The immunoglobulin D-binding protein MID from Moraxella catarrhalis is also an adhesin - PubMed (original) (raw)

The immunoglobulin D-binding protein MID from Moraxella catarrhalis is also an adhesin

Arne Forsgren et al. Infect Immun. 2003 Jun.

Abstract

The Moraxella catarrhalis immunoglobulin D (IgD)-binding protein (MID) is a 200-kDa outer membrane protein displaying a unique and specific affinity for human IgD. MID is found in the majority of M. catarrhalis strains. In the present paper, we show that MID-expressing M. catarrhalis strains agglutinate human erythrocytes and bind to type II alveolar epithelial cells. In contrast, M. catarrhalis isolates with low MID expression levels and two mutants deficient in MID, but with readily detectable UspA1 expression, do not agglutinate erythrocytes and have a 50% lower adhesive capacity. To examine the adhesive part of MID, the protein was dissected into nine fragments covering the entire molecule. The truncated MID proteins were expressed in Escherichia coli, purified, and used for raising polyclonal antibodies in rabbits. Interestingly, by using recombinant fragments, we show that the hemagglutinating and adhesive part of MID is localized within the 150-amino-acid fragment MID(764-913). In addition, antibodies against full-length MID, MID(764-913), or a 30-amino-acid consensus sequence (MID(775-804)) inhibited adhesion to alveolar epithelial cells. Antibodies against UspA1, an outer membrane protein expressed in essentially all M. catarrhalis strains, also inhibited adhesion, suggesting that both MID and UspA1 are needed for optimal attachment to epithelial cells. Taken together, in addition to MID-dependent IgD binding, we have demonstrated that the outer membrane protein MID is a novel adhesin that would be a suitable target for a future vaccine against M. catarrhalis.

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Figures

FIG. 1.

FIG. 1.

Flow cytometry profiles of M. catarrhalis clinical isolates expressing MID at high or low intensity. IgD binding is shown for the high-intensity MID-expressing strain Bc5 (A and B) and the low-intensity MID-expressing strain BBH17 (C and D). MID expression intensity was defined according to the work of Möllenkvist et al. (22). Bacteria were incubated with human myeloma protein IgD(κ) followed by FITC-conjugated anti-IgD pAbs. Thereafter, fluorescence was analyzed by flow cytometry. Controls incubated with the FITC-conjugated anti-IgD pAb without preincubation with IgD are included (A and C).

FIG. 2.

FIG. 2.

High-intensity-MID-expressing M. catarrhalis isolates strongly adhere to epithelial cells, whereas low-intensity-MID-expressing strains and MID mutants do not. (A) Adhesion of six different M. catarrhalis strains with high or low expression of the outer membrane protein MID was studied. (B) Two different strains with mutated MID are compared to the corresponding wild-type strains. All clinical isolates expressed UspA1 as revealed by Western blotting. The M. catarrhalis strains were added to the type II alveolar epithelial cell line A549. After centrifugation, cells and bacteria were incubated for 30 min at 37°C, followed by extensive washes. Cells were treated with trypsin-EDTA, and suspensions were plated on blood agar plates and incubated overnight. CFU was counted, and attachment was calculated as a percentage of the inoculum, as follows: (CFU adherent)/(CFU added) × 100. Data are means from four independent experiments with duplicates. Error bars, standard errors of the means. The adhesion of the three high-intensity-MID-expressing strains was statistically different (P ≤ 0.01) from the adhesion of the three low-intensity-MID-expressing isolates.

FIG. 3.

FIG. 3.

MID binds to human erythrocytes, and the active domain is located between amino acid residues 764 and 913 (fragment E). (A) A series of truncated MID proteins (designated A to I) were manufactured and then used to immunize rabbits. The asterisk indicates the IgD-binding part of the MID molecule (23). (B) The resulting rabbit pAbs were equilibrated for antibody content as described in Materials and Methods. (C) The various truncated MID fragments were analyzed for binding to human erythrocytes by using the specific rabbit pAbs. Recombinant truncated proteins were produced in E. coli and purified by affinity chromatography as described in detail in Materials and Methods. On three occasions, rabbits were immunized with recombinant MID fragments. Microtiter plates were coated with a freshly isolated human erythrocyte membrane fraction, and the recombinant MID fragments were then added at the indicated concentrations (4.0, 0.5, or 0.125 μg/ml). Thereafter, specific rabbit anti-MID polyclonal antisera at dilutions equilibrated for specific antibody activity were added. After washes, plates were developed with HRP-conjugated goat anti-rabbit pAbs.

FIG. 4.

FIG. 4.

125I-labeled recombinant MID764-913 (fragment E) is specifically bound to erythrocytes and epithelial cells. 125I-labeled MID fragments (fragments A to I) were added to human erythrocytes (A) or to epithelial cells (B). All truncated MID proteins were produced in E. coli and purified by affinity chromatography. The resulting recombinant proteins were labeled with 125I and added to erythrocytes or the epithelial cell line A549. After several washes, bound radioactivity was measured in a γ-counter. Data are means from two experiments with duplicates. Error bars, standard deviations.

FIG. 5.

FIG. 5.

Adhesion of MID-expressing M. catarrhalis to epithelial cells depends on amino acid residues 764 to 913 (fragment E). Decreased adhesion to epithelial cells was observed with MID-expressing bacteria coated with a rabbit anti-MID1-2139, anti-MID764-913 (fragment E), anti-MID775-804, or anti-UspA1 pAb versus a preimmune serum or an anti-MID367-578 (fragment C) pAb. Bacteria were preincubated with the preimmune sera or specific antisera (dilution, 1/250), or with an anti-MID775-804 pAb (final concentration, 4.0 μg/ml), for 1 h at 4°C. Thereafter, bacteria were added to the epithelial cells, followed by centrifugation and incubation for 30 min at 37°C. After washes, cells were treated with trypsin-EDTA, and suspensions were plated onto blood agar plates. CFU were counted after an overnight incubation. The attachment ratio [(CFU adherent/CFU added) × 100] was calculated. All values were related to the adherence ratio obtained with the preimmune control serum (92% ± 4.3% [mean ± standard deviation] of the ratio for the control without any antisera), which was set to 100%. Data are means from four separate experiments with duplicates. Error bars, standard deviations. ***, P ≤ 0.001; **, P ≤ 0.01; *, P ≤ 0.05.

FIG. 6.

FIG. 6.

The MID molecule contains a coiled-coil structure within MID764-913 (fragment E). The full-length Bc5 MID1-2139 protein was analyzed by using Coils, version 2.2, software (19).

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