Carcinoembryonic antigen family receptor specificity of Neisseria meningitidis Opa variants influences adherence to and invasion of proinflammatory cytokine-activated endothelial cells - PubMed (original) (raw)
Carcinoembryonic antigen family receptor specificity of Neisseria meningitidis Opa variants influences adherence to and invasion of proinflammatory cytokine-activated endothelial cells
P Muenzner et al. Infect Immun. 2000 Jun.
Abstract
The carcinoembryonic antigen (CEA) family member CEACAM1 (previously called biliary glycoprotein or CD66a) was previously shown to function as a receptor that can mediate the binding of Opa protein-expressing Neisseria meningitidis to both neutrophils and epithelial cells. Since neutrophils and polarized epithelia have both been shown to coexpress multiple CEACAM receptors, we have now extended this work to characterize the binding specificity of meningococcal Opa proteins with other CEA family members. To do so, we used recombinant Escherichia coli expressing nine different Opa variants from three meningococcal strains and stably transfected cell lines expressing single members of the CEACAM family. These infection studies demonstrated that seven of the nine Opa variants bound to at least one CEACAM receptor and that binding to each of these receptors is sufficient to trigger the Opa-dependent bacterial uptake by these cell lines. The other two Opa variants do not appear to bind to either CEACAM receptors or heparan sulfate proteoglycan receptors, which are bound by some gonococcal Opa variants, thus implying a novel class of Opa proteins. We have also extended previous studies by demonstrating induction of CEACAM1 expression after stimulation of human umbilical vein endothelial cells with the proinflammatory cytokine tumor necrosis factor alpha, which is present in high concentrations during meningococcal disease. This induced expression of CEACAM1 leads to an increased Opa-dependent bacterial binding and invasion into the primary endothelia, implying that these interactions may play an important role in the pathogenesis of invasive meningococcal disease.
Figures
FIG. 1
Recombinant Opa protein expression. Opa protein variants cloned from serogroup A or C strains of N. meningiditis were cloned and expressed in E. coli DH5 or a recombinant N. gonorrhoeae MS11 derivative that has deletions in both pilin (pilE) loci and in the opaC30 locus that encodes the heparan sulfate proteoglycan-specific Opa30. Component proteins from bacterial lysate (5 μg/lane) were separated by SDS-PAGE, and immunoblots were probed with the Opa-specific MAb 4B12/C11 (1).
FIG. 2
Opa-mediated interactions with stably transfected CEACAM-expressing cell lines. HeLa cell lines expressing either CEACAM1, CEACAM3, CEACAM5, or CEACAM6 were infected for 3 h with E. coli DH5 strains expressing the indicated Opa variants. After washing, bacteria which associated with the transfected cells were quantified by lysing the HeLa cell membranes with 1% saponin and then dilution plating (black bars; 10−4 CFU). To determine the relative levels of bacteria which were intracellular, the washed samples were instead incubated in the presence of gentamicin prior to cellular lysis (grey bars; 10−2 CFU) and quantified. None of the Opa variants tested here bound to HeLa cells transfected with the empty expression vector or to stably transfected HeLa cells expressing CEACAM8 (data not shown). Assays were performed in triplicate on at least three different occasions, and the data illustrate the mean ± standard deviation of one representative experiment.
FIG. 3
Induced pattern of CEACAM receptor expression in TNF-α-stimulated HUVECs. (A) Component proteins of lysates prepared from primary HUVECs grown in the presence (+; final concentration, 10 ng/ml) or absence (−) of TNF-α were separated by SDS-PAGE, and immunoblots were probed with either the CEACAM1-, CEACAM3-, CEACAM5-, and CEACAM6-specific MAb D14HD11, the CEACAM6-specific MAb 9A6, the CEACAM8-specific MAb 80H3, or the CEACAM3- and CEACAM5-specific MAb Col1. Coomassie blue-stained samples run in parallel to the immunoblotted samples are shown in the first panel in order to confirm that protein loading was equal in the stimulated and nonstimulated cells. (B) CEACAM1 transcript expression by TNF-α-stimulated HUVECs. Total RNA was isolated after the indicated time intervals following the addition of TNF-α to the growth media. Semiquantitative duplex RT-PCR was then performed using a combination of primer pairs for the specific amplification of CEACAM and GADPH (glyceraldehyde-3-phosphate dehydrogenase) transcript fragments from total mRNA. Resulting DNA fragments were separated by electrophoresis, and amplified fragments are labeled, with the constitutively expressed GADPH standard being used to verify consistent sample amplification and loading. Sizes are indicated in base pairs.
FIG. 4
Opa-mediated association with primary endothelial cells. HUVECs seeded on glass coverslips and grown in the presence or absence of TNF-α (10 ng/ml) were infected with recombinant E. coli DH5 strains expressing the indicated meningococcal Opa variants. After 3 h, samples were fixed and then immunofluorescently labeled for analysis by confocal laser scanning microscopy. (A and B) Fixed samples of untreated (A) and TNF-α-stimulated (B) cells that have been infected with E. coli DH5 expressing meningococcal Opa94 were stained to display actin cytoskeleton (green), total bacteria (blue), and extracellular bacteria (red). Representative intracellular and extracellular bacteria are indicated by open and closed arrows, respectively. (C) Total bacteria associated per cell. Black bars, adherence to untreated HUVECs; grey bars, adherence to TNF-α-activated HUVECs. (D) Intracellular bacteria per cell. Black bars, intracellular bacteria associated with untreated HUVECs; grey bars, intracellular bacteria associated with TNF-α-activated HUVECs.
FIG. 4
Opa-mediated association with primary endothelial cells. HUVECs seeded on glass coverslips and grown in the presence or absence of TNF-α (10 ng/ml) were infected with recombinant E. coli DH5 strains expressing the indicated meningococcal Opa variants. After 3 h, samples were fixed and then immunofluorescently labeled for analysis by confocal laser scanning microscopy. (A and B) Fixed samples of untreated (A) and TNF-α-stimulated (B) cells that have been infected with E. coli DH5 expressing meningococcal Opa94 were stained to display actin cytoskeleton (green), total bacteria (blue), and extracellular bacteria (red). Representative intracellular and extracellular bacteria are indicated by open and closed arrows, respectively. (C) Total bacteria associated per cell. Black bars, adherence to untreated HUVECs; grey bars, adherence to TNF-α-activated HUVECs. (D) Intracellular bacteria per cell. Black bars, intracellular bacteria associated with untreated HUVECs; grey bars, intracellular bacteria associated with TNF-α-activated HUVECs.
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