherichia coli traversal of the blood-brain barrier in the development of E. colimeningitis is not well understood. We have previously shown that a novel Ibe10 protein found in cerebrospinal fluid isolates of E. coli is necessary for invasion of the brain microvascular endothelial cells (BMEC) that constitute the blood-brain barrier both in vitro and in a newborn rat model of hematogenous meningitis. Here we identified a novel Ibe10 binding molecule/receptor (Ibe10R) on both bovine BMEC (HBMEC) and human BMEC (HBMEC) that is responsible for invasion by E. coli. Ibe10R, an approximately 55-kDa protein, was purified from BBMEC by Ibe10-Ni-Sepharose affinity chromatography. Bovine Ibe10R, as well as polyclonal antibodies to Ibe10R, blocked E. coli invasion of BBMEC very effectively. The N-terminal amino acid sequence of Ibe10R showed 75% homology to serum albumin. However, the amino acid sequence of an Ibe10R fragment generated by limited enzymatic digestion did not reveal homology to any other proteins, suggesting that Ibe10R represents a novel albumin-like protein. Immunocytochemical analysis of BBMEC using anti-Ibe10R antibody suggested that only a subset of cultured BBMEC express Ibe10R on their surface. Enrichment of Ibe10R-positive BBMEC by fluorescence-activated cell sorting with anti-Ibe10R antibody resulted in enhanced invasion by E. coli. The anti-Ibe10R antibody raised against bovine Ibe10R also blocked E. coli invasion of HBMEC very effectively. Interestingly, anti-Ibe10R antibody affinity chromatography of HBMEC membrane proteins revealed a smaller protein with an approximate molecular mass of 45 kDa. These results suggest that the Ibe10 of E. coli interacts with a novel BMEC surface protein, Ibe10R, for invasion of both BBMEC and HBMEC.

Download Full-text

The Gene Locus yijP Contributes to Escherichia coli K1 Invasion of Brain Microvascular Endothelial Cells

Infection and Immunity ◽

10.1128/iai.67.9.4751-4756.1999 ◽

1999 ◽

Vol 67 (9) ◽

pp. 4751-4756 ◽

Cited By ~ 59

Author(s):

Ying Wang ◽

Sheng-He Huang ◽

Carol A. Wass ◽

Monique F. Stins ◽

Kwang Sik Kim

Keyword(s):

Escherichia Coli ◽

Endothelial Cells ◽

Blood Brain Barrier ◽

Parent Strain ◽

Gene Locus ◽

Brain Barrier ◽

Microvascular Endothelial Cells ◽

Brain Microvascular Endothelial Cells ◽

E Coli ◽

Blood Brain

ABSTRACT Most cases of Escherichia coli meningitis develop as a result of hematogenous spread, but it is not clear how circulatingE. coli crosses the blood-brain barrier. A TnphoA mutant of E. coli K1 RS218 was shown to be significantly less invasive than its parent strain in bovine and human brain microvascular endothelial cells (BMEC), which constitute the blood-brain barrier. More importantly, traversal of the blood-brain barrier was significantly less with this mutant than with the parent strain in newborn rats with experimental hematogenous meningitis. A DNA segment containing the TnphoA insertion site was cloned from RS218, and the cloned DNA complemented the TnphoAmutant in invasion of BMEC. Nucleotide sequence revealed a near identity to that of a hypothetical yijP gene (also calledf577) in the E. coli K-12 genome. Sequence analysis indicated that the E. coli K1 yijPgene likely encodes a 66.6-kDa membrane protein. Deletion and complementation experiments indicated that the yijP gene was involved in E. coli K1 invasion of BMEC, i.e., the invasive ability of E. coli K1 was significantly reduced after yijP was deleted and was restored by complementation with a plasmid containing the yijP open reading frame. This is the first demonstration that the yijP gene locus plays a role in the pathogenesis of E. coli K1 meningitis.

Download Full-text

Meningitic Escherichia coli Induction of ANGPTL4 in Brain Microvascular Endothelial Cells Contributes to Blood–Brain Barrier Disruption via ARHGAP5/RhoA/MYL5 Signaling Cascade

Pathogens ◽

10.3390/pathogens8040254 ◽

2019 ◽

Vol 8 (4) ◽

pp. 254 ◽

Cited By ~ 1

Author(s):

Lu Liu ◽

Jixuan Li ◽

Dong Huo ◽

Zhong Peng ◽

Ruicheng Yang ◽

...

Keyword(s):

Escherichia Coli ◽

Endothelial Cells ◽

Bacterial Meningitis ◽

Blood Brain Barrier ◽

Brain Barrier ◽

Microvascular Endothelial Cells ◽

Signaling Cascade ◽

Brain Microvascular Endothelial Cells ◽

E Coli ◽

Blood Brain

Bacterial meningitis is currently recognized as one of the most important life-threatening infections of the central nervous system (CNS) with high morbidity and mortality, despite the advancements in antimicrobial treatment. The disruption of blood–brain barrier (BBB) induced by meningitis bacteria is crucial for the development of bacterial meningitis. However, the complete mechanisms involving in the BBB disruption remain to be elucidated. Here, we found meningitic Escherichia coli induction of angiopoietin-like 4 (ANGPTL4) in brain microvascular endothelial cells (BMECs) contributes to BBB disruption via ARHGAP5/RhoA/MYL5 signaling cascade, by the demonstration that ANGPTL4 was significantly upregulated in meningitis E. coli infection of BMECs as well as mice, and treatment of the recombinant ANGPTL4 protein led to an increased permeability of the BBB in vitro and in vivo. Moreover, we found that ANGPTL4 did not affect the expression of tight junction proteins involved in BBB disruption, but it increased the expression of MYL5, which was found to have a negative role on the regulation of barrier function during meningitic E. coli infection, through the activation of RhoA signaling pathway. To our knowledge, this is the first report demonstrating the disruption of BBB induced by ANGPTL4 through the ARHGAP5/RhoA/MYL5 pathway, which largely supports the involvement of ANGPTL4 during meningitic E. coli invasion and further expands the theoretical basis for the mechanism of bacterial meningitis.

Download Full-text

Escherichia coli K1 aslAContributes to Invasion of Brain Microvascular Endothelial Cells In Vitro and In Vivo

Infection and Immunity ◽

10.1128/iai.68.9.5062-5067.2000 ◽

2000 ◽

Vol 68 (9) ◽

pp. 5062-5067 ◽

Cited By ~ 57

Author(s):

Jill A. Hoffman ◽

Julie L. Badger ◽

Yan Zhang ◽

Sheng-He Huang ◽

Kwang Sik Kim

Keyword(s):

Escherichia Coli ◽

Endothelial Cells ◽

Blood Brain Barrier ◽

Brain Barrier ◽

Microvascular Endothelial Cells ◽

Brain Microvascular Endothelial Cells ◽

E Coli ◽

Blood Brain ◽

Microvascular Endothelial

ABSTRACT Neonatal Escherichia coli meningitis remains a devastating disease, with unacceptably high morbidity and mortality despite advances in supportive care measures and bactericidal antibiotics. To further our ability to improve the outcome of affected neonates, a better understanding of the pathogenesis of the disease is necessary. To identify potential bacterial genes which contribute toE. coli invasion of the blood-brain barrier, a cerebrospinal fluid isolate of E. coli K1 was mutagenized with TnphoA. TnphoA mutant 27A-6 was found to have a significantly decreased ability to invade brain microvascular endothelial cells compared to the wild type. In vivo, 32% of the animals infected with mutant 27A-6 developed meningitis, compared to 82% of those infected with the parent strain, despite similar levels of bacteremia. The DNA flanking the TnphoA insertion in 27A-6 was cloned and sequenced and determined to be homologous toE. coli K-12 aslA (arylsulfatase-like gene). The deduced amino acid sequence of the E. coli K1aslA gene product shows homology to a well-characterized arylsulfatase family of enzymes found in eukaryotes, as well as prokaryotes. Two additional aslA mutants were constructed by targeted gene disruption and internal gene deletion. Both of these mutants demonstrated decreased invasion phenotypes, similar to that of TnphoA mutant 27A-6. Complementation of the decreased-invasion phenotypes of these mutants was achieved whenaslA was supplied in trans. This is the first demonstration that this locus contributes to invasion of the blood-brain barrier by E. coli K1.

Download Full-text

Meningitic Escherichia coli α-hemolysin aggravates blood–brain barrier disruption via targeting TGFβ1-triggered hedgehog signaling

Molecular Brain ◽

10.1186/s13041-021-00826-2 ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Jiyang Fu ◽

Liang Li ◽

Dong Huo ◽

Ruicheng Yang ◽

Bo Yang ◽

...

Keyword(s):

Escherichia Coli ◽

Endothelial Cells ◽

Bacterial Meningitis ◽

Blood Brain Barrier ◽

Hedgehog Signaling ◽

Brain Barrier ◽

Microvascular Endothelial Cells ◽

Brain Microvascular Endothelial Cells ◽

E Coli ◽

Blood Brain

AbstractBacterial meningitis is a life-threatening infectious disease with severe neurological sequelae and a high mortality rate, in which Escherichia coli is one of the primary Gram-negative etiological bacteria. Meningitic E. coli infection is often accompanied by an elevated blood–brain barrier (BBB) permeability. BBB is the structural and functional barrier composed of brain microvascular endothelial cells (BMECs), astrocytes, and pericytes, and we have previously shown that astrocytes-derived TGFβ1 physiologically maintained the BBB permeability by triggering a non-canonical hedgehog signaling in brain microvascular endothelial cells (BMECs). Here, we subsequently demonstrated that meningitic E. coli infection could subvert this intercellular communication within BBB by attenuating TGFBRII/Gli2-mediated such signaling. By high-throughput screening, we identified E. coli α-hemolysin as the critical determinant responsible for this attenuation through Sp1-dependent TGFBRII reduction and triggering Ca2+ influx and protein kinase A activation, thus leading to Gli2 suppression. Additionally, the exogenous hedgehog agonist SAG exhibited promising protection against the infection-caused BBB dysfunction. Our work revealed a hedgehog-targeted pathogenic mechanism during meningitic E. coli-caused BBB disruption and suggested that activating hedgehog signaling within BBB could be a potential protective strategy for future therapy of bacterial meningitis.

Download Full-text

IbeA and OmpA of Escherichia coli K1 Exploit Rac1 Activation for Invasion of Human Brain Microvascular Endothelial Cells

Infection and Immunity ◽

10.1128/iai.06320-11 ◽

2012 ◽

Vol 80 (6) ◽

pp. 2035-2041 ◽

Cited By ~ 29

Author(s):

Ravi Maruvada ◽

Kwang Sik Kim

Keyword(s):

Escherichia Coli ◽

Endothelial Cells ◽

Human Brain ◽

Blood Brain Barrier ◽

Brain Barrier ◽

Microvascular Endothelial Cells ◽

Brain Microvascular Endothelial Cells ◽

Content Type ◽

E Coli ◽

Blood Brain

ABSTRACTMeningitis-causingEscherichia coliK1 internalization of the blood-brain barrier is required for penetration into the brain, but the host-microbial interactions involved inE. colientry of the blood-brain barrier remain incompletely understood. We show here that a meningitis-causingE. coliK1 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 inE. coliK1-mediated Rac1 activation. Since activated STAT3 is known to bind GTP-Rac1, the relationship between STAT3 and Rac1 was examined inE. coliK1 invasion of HBMEC. Downregulation of STAT3 resulted in significantly decreasedE. coliinvasion compared to control HBMEC, as well as a corresponding decrease in GTP-Rac1, suggesting that Rac1 activation in response toE. coliis under the control of STAT3. More importantly, twoE. colideterminants 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 specificE. colideterminants regulate a novel mechanism of STAT3 cross talk with Rac1 inE. coliK1 invasion of HBMEC.

Download Full-text

Hyperhomocysteinemia increases permeability of the blood-brain barrier by NMDA receptor-dependent regulation of adherens and tight junctions

Blood ◽

10.1182/blood-2011-02-338269 ◽

2011 ◽

Vol 118 (7) ◽

pp. 2007-2014 ◽

Cited By ~ 68

Author(s):

Richard S. Beard ◽

Jason J. Reynolds ◽

Shawn E. Bearden

Keyword(s):

Endothelial Cells ◽

Blood Brain Barrier ◽

Brain Barrier ◽

Microvascular Endothelial Cells ◽

Brain Microvascular Endothelial Cells ◽

Barrier Permeability ◽

Blood Brain Barrier Permeability ◽

Blood Brain ◽

Junction Protein ◽

Brain Barrier Permeability

Abstract Hyperhomocysteinemia (HHcy) increases permeability of the blood-brain barrier, but the mechanisms are undetermined. Homocysteine (Hcy) is an agonist of the neuronal N-methyl-D-aspartate receptor (NMDAr). We tested the hypothesis that HHcy disrupts the blood-brain barrier by an NMDAr-dependent mechanism in endothelium. In brain microvascular endothelial cells, there was no change in expression of the adherens junction protein VE-cadherin with Hcy treatment, but there was a significant decrease in the amount of β-catenin at the membrane. Moreover, Hcy caused nuclear translocation of β-catenin and attachment to the promoter for the tight junction protein claudin-5, with concomitant reduction in claudin-5 expression. Using a murine model of HHcy (cbs+/−), treatment for 2 weeks with an NMDAr antagonist (memantine) rescued cerebrovascular expression of claudin-5 and blood-brain barrier permeability to both exogenous sodium fluorescein and endogenous IgG. Memantine had no effect on these parameters in wild-type littermates. The same results were obtained using an in vitro model with brain microvascular endothelial cells. These data provide the first evidence that the NMDAr is required for Hcy-mediated increases in blood-brain barrier permeability. Modulating cerebral microvascular NMDAr activity may present a novel therapeutic target in diseases associated with opening of the blood-brain barrier in HHcy, such as stroke and dementia.

Download Full-text

Anthropogenic Iron Oxide Nanoparticles Induce Damage to Brain Microvascular Endothelial Cells Forming the Blood-Brain Barrier

Advances in Alzheimer’s Disease - Alzheimer’s Disease and Air Pollution ◽

10.3233/aiad210010 ◽

2021 ◽

Author(s):

Lorena Gárate-Vélez ◽

Claudia Escudero-Lourdes ◽

Daniela Salado-Leza ◽

Armando González-Sánchez ◽

Ildemar Alvarado-Morales ◽

...

Keyword(s):

Endothelial Cells ◽

Blood Brain Barrier ◽

Brain Barrier ◽

Microvascular Endothelial Cells ◽

Brain Microvascular Endothelial Cells ◽

Blood Brain ◽

Fe3o4 Nps ◽

Microvascular Endothelial ◽

The Brain

Background: Iron nanoparticles, mainly in magnetite phase (Fe3O4 NPs), are released to the environment in areas with high traffic density and braking frequency. Fe3O4 NPs were found in postmortem human brains and are assumed to get directly into the brain through the olfactory nerve. However, these pollution-derived NPs may also translocate from the lungs to the bloodstream and then, through the blood-brain barrier (BBB), into the brain inducing oxidative and inflammatory responses that contribute to neurodegeneration. Objective: To describe the interaction and toxicity of pollution-derived Fe3O4 NPs on primary rat brain microvascular endothelial cells (rBMECs), main constituents of in vitro BBB models. Methods: Synthetic bare Fe3O4 NPs that mimic the environmental ones (miFe3O4) were synthesized by co-precipitation and characterized using complementary techniques. The rBMECs were cultured in Transwell® plates. The NPs-cell interaction was evaluated through transmission electron microscopy and standard colorimetric in vitro assays. Results: The miFe3O4 NPs, with a mean diameter of 8.45 ± 0.14 nm, presented both magnetite and maghemite phases, and showed super-paramagnetic properties. Results suggest that miFe3O4 NPs are internalized by rBMECs through endocytosis and that they are able to cross the cells monolayer. The lowest miFe3O4 NPs concentration tested induced mid cytotoxicity in terms of 1) membrane integrity (LDH release) and 2) metabolic activity (MTS transformation). Conclusion: Pollution-derived Fe3O4 NPs may interact and cross the microvascular endothelial cells forming the BBB and cause biological damage.

Download Full-text