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. 2002 Aug;70(8):4556-63.
doi: 10.1128/IAI.70.8.4556-4563.2002.

VSports手机版 - Identification of Escherichia coli outer membrane protein A receptor on human brain microvascular endothelial cells

Nemani V Prasadarao  1
Affiliations

Identification of Escherichia coli outer membrane protein A receptor on human brain microvascular endothelial cells

Nemani V Prasadarao. Infect Immun. 2002 Aug.

Erratum in (V体育平台登录)

  • Infect Immun 2002 Nov;70(11):6513

Abstract

Neonatal Escherichia coli meningitis continues to be a diagnostic and treatment challenge despite the availability of active antibiotics. Our earlier studies have shown that outer membrane protein A (OmpA) is one of the major factors responsible for Escherichia coli traversal across the blood-brain barrier that constitutes a lining of brain microvascular endothelial cells (BMEC). In this study we showed that OmpA binds to a 95-kDa human BMEC (HBMEC) glycoprotein (Ecgp) for E. coli invasion VSports手机版. Ecgp was partially purified by wheat germ agglutinin and Maackia amurensis lectin (MAL) affinity chromatography. The MAL affinity-purified HBMEC proteins bound to OmpA(+) E. coli but not to OmpA(-) E. coli. In addition, the deglycosylated MAL-bound proteins still interact with OmpA(+) E. coli, indicating the role of protein backbone in mediating the OmpA binding to HBMEC. Interestingly, the MAL affinity-bound fraction showed one more protein, a 65-kDa protein that bound to OmpA(+) E. coli in addition to Ecgp. Further, the 65-kDa protein was shown to be a cleavage product of Ecgp. Immunocytochemistry of HBMEC infected with OmpA(+) E. coli by using anti-Ecgp antibody suggests that Ecgp clusters at the E. coli entry site. Anti-Ecgp antibody also reacted to microvascular endothelium on human brain tissue sections, indicating the biological relevance of Ecgp in E. coli meningitis. Partial N-terminal amino acid sequence of Ecgp suggested that it has 87% sequence homology to gp96, an endoplasmic reticulum-resident molecular chaperone that is often expressed on the cell surface. In contrast, the 65-kDa protein, which could be the internal portion of Ecgp, showed 70% sequence homology to an S-fimbria-binding sialoglycoprotein reported earlier. These results suggest that OmpA interacts with Ecgp via the carbohydrate epitope, as well as with the protein portion for invading HBMEC. .

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Figures

FIG. 1.
FIG. 1.
Binding of HBMEC proteins to OmpA+ E. coli. Confluent monolayers of HBMEC were biotinylated, and membrane proteins were prepared and incubated with OmpA+ and OmpA− E. coli strains as described in Materials and Methods. Biotinylated BSA was used as a control. In some experiments, the HBMEC membrane proteins were preincubated with either outer membrane proteins of E44 (OmpA-Lipo.) or E91 (E91-Lipo.) reconstituted in liposomes for 30 min on ice, followed by incubation with E44. Similarly biotinylated HUVEC and CEC membranes were also analyzed by immunoblotting with streptavidin-peroxidase for their binding capacity to E44. The bound proteins were identified by diaminobenzidene and hydrogen peroxide. The molecular weight markers are indicated on the right.
FIG. 2.
FIG. 2.
Identification of OmpA-bound proteins from MAL affinity chromatography-purified proteins. HBMEC membrane proteins either unbiotinylated or biotinylated were initially subjected to WGA affinity chromatography, and the WGA-bound proteins were further subjected to MAL chromatography. The lectin-bound protein fractions were separated by SDS-10%PAGE. The unbiotinylated proteins were stained with Coomassie brilliant blue (A), and the biotinylated proteins were transferred to nitrocellulose, followed by probing with streptavidin-peroxidase (B). In addition, the 95-kDa protein from the unbiotinylated MAL-bound fraction was electroeluted, frozen at −20°C overnight, thawed, and subjected to SDS-PAGE (Ecgp-elect.).
FIG. 3.
FIG. 3.
Western analysis of various HBMEC fractions with anti-Ecgp antibody. HBMEC membrane proteins were subjected to WGA and MAL affinity chromatography as described in Materials and Methods. The MAL-bound fraction (0.5 mg) was incubated with E44 (E44+MAL-bound) or E91 (E91+MAL-bound), the bound proteins were released and separated by SDS-PAGE. After the proteins were transferred to nitrocellulose, the blot was subjected to immunoblotting with anti-Ecgp antibody. Similarly, deglycosylated Ecgp (Deglyc-Ecgp), MAL-bound either HUVEC (E44+HUVEC), or CEC (E44+CEC) membrane proteins were also analyzed as described. WGA-bound and MAL-bound fractions were also loaded onto the gel to examine the protein pattern. The molecular weight markers are indicated on the left.
FIG. 4.
FIG. 4.
Inhibition of E. coli with WGA- and MAL-bound fractions. OmpA+ E. coli (107 CFU in 100 μl) samples were incubated with either 5 μg (MAL-1) or 10 μg (MAL-2) of MAL-bound HBMEC fractions or deglycosylated MAL-bound fraction (D.Ecgp) for 1 h on ice and then added to HBMEC monolayers. In some experiments, WGA-bound either HBMEC (E44+WGA) or HUVEC (E44+HUVEC) fractions were incubated with OmpA+ E. coli strain. The invasion assays were carried out as described in Materials and Methods. Fetuin, a control protein, was used at a 100-μg concentration. The experiments were carried out at least three times in triplicate and expressed as a percentage of control with no protein (10,750 ± 1250 CFU/well). The error bars represent the standard deviation. The inhibition of E. coli invasion by WGA- and MAL-bound fractions and deglycosylated Ecgp was statistically significant compared to the control (P < 0.05 by two-tailed, unpaired t test).
FIG. 5.
FIG. 5.
Anti-Ecgp antibody recognizes HBMEC surface proteins. (A) Biotinylated HBMEC proteins (0.2 mg) were preincubated with 1 ml of agarose to preclear the nonspecific binding proteins. The supernatant was incubated with 1 μg of anti-Ecgp antibody overnight at 4°C, followed by incubation with protein A-agarose. The immunocomplexes were released by Laemmli buffer, separated by SDS-PAGE, and probed with streptavidin-peroxidase. An unrelated antibody was used as a control (Cont-Ab). (B) HBMEC and HUVEC membrane proteins were subjected to Ecgp-Ab affinity chromatography as described in Materials and Methods. The bound proteins were eluted and subjected to immunoblotting with Ecgp-Ab.
FIG. 6.
FIG. 6.
Inhibition of E. coli invasion of HBMEC by Ecgp-Ab and Ecgp-Ab affinity-purified proteins. Confluent monolayers of HBMEC were incubated with either 10 μg (Ab-1) or 20 μg (Ab-2) of Ecgp-Ab or control antibody (C-Ab) for 1 h at 37°C prior to infection with the bacteria. In some experiments, Ecgp-Ab affinity-purified proteins from HBMEC (10 μg of Ab-affini-2), HUVEC (10 μg of Ab-HUVEC), CEC (10 μg of Ab-CEC), or BSA (10 μg) were incubated with E44 for 1 h on ice before adding to the HBMEC monolayers. Invasion assays were carried out as described in Materials and Methods. The experiments were carried out at least two times in triplicate and are expressed as a percentage of control with no protein (10,400 ± 2,000 CFU/well). The error bars represent the standard deviation. The inhibition of E. coli invasion by Ecgp-Ab and Ecgp-Ab affinity-purified proteins was statistically significant compared to the control (P < 0.05 by two-tailed, unpaired t test).
FIG. 7.
FIG. 7.
Immunocytochemistry of HBMEC and human brain sections. Confluent untreated HBMEC monolayers (A and B) were treated with Ecgp-Ab (1:500), followed by infection with either OmpA+ E. coli (C and D) or OmpA− E. coli (E and F). The monolayers were washed and fixed with 2% paraformaldehyde. The bound Ecgp-Ab was identified by Cy3-conjugated secondary antibody, followed by visualization with laser confocal microscope. The sections of brain embedded in paraffin were stained with either Ecgp-Ab (G) or control rabbit IgG (H), followed by the addition of horseradish peroxidase-conjugated secondary antibody. The brown color was developed with diaminobenzidene and hydrogen peroxide. The pictures were edited and labeled using Adobe Photoshop 6.0. The arrows indicate either the location of invading bacteria or the clusterization of Ecgp.
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