Structural and Functional Analysis of Cell Wall-anchored Polypeptide Adhesin BspA in Streptococcus agalactiae

Abstract

Streptococcus agalactiae (group B Streptococcus, GBS) is the predominant cause of early-onset infectious disease in neonates and is responsible for life-threatening infections in elderly and immunocompromised individuals. Clinical manifestations of GBS infection include sepsis, pneumonia, and meningitis. Here, we describe BspA, a deviant antigen I/II family polypeptide that confers adhesive properties linked to pathogenesis in GBS. Heterologous expression of BspA on the surface of the non-adherent bacterium Lactococcus lactis confers adherence to scavenger receptor gp340, human vaginal epithelium, and to the fungus Candida albicans Complementary crystallographic and biophysical characterization of BspA reveal a novel β-sandwich adhesion domain and unique asparagine-dependent super-helical stalk. Collectively, these findings establish a new bacterial adhesin structure that has in effect been hijacked by a pathogenic Streptococcus species to provide competitive advantage in human mucosal infections.

Keywords: AgI/II family polypeptide; Candida albicans; Streptococcus; adhesin; bacterial adhesion; bacterial pathogenesis; protein conformation; structural model; structure-function; x-ray crystallography.

FIGURE 1.

A, model structure of Bsp family proteins based on the proposed domain organization of other AgI/II polypeptides (8, 9). This comprises a stalk consisting of the α-helical A domain and the polyproline II (PPII) helical P domain, separating the V domain and the C-terminal domains. The C-terminal domain is followed by the LPXTG motif required for cell wall anchorage. B, amino sequence alignment of BspA, BspB, BspC, and BspD. Structural regions are colored as in A with amino acids conserved in all four proteins highlighted in black.

FIGURE 2.

Interactions of BspA with immobilized gp340. A, dot immunoblot to verify the expression of BspA on the surface of L. lactis NZ9800. Lactococci were cultured for 16 h in the absence (uninduced) or presence of 10 or 100 ng/ml nisin to induce BspA expression. Lactococcal suspensions (A_{600 nm} 2.0) were serially 2-fold diluted and spotted onto nitrocellulose membrane. The blot was probed with α-Spy1325 mid antibodies to the V region of AspA (25). B, binding of L. lactis pMSP.bspA⁺ or vector control (pMSP) to immobilized gp340. Induced or uninduced cells were then incubated in microwells coated with 50 ng of gp340 (black column) or blocking agent BSA (gray column) for 2 h at 37 °C. Nonadherent cells were removed, and total biomass was measured by crystal violet staining. Values given represent mean ± S.D. of three independent experiments performed in triplicate. *, p < 0.005 relative to vector control, calculated using an unpaired Student's t test.

FIGURE 3.

Antibody inhibition binding studies. A, affinity purification of α-rV.BspA antibodies. Antibodies reactive against recombinant BspA-V (α-rV.BspA) were affinity-purified from antibodies raised against AspA (α-Spy1325mid). Purified α-rV.BspA antibodies were used to probe dot immunoblots of serially diluted (1:2) recombinant proteins corresponding to BspA-V, -P, and -C domains. Purified antibodies reacted only with BspA-rV. B, effects of affinity-purified antibody α-rV.BspA on adherence of L. lactis expressing BspA to immobilized gp340. L. lactis pMSP.bspA⁺ cells were FITC-labeled, preincubated without antibody or with 2.4 μg α-rV.BspA, and then incubated in microwells coated with 50 ng of gp340 for 2 h at 37 °C. Nonadherent cells were removed, and relative numbers of attached cells measured in a fluorescence plate reader. L. lactis pKS80.sspB⁺ expressing S. gordonii SspB was included as a control. Values given represent the mean ± S.D. of two independent experiments performed in duplicate. *, p < 0.0001 relative to no antiserum (A/S) control, calculated using an unpaired Student's t test.

FIGURE 4.

Interactions of GBS or lactococci expressing BspA with C. albicans. A, GBS NEM316 cells; B, L. lactis pMSP vector control; or C, L. lactis pMSP.bspA⁺. L. lactis strains were cultured for 16 h in the absence (uninduced) or presence of 100 ng/ml nisin to induce BspA expression. GBS or L. lactis cells were FITC-labeled and incubated in suspension with hypha-forming cells of C. albicans stained with calcofluor white. Aggregates were visualized by fluorescence microscopy. Scale bars, 20 μm.

FIGURE 5.

Interactions of L. lactis pMSP.bspA⁺ or pMSP vector control with VK2/E6E7 vaginal epithelial cell line. Lactococci (multiplicity of infection 5) were incubated with monolayers of VK2/E6E7 cells and numbers of associated CFU enumerated from serial dilutions of recovered cell lysates. Values given represent mean ± S.D. of four independent experiments performed in triplicate. *, p < 0.05 relative to vector control, calculated using a paired Student's t test.

FIGURE 6.

Crystal structures of BspA-V and -C domains. A, superposition of the crystal structures of BspA-C_G744D (green) and BspC (blue). The location of the two isopeptide bonds are highlighted. B, crystal structure of the BspA-V strand-swapped homodimer. C, crystal structure of a BspA-V monomer. The domain adopts a β-sandwich fold, consisting of two anti-parallel sheets made up of five and seven strands, respectively. The N- and C-terminal strands meet to connect the V domain with A and P domains, respectively. A close-up view of the BspA-V target binding pocket is provided as an inset. The putative gating loop is labeled. For clarity, residues within the gating loop for which compelling electron density was not observed (Asp-298 and Asn-299) have been excluded from the structure. D, electrostatic surface representation of BspA-V mirroring the views of the protein shown in C. Electrostatic potentials were calculated using APBS (69).

FIGURE 7.

Biophysical characterization of BspA-A·P complex formation. A, CD spectroscopy of BspA-A, BspA-P, and an equimolar mixture of both proteins. B, ITC analysis monitoring the titration of BspA-A into BspA-P. C, SEC elution profiles of BspA-A, BspA-P, and the BspA-A·P complex. D, SEC column calibration curve calculated using Bio-Rad protein standards (thyroglobulin, 670 kDa; γ-globulin, 158 kDa; ovalbumin, 44 kDa; myoglobin, 17 kDa; vitamin B₁₂, 1.35 kDa). E, molecular model of the BspA-A·P complex highlighting the role of BspA-A domain (blue) asparagine residues in mediating interaction with BspA-P (green). F, CD spectroscopy of BspA-A_ΔAsn, BspA-P, and an equimolar mixture of both proteins. G, SEC elution profiles of BspA-A_ΔAsn, BspA-P, and an equimolar mixture of both proteins.

FIGURE 8.

Molecular dynamics simulations of the model of BspA-A·P, BspA-A, and BspA-P. A, structures at time points during the simulations. The dimeric BspA-A·P model behaves as a semi-rigid rod during the simulation, whereas the BspA-A α-helix shows kinking and partial unfolding of the helix. The PPII BspA-P rapidly collapses into a random coil. Ribbons are rainbow colored blue N terminus through red C terminus. B, initial model. The alignment of most of the asparagine residues in BspA-A toward the backbone of BspA-P is shown. Asn residues, pink spheres; Pro residues, magenta spheres. C, plots of radius of gyration with respect to time. BspA-A·P, red; BspA-A, green; BspA-P, blue.