Carol A. Wass

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 Δp85 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 ofE. coli meningitis.

Involvement of Focal Adhesion Kinase in Escherichia 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.

Application of signature-tagged mutagenesis for identification of escherichia coli K1 genes that contribute to invasion of human brain microvascular endothelial cells

ABSTRACT 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 identifiedtraJ 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, atraJ 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.

Blood-brain barrier permeability during the development of experimental bacterial meningitis in the rat

In an attempt to examine whether routes of bacterial entry into the central nervous system have any bearing on subsequent changes in blood-brain barrier permeability, we examined cerebrospinal fluid (CSF) penetration of circulating 125I-albumin in two different models of experimental meningitis due to K1 Escherichia coli, type III group B streptococcus, or Haemophilus influenzae type b in infant rats: hematogenous meningitis subsequent to subcutaneous inoculation of bacteria vs meningitis induced by direct inoculation of bacteria into the CSF via the cisterna magna. In the model of hematogenous meningitis, the mean CSF penetration was significantly greater in animals with H. influenzae type b meningitis than in those with meningitis due to K1 E. coli or type III group B streptococcus. In contrast, the mean CSF penetration was significantly enhanced in all animals with meningitis induced by intracisternal inoculation regardless of infecting pathogens. Tumor necrosis factor activity in CSF appeared to correlate with the functional penetration of circulating albumin across the blood-brain barrier in both models of experimental meningitis. These findings suggest that the alterations of blood-brain barrier permeability during development of experimental meningitis may vary for different models of inducing meningitis and that the mechanisms responsible for these different permeability changes may be multifactorial.

Demonstration of Opsonic and Protective Activity of Human Cord Sera against Type III Group B Streptococcus that Are Independent of Type-Specific Antibody

ABSTRACT: In an effort to further understand the host defense against group B streptococcus (GBS), we examined 71 human cord sera for their content of type III GBS IgG antibody by enzyme-linked immunosorbent assay and correlated the results with opsonic and protective activity against type III GBS. Most cord sera (67%) containing >0.1 μg/ml of type III GBS IgG antibody promoted phagocytosis and killing in vitro and protection against type III GBS in neonatal rats. However, 26% of cord sera containing <0.1 tig/ml of type III IgG antibody exhibited similar activity in vitro and in vivo against type III GBS. This opsonic and protective activity was retained in IgG fraction of whole serum, and was not directly associated with complement activity or with fibronectin. Further studies are needed to understand the mechanisms responsible for the opsonic and protective activity of some cord sera against type III GBS that may be independent of antibody to the type-specific polysaccharide antigen.

A human IgG 3 is opsonic in vitro against type III group B streptococci

Preparations of IgG 3 isolated by absorption of IgG 1, IgG 2, and IgG 4 from a human iv immunoglobulin with protein A-Sepharose were evaluated for their opsonic activities against type III group B streptococcal (GBS) strains. The resulting preparations were free of IgG 1 and IgG 2 and contained only trace amounts of IgG 4 (<2% of total IgG). These IgG 3 preparations exhibited excellent opsonic activities against type III GBS strains, similar to those of the unfractionated iv immunoglobulin (based on total IgG concentrations in the opsonic assays). In contrast, preparations of IgG 1, 2, and 4 eluted from protein A-Sepharose with 2M acetic acid and 7M urea were significantly less effective in enhancing phagocytosis and killing of type III GBS than IgG 3 preparations or iv immunoglobulin. The reasons for excellent opsonic activity of IgG 3 preparations as well as for decreased opsonic activity of IgG 1, 2, and 4 preparations are not clear. Perhaps alteration of IgG by lower pH and high concentrations of urea may have impaired the functional activity of IgG 1, 2, and 4 preparations. The significant finding of this study is the first demonstration of the excellent opsonic activity of IgG 3, emphasizing the importance of having intact IgG 3 in commercial immunoglobulin preparations used in prophylaxis or treatment of GBS infections.

Mechanisms of Cytotoxic Necrotizing Factor-1 in Escherichia Coli K1 Invasion of the Central Nervous System

Primary microcephaly (OMlM 251200) is an autosomal recessive neurodevelopmental condition in which there is a global reduction in cerebral cortex volume to a size comparable with that of early hominids. This marked reduction in human brain size (e.g. 430g compared with 1450g in the normal adult) raises the question as to whether genes mutated in microcephaly may provide insights into the mechanisms by which brain size has dramatically increased during evolution. We have identified a gene encoding a BRCAl C-terminal domaincontaining protein, mutated in MCPHl families sharing an ancestral 8p23 haplotype. This gene, microcepkalin, is expressed in the developing cerebral cortex of the fetal brain. Sequence homologies suggest a role in DNA repair or cell cycle regulation. The microceplialin gene has been present throughout vertebrate evolution, and we have characterised fish, rodent and primate orthologs. All contain the angiopoeifin-2 gene on the reverse strand in intron 12 of microcephalin. Protein sequence homology is strikingly low, with 57% sequence identity between mouse and human orthologs (c.f. mean human-mouse protein sequence identity, 85%; angiopoeitin-2, 88%). This suggests the potential for significant functional changes during recent evolution. Further studies of microcephalin in different species should provide insights into the regulation of cortical neural cell number, potentially aid understanding of neural progenitor/stem cell regulation and shed light on the evolution of the cerebral cortex.

1122 TREATMENT OF EXPERIMENTAL E. COLI (EC) INFECTION WITH THE COMBINATION OF CHEMOTHERAPY AND IMMUNOTHERAPY

The morbidity and mortality associated with neonatal EC infection have remained significant despite advances in antimicrobial chemotherapy. In a search for more effective therapy, we evaluated combination therapy against a K1 EC with cefotaxime (Ct) and two different antibody (Ab) preparations, rabbit J5 antiserum (J5 Ab) and murine IgM monoclonal antibody to the lipopolysaccharide of the infecting EC serotype (LPS Ab). EC bacteremia and meningitis.were induced in 5-day-old rats by sc inoculation. At 6 days of age, each litter was divided into 3 groups to receive Ct (50 mg/kg sc twice daily), one of the Ab preparations (50 μl/10 gm ip) or combination therapy. A control rat from each litter received saline. Bacterial counts in blood and CSF were determined daily before and during therapy and mortality recorded. All animals receiving saline died. Bacterial clearance was inversely related to the mortality, which is summarized below:As noted, LPS Ab enhanced the efficacy of Ct while J5 Ab was not beneficial. These findings suggest that the combination of antibiotics and LPS Ab have enhanced efficacy in this model.