Outer membrane protein A of Escherichia coli contributes to invasion of brain microvascular endothelial cells

Abstract

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.

Endocytosis ofCandida albicansby vascular endothelial cells is associated with tyrosine phosphorylation of specific host cell proteins

“…Third, E. coli stimulates the tyrosine phosphorylation of endothelial cell proteins and is endocytosed after 10-20 min of infection (Prasadarao et al, 1997;, compared with C. albicans which induces tyrosine phosphorylation and its own endocytosis only after 60 min of contact with the endothelial cells. Finally, the uptake of E. coli by brain microvascular endothelial cells is associated with the tyrosine phosphorylation of a MAP kinase (Prasadarao et al, 1997). Candida albicans did not induce any detectable phosphorylation of MAP kinases, which would have been discernible in the Triton X-100 soluble fraction.…”

IQGAP1 mediates the disruption of adherens junctions to promoteEscherichia coliK1 invasion of brain endothelial cells

“…Our previous observations showed that actin polymerization is necessary for OmpA+ E. coli to invade HBMEC (Prasadarao et al, 1996). Therefore, to examine the possible role of IQGAP1 in actin remodelling, HBMEC were infected with OmpA+ E. coli and examined for actin and IQGAP1 localization using rhodamine-phalloidin and antimyc antibody, respectively at different time points.…”

“…Escherichia coli E44, a rifampin-resistant mutant of E. coli K1 strain RS 218 (serotype O18:K1:H7), was previously isolated from the cerebrospinal fluid of a neonate with meningitis and invades HBMEC. E91, a derivative of E44 in which the ompA gene was disrupted by allelic exchange, has been described earlier (Prasadarao et al, 1996). All bacteria were grown in Luria-Bertani broth with appropriate antibiotics.…”

“…Ninety-seven per cent of the cells used in this study were positive for Factor VIII-rag, carbonic anhydrase IV, acetylated LDL uptake and 100% negative for GFAP as assessed by flow cytometry. Total cell-associated and invasion assays using OmpA+ E. coli and OmpA-E. coli were performed as described previously (Prasadarao et al, 1996). Briefly, HBMEC grown in 24-well cell culture plates to 95% confluence were infected with 10 7 cfu of E. coli K1 strains in an experimental medium (1:1 mixture of Ham's F-12 and M-199 containing 5% heat-inactivated fetal bovine serum) and incubated for 90 min at 37°C in an atmosphere containing 5% CO2.…”

Escherichia coliinteraction with human brain microvascular endothelial cells induces signal transducer and activator of transcription 3 association with the C-terminal domain of Ec-gp96, the outer membrane protein A receptor for invasion

“…The E. coli strains E44 (OmpA+) and E91 (OmpA-) used in this study are derivatives of the E. coli K1 strain RS218 (serotype 018 : K1 : H7) (Prasadarao et al, 1996a). Strain E44 is a spontaneous rifampicin-resistant mutant and invades HBMEC.…”