Escherichia coli K1 invasion of brain microvascular endothelial cells (BMECs) is a prerequisite for penetration into the central nervous system and requires actin cytoskeletal rearrangements. Here, we demonstrate that E. coli K1 invasion of BMECs requires RhoA activation. In addition, we show that cytotoxic necrotizing factor-1 (CNF1) contributes to E. coli K1 invasion of brain endothelial cells in vitro and traversal of the blood-brain barrier in the experimental hematogenous meningitis animal model. These in vitro and in vivo effects of CNF1 were dependent upon RhoA activation as shown by (a) decreased invasion and RhoA activation with the ⌬cnf1 mutant of E. coli K1 and (b) restoration of invasion frequency of the ⌬cnf1 mutant to the level of the parent E. coli K1 strain in BMECs with constitutively active RhoA. In addition, CNF1-enhanced E. coli invasion of brain endothelial cells and stress fiber formation were independent of focal adhesion kinase and phosphatidylinositol 3-kinase activation. This is the first demonstration that CNF1 contributes to E. coli K1 invasion of BMECs.Inadequate knowledge of the pathogenesis associated with bacterial entry into the central nervous system contributes to considerable mortality and morbidity associated with bacterial meningitis. For example, most cases of bacterial meningitis occur as a result of hematogenous spread, but it is unclear how circulating bacteria cross the blood-brain barrier (1). Escherichia coli is the most common Gram-negative microorganism causing meningitis in the neonatal period. We have previously shown that E. coli K1 crossing of the blood-brain barrier in vivo requires a threshold level of bacteremia and invasion of brain microvascular endothelial cells (BMECs) 1 and identified several E. coli determinants (OmpA, Ibe proteins, AslA, and TraJ) contributing to BMEC invasion in vitro and in vivo (2-7). We have also demonstrated that host cell actin cytoskeletal rearrangements are required for E. coli K1 invasion of BMECs, as shown by invasive E. coli K1-associated F-actin condensation and blockade of E. coli K1 invasion of BMECs by the microfilament-disrupting agents cytochalasin D and latrunculin A (8); but the specific host cell signaling pathways involved in E. coli K1 invasion and actin cytoskeletal rearrangements remain incompletely understood.Our recent studies have shown that tyrosine phosphorylation of several host cell signaling molecules is involved in E. coli K1 invasion of BMECs, as treatment of BMECs with genistein, a protein-tyrosine kinase inhibitor, blocks E. coli K1 invasion of BMECs (9). In addition, focal adhesion kinase (FAK) and phosphatidylinositol 3-kinase (PI3K) are crucial signaling pathways contributing to E. coli K1 invasion of BMECs (9, 10), but the basis of E. coli K1 activation of FAK and PI3K has yet to be defined. It is also unclear which microbial factors contribute to actin cytoskeletal rearrangements in BMECs.Cytotoxic necrotizing factor-1 (CNF1) is a dermonecrotic protein toxin produced by human and animal isolates...

Escherichia coli is the most common gram-negative bacteria causing meningitis during the neonatal period, but it is unclear what microbial factors mediate traversal of E. coli across the blood-brain barrier. Outer membrane protein A (OmpA), a highly conserved 35-kDa protein, was examined for its role in E. coli K1 invasion of brain microvascular endothelial cells (BMEC). The invasive capability of the OmpA ؉ strains was 25-to 50-fold greater than that of OmpA ؊ strains, and the invasive capability of OmpA ؊ strains was restored to the level of the OmpA ؉ strain by complementation with the ompA gene. Purified OmpA proteins and polyclonal anti-OmpA antibodies inhibited the invasion of OmpA ؉ E. coli into BMEC. Two short synthetic peptides (a hexamer, Asn-27-Glu-32, and a pentamer, Gly-65-Asn-69) generated from the N-terminal amino acid sequence of OmpA exhibited significant inhibition of OmpA ؉ E. coli invasion, suggesting that these two sequences represent the OmpA domains involved in E. coli invasion of BMEC. These findings suggest that OmpA is the first microbial structure identified to enhance E. coli invasion of BMEC, an important event in the pathogenesis of E. coli meningitis.

Most cases of neonatal Escherichia coli meningitis develop as a result of hematogenous spread, but it is not clear how circulating E. coli crosses the blood-brain barrier. In an attempt to identify E. coli structures contributing to invasion into the central nervous system (CNS), TnphoA mutagenesis was performed with an invasive CSF isolate of E. coli K1 strain RS218 (O18:K1:H7), and TnphoA mutants were examined for their noninvasive capability in brain microvascular endothelial cells (BMEC). The noninvasive mutants exhibited the invasive ability of <1% of the parent strain. One of the noninvasive mutants (10A-23) with a single TnphoA insertion and no changes in phenotypic characteristics was found to be significantly less invasive into the CNS in the newborn rat model of hematogenous E. coli meningitis. The TnphoA inserts with flanking sequences were cloned and sequenced. A novel open reading frame (8.2 kDa) was identified. Open reading frame analysis indicated that the 8.2-kDa protein (Ibe10) contained multiple transmembrane domains. ibe10 was cloned into an expression vector, pQE30, and the purified Ibe10 was shown to inhibit invasion of BMEC by strain RS218. These findings indicate that ibe10 is one of the E. coli genes involved in the invasion of BMEC in vitro and in vivo.

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 ⌬p85 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.In neonates, Escherichia coli is the most common Gramnegative bacterium that causes meningitis, a serious disease affecting the central nervous system. The most distressing aspect of neonatal Gram-negative meningitis is high morbidity and mortality despite advances in antimicrobial chemotherapy and supportive care. Increased understanding of the pathogenesis and pathophysiology of this disease can lead to improved outcome. Intravascular survival and penetration of the bloodbrain barrier by circulating bacteria represent the most critical events in the development of bacterial meningitis (1). Invasion of brain microvascular endothelial cells (BMEC)1 is a requirement for E. coli crossing of the blood-brain barrier, and it involves attachment of E. coli to BMEC through interaction of bacterial ligands with corresponding receptors present on BMEC cell surface (2). Several E. coli determinants (OmpA, IbeA, Ibe B, and YijP) involved in the invasion of BMEC have been identified (3-6). Receptors on the surface of BMEC for two of these E. coli determinants (OmpA and IbeA) have been characterized biochemically (7,8). We have recently demonstrated that E. coli invasion of BMEC occurs via a zipper-like mechanism and requires cytoskeletal rearrangements in the host cell (9). We have further characterized in BMEC that E. coli K1 induces tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin, a cytoskeletal protein known to associate with FAK (10). Furthermore, using FAK dominantnegative mutants we have shown that FAK kinase activity and its autophosphorylation site tyrosine 397 (Tyr-397) are critical for E. coli K1 invasion of HBMEC (10). These results establish that FAK signaling is e...

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SummaryMost cases of Escherichia coli K1 meningitis arise as a result of haematogenous spread, however there is a limited understanding of the mechanisms by which circulating E. coli K1 cross the blood±brain barrier. We have previously shown that environmental growth conditions both positively and negatively influence the capabilities of E. coli K1 to invade brain microvascular endothelial cells (BMEC), for example growth in media supplemented with 50% newborn bovine serum (NBS) increased BMEC invasion, whereas growth in media supplemented with 0.2 M NaCl repressed invasion in vitro and in vivo. In this study, differential fluorescence induction (DFI) was used to identify E. coli K1 genes involved in this differentially expressed invasion phenotype. E. coli K1 promoter libraries were constructed and screened for gfp expression in a manner analogous to the above growth conditions. Twentyfour clones were isolated that showed fluorescence induction when grown under the invasion-enhancing condition (i.e. NBS). Four of these clones also demonstrated repression or no induction of fluorescence when grown under the invasion-repressing condition (i.e. 0.2 M NaCl). One such clone, containing a ygdP promoter and an open reading frame (ORF), showed significant homology to Bartonella bacilliformis IalA (invasion associated locus). Among the other NBSinducing loci, finPtraJ was identified as well as several clones with no homology to other known genes. When ygdP, finPtraJ and several of the unique loci were disrupted in E. coli K1, there was a significant decrease in human BMEC (HBMEC) invasion. RNA transcript analysis determined that these newly identified invasion loci were differentially regulated at the transcriptional level. This is the first demonstration of using DFI to identify E. coli K1 genes contributing to HBMEC invasion.

Escherichia coli K1 is the leading cause of gram-negative bacterial meningitis in neonates. It is principally due to our limited understanding of the pathogenesis of this disease that the morbidity and mortality rates remain unacceptably high. To identify genes required for E. coli K1 penetration of the blood-brain barrier (BBB), we used the negative selection strategy of signature-tagged transposon mutagenesis (STM) to screen mutants for loss or decreased invasion of human brain microvascular endothelial cells (HBMEC) which comprise the BBB. A total of 3,360 insertion mutants of E. coli K1 were screened, and potential HBMEC invasion mutants were subjected to a secondary invasion screen. Those mutants that failed to pass the serial invasion screens were then tested individually. Seven prototrophic mutants were found to exhibit significantly decreased invasive ability in HBMEC. We identified traJ and five previously uncharacterized loci whose gene products are necessary for HBMEC invasion by E. coli K1. In addition, cnf1, a gene previously shown to play a role in bacterial invasion, was identified. More importantly, a traJ mutant was attenuated in penetration of the BBB in the neonatal rat model of experimental hematogenous meningitis. This is the first in vivo demonstration that traJ is involved in the pathogenesis of E. coli K1 meningitis.In order for meningitic bacteria to cause disease, the pathogen must invade the normally nonpenetrable blood-brain barrier (BBB). The two principal causes of bacterial meningitis in the neonatal period are Escherichia coli K1 and group B streptococci (10). Earlier work has shown that E. coli K1 and group B streptococci invade brain microvascular endothelial cells (BMEC) and cross the BBB in the newborn rat model of experimental hematogenous meningitis (1,15,16,20). Our previous studies have identified few E. coli K1 gene products that contribute to bacterial invasion from the circulating blood to the central nervous system (CNS). ibeA, ibeB, and outer membrane protein A (OmpA) have been demonstrated to be involved in E. coli K1 BMEC invasion in vitro and causing meningitis in the newborn rat (15,16,22,23). In addition, the K1 capsular polysaccharide has been shown to play a role in bacterial survival during penetration of the BBB (14, 17). Although these factors have been identified to be necessary for efficient E. coli K1 penetration of BMEC in vitro and in vivo, they have not been shown to be sufficient. This suggests that there are yet to be identified genes that are involved in the E. coli K1 penetration of the BBB.To facilitate the identification of E. coli K1 genes contributing to human BMEC (HBMEC) invasion, the recently developed technique of signature-tagged transposon mutagenesis (STM) was used (13). In STM, a unique DNA tag is incorporated into a transposon that enables each transposon mutant to be distinguished from others. Detection of these tags by hybridization allows for a large number of insertion mutants of E. coli K1 to be simultaneously subjected to a selec...

Inadequate knowledge of pathogenesis and pathophysiology has contributed to the high mortality and morbidity associated with neonatal Escherichia coli meningitis. We have shown previously that outer membrane protein A (OmpA) contributes to E. coli K1 invasion of brain microvascular endothelial cells. In this study we report that this OmpA ؉ K1 E. coli invasion of brain microvascular endothelial cells was inhibited by wheat germ agglutinin and chitooligomers prepared from the polymer of 1,4-linked GlcNAc, chitin. The specificity of the interaction between OmpA and GlcNAc␤1-4GlcNAc epitopes was verified by the demonstration that chitotriose-bound OmpA and wheat germ agglutinin-bound brain microvascular endothelial cell membrane proteins inhibit E. coli K1 invasion. Of interest, OmpA ؉ E. coli invasion into systemic endothelial cells did not occur, but invasion similar to that of brain microvascular endothelial cells was observed when systemic cells were treated with ␣-fucosidase, suggesting that the GlcNAc␤1-4GlcNAc moieties might be substituted with L-fucose on these cells. More importantly, the chitooligomers prevented entry of E. coli K1 into the cerebrospinal fluid of newborn rats with experimental hematogenous E. coli meningitis, suggesting that the GlcNAc␤1-4GlcNAc epitope of brain microvascular endothelial cells indeed mediates the traversal of E. coli K1 across the blood-brain barrier. A novel strategy with the use of soluble receptor analog(s) may be feasible in the prevention of devastating neonatal E. coli meningitis.