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 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 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 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.

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

1999 ◽  
Vol 67 (3) ◽  
pp. 1131-1138 ◽  
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.

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.

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