Mapping of the Neisseria meningitidis NadA cell-binding site: relevance of predicted {alpha}-helices in the NH2-terminal and dimeric coiled-coil regions

Abstract

NadA is a trimeric autotransporter protein of Neisseria meningitidis belonging to the group of oligomeric coiled-coil adhesins. It is implicated in the colonization of the human upper respiratory tract by hypervirulent serogroup B N. meningitidis strains and is part of a multiantigen anti-serogroup B vaccine. Structure prediction indicates that NadA is made by a COOH-terminal membrane anchor (also necessary for autotranslocation to the bacterial surface), an intermediate elongated coiled-coil-rich stalk, and an NH(2)-terminal region involved in cell interaction. Electron microscopy analysis and structure prediction suggest that the apical region of NadA forms a compact and globular domain. Deletion studies proved that the NH(2)-terminal sequence (residues 24 to 87) is necessary for cell adhesion. In this study, to better define the NadA cell binding site, we exploited (i) a panel of NadA mutants lacking sequences along the coiled-coil stalk and (ii) several oligoclonal rabbit antibodies, and their relative Fab fragments, directed to linear epitopes distributed along the NadA ectodomain. We identified two critical regions for the NadA-cell receptor interaction with Chang cells: the NH(2) globular head domain and the NH(2) dimeric intrachain coiled-coil α-helices stemming from the stalk. This raises the importance of different modules within the predicted NadA structure. The identification of linear epitopes involved in receptor binding that are able to induce interfering antibodies reinforces the importance of NadA as a vaccine antigen.

FIG. 1.

Analysis of NadA binding regions using deletion mutants expressed in E. coli. (A) Proposed three-dimensional organization of NadA protein. Portions of the extracellular passenger domain, which are predicted to form dimeric and trimeric coiled coils, are shown in blue and red, respectively. The profile of different propensities to form dimeric and trimeric coiled-coil supersecondary structures has been calculated using Multicoil software (http://multicoil.lcs.mit.edu/cgibin/multicoil). The NadA globular head is shown in yellow. The α-helix linker region (L2L1) and β-barrel parts within the integral outer membrane translocator domains are shown in orange and green, respectively (15). (B) Schematic representation of the various NadA mutants used in this study. The dimeric and trimeric coiled coils, the α-helix linker, and the beta region are indicated according to the color scheme of panel A. The leader peptide is not indicated. (C) FACS analysis on whole-cell bacteria expressing NadA mutants using anti-NadA_Δ351-405 serum. (D) Western blot analysis of the expression of NadA in E. coli. Total cell lysates of the indicated deletion mutants were assayed using an anti-NadA_Δ351-405 serum. The mutant NadA_Δ30-87 was previously demonstrated to be surface exposed in a trimeric form (2). (E) Adhesion of NadA mutants. Chang monolayers were infected with E. coli expressing full-length NadA or each single deletion mutant (MOI of 100). E. coli-pET, carrying the vector alone, was used as a negative control. Results are reported as the number of CFU per well, and values represent the mean and standard deviation of several experiments performed in triplicate.

FIG. 2.

Cross-reaction of rabbit antisera to linear epitopes of NadA with native NadA in solution and on the E. coli outer membrane. (A) The diagram reports the sequence of NadA linear peptides used to immunize rabbits and their approximate location along the primary structure. In the scheme the tripartite structure of the protein is indicated: the COOH-terminal anchor to the outer membrane with the linker region, the putative coiled-coil intermediate stalk, and the NH₂-terminal globular head. Within the last, the three subdomains previously deleted with loss of cell-binding capacity (2) are also indicated as I (aa 24 to 42), II (aa 43 to 70), and III (aa 71 to 87). Antiserum to NadA_25-33 (residues in brackets) was also used in this study in addition to antiserum to NadA_25-39. The two sera were very similar, and data shown later are therefore primarily relative to antibodies obtained with the sequence at aa 25 to 39. (B) Antisera to linear epitopes of NadA bind to the surface of NadA-expressing E. coli as assessed by FACS analysis. NadA binding antibodies were revealed by PE-labeled secondary anti-rabbit IgG antibodies. (C) ELISA showing the titers of anti-peptide sera, using NadA_Δ351-405 or wild-type E. coli NadA as capturing antigens. Columns represent the reciprocal of the serum dilution giving the half-maximal optical density. The location of the sequence targeted within the predicted structure of the adhesin is schematically indicated.

FIG. 3.

Binding of affinity-purified Ab/Fab specific for NadA peptides to soluble or membrane-associated native NadA. (A) Characterization of affinity-purified Abs and Fab fragments. Coomassie staining after SDS-PAGE of a representative example of affinity-purified antipeptide antibody (aa 52 to 70) and after its cleavage by matrix-linked papain to produce Fc and Fab fragments (lanes 1, 2, and 3), and Western blotting of purified Fab fragments with anti-IgG(H+L) antibodies (lanes 4 and 5). (B) ELISAs performed using purified NadA_Δ351-405 or E. coli NadA as capturing antigens and different concentrations of affinity-purified Abs and Fab fragments to the indicated NadA peptides. Bound Ab/Fab fragments were revealed by secondary Ab to rabbit IgG conjugated to horseradish peroxidase. After colorimetric development, the reciprocal of the Ab/Fab fragment nanomolar concentration giving an optical density (OD) of 0.75 was calculated from the graphs and plotted.

FIG. 4.

Neutralization of NadA-mediated E. coli adhesion to Chang cells by anti-NadA peptide Abs/Fab fragments. Adherence of E. coli NadA to Chang cells was inhibited by the addition of 45 nM (A) or increasing concentrations (B) of affinity-purified Abs/Fab fragments against the specified NadA peptide. Adhesion efficacy is expressed with respect to control sample (E. coli NadA infecting Chang cells in the absence of Ab/Fab fragments), arbitrarily fixed at 100. Data are the means ± standard deviations from several experiments run in triplicate.

FIG. 5.

Efficacy of antisera raised against NadA linear peptides to inhibit N. meningitidis adhesion to Chang cells. Adherence to epithelial cells of N. meningitidis serogroup B strain M58 was inhibited using different dilutions of the indicated rabbit sera. Data are expressed compared to control sample (MC58 strain in the absence of serum) arbitrarily fixed at 100. Values represent the means and standard deviations of one representative experiment performed in triplicate. p.i., preimmune serum.

FIG. 6.

Model of NadA-NadA receptor interaction. The picture shows the predicted globular head (aa 24 to 42, subdomain I; aa 43-87, subdomains II and III) and the neighboring intrachain coiled-coil domain (aa 88-133, ccD). It is proposed that the regions around sequences of aa 24 to 39 and 94 to 121 form the surface interacting with the NadA receptor (R), while aa 42 to 88 are not directly associated with NadA receptor. The possible interaction with specific Fab fragments and NadA receptor is also depicted to account for competition studies. A single polypeptide chain of the three forming the adhesin oligomer is shown here for clarity.