Cloning, expression, and sequencing of a cell surface antigen containing a leucine-rich repeat motif from Bacteroides forsythus ATCC 43037

Abstract

Bacteroides forsythus is a recently recognized human periodontopathogen associated with advanced, as well as recurrent, periodontitis. However, very little is known about the mechanism of pathogenesis of this organism. The present study was undertaken to identify the surface molecules of this bacterium that may play roles in its adherence to oral tissues or triggering of a host immune response(s). The gene (bspA) encoding a cell surface-associated protein of B. forsythus with an apparent molecular mass of 98 kDa was isolated by immunoscreening of a B. forsythus gene library constructed in a lambda ZAP II vector. The encoded 98-kDa protein (BspA) contains 14 complete repeats of 23 amino acid residues that show partial homology to leucine-rich repeat motifs. A recombinant protein containing the repeat region was expressed in Escherichia coli, purified, and utilized for antibody production, as well as in vitro binding studies. The purified recombinant protein bound strongly to fibronectin and fibrinogen in a dose-dependent manner and further inhibited the binding of B. forsythus cells to these extracellular matrix (ECM) components. In addition, adult patients with B. forsythus-associated periodontitis expressed specific antibodies against the BspA protein. We report here the cloning and expression of an immunogenic cell surface-associated protein (BspA) of B. forsythus and speculate that it mediates the binding of bacteria to ECM components and clotting factors (fibronectin and fibrinogen, respectively), which may be important in the colonization of the oral cavity by this bacterium and is also a target for the host immune response.

FIG. 1

(a) Restriction maps of positive clone pBBf4 and ExoIII-S1 nuclease deletion clones. The cloned fragment in positive clone pBBf4 is 4.2 kbp. The portions of the plasmid sequence flanking the cloned insert are shown as filled closed boxes. The ORF for the bspA gene in pBBf4 and the lacZ-bspA fusions in deletion clones are shown as dotted lines. H, HincII. (b) Immunoblot analysis of the positive clone and the ExoIII deletions. E. coli extracts harboring positive clone pBBf4 and the 5′ deletions of the insert were transferred onto nitrocellulose membranes after separation by SDS-PAGE. Membranes were probed with rabbit anti-B. forsythus serum followed by goat anti-rabbit IgG coupled to HRP. Membranes were developed with the color developing reagent TMB. Lanes: 1, total B. forsythus cell extract; 2, pBBf4 (positive clone); 3, pBluescriptSK (negative control); 4, pBBf4-D3; 5, pBBf4-D5a; 6, pBBf4-5b; 7, pBBf4-D5c; 8, pBBf4-D7; 9, pBBf4-D9. The positions of molecular size markers (sizes are in kilodaltons) are shown on the left.

FIG. 2

SDS-PAGE analysis of recombinant protein rBsp70. Lanes: 1, E. coli cells harboring pGEX-2T; 2, E. coli cells harboring pGEX-2.1 HincII; 3, purified recombinant protein rBsP70. The values on the left are molecular sizes in kilodaltons.

FIG. 3

Reactivity of rBsp70 with anti-B. forsythus (Bf) and anti-rBsp70 antibodies as determined by ELISA. Microtiter plate wells coated with purified protein rBsp70 were incubated with 1:5,000-diluted rabbit anti-B. forsythus or rabbit anti-rBsp70 serum and then incubated with goat anti-rabbit IgG coupled to HRP, and color was developed with the substrate reagent TMB. Absorbance was plotted against rBsp70 for each of the two antibodies.

FIG. 4

Nucleotide sequence and the deduced amino acid sequence of the BspA protein. The potential leader peptide sequence is underlined, and the possible cleavage site is shown by a downward-pointing arrow. The LRR region is underlined with double-head arrows. A putative ribosomal binding site (rbs) and two restriction sites (HincII and StyI) are shown. A possible terminator is indicated by the back-to-back arrows.

FIG. 5

Kyte-Doolittle hydropathy plot of the deduced amino acid sequence encoded by the bspA gene of B. forsythus. Hydrophobic regions are above the line, and the hydrophobic index is indicated on the vertical axis. Numbers on the horizontal axis refer to positions in the ORF. A putative signal sequence is indicated by the arrow.

FIG. 6

Alignment of the 23-aminno-acid repeats of the BspA protein and the T. pallidum LRR protein (TpLRR). The amino acid positions of the BspA protein are indicated on the left. The one-letter amino acid code is used. An amino acid is included in the consensus if it is present at that position in more than half of the repeats. Amino acid positions with identical or a similar amino acid substitutions between the BspA consensus and the TpLRR consensus are underlined. X, any amino acid; a, alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, or methionine; N, asparagine, cysteine, or threonine.

FIG. 7

Overlay assay. (a) Binding of rBsp70 to surface-bound protein ligands. One micogram of each of the proteins (from the left: 1, fibronectin (Fb); 2, fibrinogen (Fn); 3, type I collagen (Coll. I); 4, BSA; 5, saline blank) was blotted onto a nitrocellulose membrane. The membrane was probed with biotin-labeled rBsp70, followed by a streptavidin-peroxidase conjugate, and color was developed with the substrate TMB. The membranes in panel b were blotted with twofold serial dilutions of 4 μg of fibrinogen (or fibronectin) mixed with 4 μg of BSA. The membranes were then probed with biotin-labeled rBsp70, followed by a streptavidin-peroxidase conjugate, and color was developed with TMB as described above.

FIG. 8

Binding inhibition assay. Inhibition of B. forsythus binding to surface-coated matrix components by rBsp70. Microtiter plate wells coated with matrix components were incubated with B. forsythus cells in the presence of increasing concentrations of the rBsp70 protein. Bound cells were detected by an anti-B. forsythus antibody, followed by a second antibody coupled to peroxidase, and color development with TMB. Percent inhibition of B. forsythus cell binding to each matrix component was plotted against the rBsp70 concentration. The results shown are means of duplicate samples that are representative of several experiments.

FIG. 9

Immunoblot analysis of the B. forsythus membrane fraction. SDS-PAGE-separated proteins were transferred onto nitrocellulose membranes and probed with a rabbit anti-rBsp70 antibody and goat anti-rabbit IgG coupled to HRP. The enzyme reaction was developed with the membrane substrate TMB. Lanes: 1, rBsp70; 2, E. coli cell extract containing pBluescrioptSK⁻ (used as negative control); 3, E. coli extract harboring pBBf4 (positive clone); 4, B. forsythus membrane fraction; 5, B. forsythus whole-cell extract; 6, culture medium of actively growing B. forsythus cells. The values on the left are molecular sizes in kilodaltons.

FIG. 10

Immunofluorescence of B. forsythus cells. B. forsythus cells were incubated with rabbit anti-rBsp-70 serum (B) or preimmune rabbit serum (A), followed by goat anti-rabbit IgG coupled to fluorescein isothiocyanate. Cells reacted with the anti-rBsp70 antibody show positive fluorescence. Original magnification, ×425.

FIG. 11

Human antibody response. The thrombin-cleaved product (rBsp70) of the GST fusion protein was separated by SDS-PAGE and transferred onto a nitrocellulose membrane. Following blocking with BSA, individual membrane strips were cut out and each was incubated with individual patient and control human sera (1:250 dilution in PBS-BSA). The membrane was then developed with HRP-coupled with goat anti-human IgG and the membrane substrate reagent TBM. Membrane strips 1 to 5 were incubated with individual patient sera positive for B. forsythus, whereas strip 6 was incubated with the serum of a healthy individual (no periodontal disease). The values on the left are molecular sizes in kilodaltons.