scholarly journals Prevention of Escherichia coli K1 Penetration of the Blood-Brain Barrier by Counteracting the Host Cell Receptor and Signaling Molecule Involved in E. coli Invasion of Human Brain Microvascular Endothelial Cells

2010 ◽  
Vol 78 (8) ◽  
pp. 3554-3559 ◽  
Author(s):  
Longkun Zhu ◽  
Donna Pearce ◽  
Kwang Sik Kim
Keyword(s):  
Escherichia Coli ◽  
Endothelial Cells ◽  
Human Brain ◽  
Blood Brain Barrier ◽  
Brain Barrier ◽  
Microvascular Endothelial Cells ◽  
Brain Microvascular Endothelial Cells ◽  
Signaling Molecule ◽  
E Coli ◽  
The Brain

ABSTRACT Escherichia coli meningitis is an important cause of mortality and morbidity, and a key contributing factor is our incomplete understanding of the pathogenesis of E. coli meningitis. We have shown that E. coli penetration into the brain requires E. coli invasion of human brain microvascular endothelial cells (HBMEC), which constitute the blood-brain barrier. E. coli invasion of HBMEC involves its interaction with HBMEC receptors, such as E. coli cytotoxic necrotizing factor 1 (CNF1) interaction with its receptor, the 67-kDa laminin receptor (67LR), and host signaling molecules including cytosolic phospholipase A2α (cPLA2α). In the present study, we showed that treatment with etoposide resulted in decreased expression of 67LR on HBMEC and inhibited E. coli invasion of HBMEC. Pharmacological inhibition of cysteinyl leukotrienes, lipoxygenated products of arachidonic acid released by cPLA2α, using montelukast (an antagonist of the type 1 cysteinyl leukotriene receptor) also inhibited E. coli invasion of HBMEC. E. coli penetration into the brain was significantly decreased by etoposide as well as by montelukast, and a combination of etoposide and montelukast was significantly more effective in inhibiting E. coli K1 invasion of HBMEC than single agents alone. These findings demonstrate for the first time that counteracting the HBMEC receptor and signaling molecule involved in E. coli invasion of HBMEC provides a novel approach for prevention of E. coli penetration into the brain, the essential step required for development of E. coli meningitis.

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

2012 ◽  
Vol 80 (6) ◽  
pp. 2035-2041 ◽  
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.

scholarly journals Environmental Growth Conditions Influence the Ability of Escherichia coli K1 To Invade Brain Microvascular Endothelial Cells and Confer Serum Resistance

1998 ◽  
Vol 66 (12) ◽  
pp. 5692-5697 ◽  
Author(s):  
Julie L. Badger ◽  
Kwang Sik Kim
Keyword(s):  
Escherichia Coli ◽  
Endothelial Cells ◽  
Blood Brain Barrier ◽  
Bacterial Growth ◽  
Growth Conditions ◽  
Brain Barrier ◽  
Microvascular Endothelial Cells ◽  
Serum Resistance ◽  
Brain Microvascular Endothelial Cells ◽  
E Coli

ABSTRACT A major limitation to advances in prevention and therapy of neonatal meningitis is our incomplete understanding of the pathogenesis of this disease. In an effort to understand the pathogenesis of meningitis due to Escherichia coli K1, we examined whether environmental growth conditions similar to those that the bacteria might be exposed to in the blood could influence the ability ofE. coli K1 to invade brain microvascular endothelial cells (BMEC) in vitro and to cross the blood-brain barrier in vivo. We found that the following bacterial growth conditions enhanced E. coli K1 invasion of BMEC 3- to 10-fold: microaerophilic growth, media buffered at pH 6.5, and media supplemented with 50% newborn bovine serum (NBS), magnesium, or iron. Growth conditions that significantly repressed invasion (i.e., 2- to 250-fold) included iron chelation, a pH of 8.5, and high osmolarity. More importantly, E. coli K1 traversal of the blood-brain barrier was significantly greater for the growth condition enhancing BMEC invasion (50% NBS) than for the condition repressing invasion (osmolarity) in newborn rats with experimental hematogenous meningitis. Of interest, bacterial growth conditions that enhanced or repressed invasion also elicited similar serum resistance phenotype patterns. This is the first demonstration that bacterial ability to enter the central nervous system can be affected by environmental growth conditions.

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

1999 ◽  
Vol 67 (9) ◽  
pp. 4751-4756 ◽  
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.

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

Pathogens ◽  
2019 ◽  
Vol 8 (4) ◽  
pp. 254 ◽  
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.

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

2000 ◽  
Vol 68 (9) ◽  
pp. 5062-5067 ◽  
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.

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

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.

scholarly journals Identification and Characterization of an Escherichia coli Invasion Gene Locus, ibeB, Required for Penetration of Brain Microvascular Endothelial Cells

1999 ◽  
Vol 67 (5) ◽  
pp. 2103-2109 ◽  
Author(s):  
Sheng-He Huang ◽  
Yu-Hua Chen ◽  
Qi Fu ◽  
Monique Stins ◽  
Ying Wang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Endothelial Cells ◽  
Blood Brain Barrier ◽  
Parent Strain ◽  
Deletion Mutant ◽  
Brain Barrier ◽  
Microvascular Endothelial Cells ◽  
Brain Microvascular Endothelial Cells ◽  
E Coli

ABSTRACT Escherichia coli K1 is the most common gram-negative organism causing neonatal meningitis, but the mechanism by whichE. coli K1 crosses the blood-brain barrier is incompletely understood. We have previously described the cloning and molecular characterization of a determinant, ibeA (also called ibe10), from the chromosome of an invasive cerebrospinal fluid isolate of E. coli K1 strain RS218 (O18:K1:H7). Here we report the identification of another chromosomal locus, ibeB, which allows RS218 to invade brain microvascular endothelial cells (BMEC). The noninvasive TnphoA mutant 7A-33 exhibited <1% the invasive ability of the parent strain in vitro in BMEC and was significantly less invasive in the central nervous system in the newborn rat model of hematogenousE. coli meningitis than the parent strain. The TnphoA insert with flanking sequences was cloned and sequenced. A 1,383-nucleotide open reading frame (ORF) encoding a 50-kDa protein was identified and termed ibeB. This ORF was found to be 97% identical to a gene encoding a 50-kDa hypothetical protein (p77211) and located in the 13-min region of the E. coli K-12 genome. However, no homology was observed between ibeB and other known invasion genes when DNA and protein databases in GenBank were searched. Like the TnphoA insertion mutant 7A-33, an isogenic ibeBdeletion mutant (IB7D5) was unable to invade BMEC. A 7.0-kb locus containing ibeB was isolated from a LambdaGEM-12 genomic library of E. coli RS218 and subcloned into a pBluescript KS vector (pKS7-7B). pKS7-7B was capable of completely restoring the BMEC invasion of the noninvasive TnphoA mutant 7A-33 and the ibeB deletion mutant IB7D5 to the level of the parent strain. More importantly, the ibeB deletion mutant IB7D5 was fully complemented by pFN476 carrying the ibeB ORF (pFN7C), indicating thatibeB is required for E. coli K1 invasion of BMEC. Taken together, these findings indicate that severalE. coli determinants, including ibeA andibeB, contribute to crossing of the blood-brain barrier.

scholarly journals Citrobacter freundii Invades and Replicates in Human Brain Microvascular Endothelial Cells

1999 ◽  
Vol 67 (8) ◽  
pp. 4208-4215 ◽  
Author(s):  
Julie L. Badger ◽  
Monique F. Stins ◽  
Kwang Sik Kim
Keyword(s):  
Endothelial Cells ◽  
Human Brain ◽  
Brain Abscess ◽  
Blood Brain Barrier ◽  
Brain Barrier ◽  
Microvascular Endothelial Cells ◽  
Brain Microvascular Endothelial Cells ◽  
Intracellular Replication ◽  
E Coli

ABSTRACT Neonatal bacterial meningitis remains a disease with unacceptable rates of morbidity and mortality despite the availability of effective antimicrobial therapy. Citrobacter spp. cause neonatal meningitis but are unique in their frequent association with brain abscess formation. The pathogenesis of Citrobacter spp. causing meningitis and brain abscess is not well characterized; however, as with other meningitis-causing bacteria (e.g.,Escherichia coli K1 and group B streptococci), penetration of the blood-brain barrier must occur. In an effort to understand the pathogenesis of Citrobacter spp. causing meningitis, we have used the in vitro blood-brain barrier model of human brain microvascular endothelial cells (HBMEC) to study the interaction between C. freundii and HBMEC. In this study, we show thatC. freundii is capable of invading and trancytosing HBMEC in vitro. Invasion of HBMEC by C. freundii was determined to be dependent on microfilaments, microtubules, endosome acidification, and de novo protein synthesis. Immunofluorescence microscopy studies revealed that microtubules aggregated after HBMEC came in contact with C. freundii; furthermore, the microtubule aggregation was time dependent and seen with C. freundii but not with noninvasive E. coli HB101 and meningitic E. coli K1. Also in contrast to other meningitis-causing bacteria, C. freundii is able to replicate within HBMEC. This is the first demonstration of a meningitis-causing bacterium capable of intracellular replication within BMEC. The important determinants of the pathogenesis of C. freundii causing meningitis and brain abscess may relate to invasion of and intracellular replication in HBMEC.

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

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.

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

2000 ◽  
Vol 68 (11) ◽  
pp. 6423-6430 ◽  
Author(s):  
Marpadga A. Reddy ◽  
Carol A. Wass ◽  
Kwang Sik Kim ◽  
David D. Schlaepfer ◽  
Nemani V. Prasadarao
Keyword(s):  
Endothelial Cells ◽  
Focal Adhesion Kinase ◽  
Tyrosine Phosphorylation ◽  
Blood Brain Barrier ◽  
Focal Adhesion ◽  
Brain Barrier ◽  
Microvascular Endothelial Cells ◽  
Brain Microvascular Endothelial Cells ◽  
E Coli ◽  
Blood Brain

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.

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