Fibronectin binding protein BBK32 of the Lyme disease spirochete promotes bacterial attachment to glycosaminoglycans

Abstract

Borrelia burgdorferi, the agent of Lyme disease, causes a multisystemic illness that can affect the skin, heart, joints, and nervous system and is capable of attachment to diverse cell types. Among the host components recognized by this spirochete are fibronectin and glycosaminoglycans (GAGs). Three surface-localized GAG-binding bacterial ligands, Bgp, DbpA, and DbpB, have been previously identified, but recent studies suggested that at least one additional GAG-binding ligand is expressed on the spirochetal surface when the spirochete is adapted to the mammalian host environment. BBK32 is a surface lipoprotein that is produced during infection and that has been shown to bind to fibronectin. In this study, we show that, when BBK32 was produced from a shuttle vector in an otherwise nonadherent high-passage B. burgdorferi strain, the protein localized on the bacterial surface and conferred attachment to fibronectin and to mammalian cell monolayers. In addition, the high-passage strain producing BBK32 bound to purified preparations of the GAGs dermatan sulfate and heparin, as well as to these GAGs on the surfaces of cultured mammalian cells. Recombinant BBK32 recognized purified heparin, indicating that the bacterial attachment to GAGs was due to direct binding by BBK32. This GAG-binding activity of BBK32 is apparently independent of fibronectin recognition, because exogenous heparin had no effect on BBK32-mediated bacterial binding to fibronectin.

FIG. 1.

BBK32 is expressed on the surface of a nonadherent B. burgdorferi strain. (A) Lysates of low-passage B. burgdorferi B31, B. burgdorferi B314 harboring pBBK32 (“B314/pBBK32”), or the control vector pJF21 (“B314/vector”) were separated by 10% SDS-PAGE and immunoblotted with the indicated antibodies (α, anti) or far-Western blotted with fibronectin (“Fn/αFn”). (B) Intact spirochetes were briefly digested with proteinase K, and lysates prepared from these spirochetes were separated by 10% SDS-PAGE and immunoblotted with the indicated antibodies.

FIG. 2.

Expression of BBK32 in a nonadherent B. burgdorferi strain promotes efficient attachment to fibronectin. Radiolabeled strains B31, B314/pJF21 (“B314/vector”), and B314/pBBK32 were added to mock-coated wells or wells coated with fibronectin. The percentage of cells stably bound was determined by liquid scintillation counting. Each bar represents the mean of four independent determinations ± the standard deviation. Asterisk indicates that binding of B314/pBBK32 to fibronectin was significantly greater (P < 0.05) than binding of B314/vector.

FIG. 3.

BBK32 promotes spirochetal attachment to cultured epithelial cells independently of fibronectin. Radiolabeled strains B31, B314/pJF21 (“vector”), and B314/pBBK32 were added to wells containing monolayers of 293 epithelial, EAhy-926 endothelial, C6 glial, or HEp-2 epithelial cells (Fn deficient), and the percentage of cells stably bound was determined. Each bar represents the mean of four independent determinations ± the standard deviation. Asterisks indicate that binding of B314/pBBK32 to the denoted cell lines was significantly greater (P < 0.05) than binding by B314/vector.

FIG. 4.

B. burgdorferi producing BBK32 binds to GAGs on the surface of epithelial cells. (A) Radiolabeled strains B31, B314/pJF21 (“B314/vector”), and B314/pBBK32 were added to wells containing monolayers of 293 epithelial cells that had been mock treated or treated with heparinase, heparitinase, or chondroitinase ABC. The percentage of cells stably bound was determined by scintillation counting. Hep., heparinase digestion; Hpt., heparitinase digestion; Chon. ABC, chondroitinase ABC digestion. Asterisks indicate that binding of B314/pBBK32 to cells treated with the denoted lyases or combination of lyases was significantly (P < 0.05) less than binding to mock-treated cells. In addition, cell binding after treatment with a combination of lyases was significantly (P < 0.05) less than binding after any single lyase treatment. (B) Radiolabeled strains B31 and B314/pBBK32 were incubated with PBS alone or 2 mg/ml of the indicated GAG for 30 min prior to incubation with monolayers of 293 epithelial cells, and the percent of cells stably bound was determined. “Derm SO₄,” dermatan sulfate; “Chon-6-SO₄,” chondroitin-6-sulfate. Each bar represents the mean of four independent determinations ± the standard deviation (SD). Incubation of B314/pBBK32 with dermatan sulfate or heparin significantly reduced (P < 0.05; asterisks) spirochete attachment to epithelial cells compared to incubation with chondroitin-6-sulfate. Binding of B314/pBBK32 to cells treated with multiple lyases significantly reduced (P < 0.05) binding compared to spirochetes binding to cells treated with the indicated single lyases. (C) Radiolabeled strains B31, B314/pJF21 (“vector”), and B314/pBBK32 were added to mock-coated wells or wells coated with the indicated GAG or fibronectin, and the percentage of cells stably bound was determined. Each bar represents the mean of four independent determinations ± SD. Asterisks indicate that binding of B314/pBBK32 to wells coated with dermatan sulfate, heparin, or fibronectin was significantly greater (P < 0.05) than binding of B314/vector.

FIG. 5.

BBK32 binds to heparin. Recombinant MBP-BBK32 or MBP proteins were added to fibronectin- or GAG-coated wells, and bound protein was quantitated by measuring absorbance at 650 nm after ELISA using MBP antiserum and an anti-rabbit horseradish peroxidase-conjugated secondary antibody. “Chon-6-SO₄,” chondroitin-6-sulfate. Each bar represents the mean of four independent determinations ± the standard deviation. Asterisks indicate that recombinant MBP-BBK32 bound wells coated with fibronectin or heparin significantly better (P < 0.05) than MBP.

FIG. 6.

Attachment of B. burgdorferi producing BBK32 to fibronectin is not inhibited by exogenous heparin. Radiolabeled B314/pBBK32 was incubated with the indicated 2 mg/ml heparin prior to addition to fibronectin- or heparin-coated wells, and the percentage of cells stably bound was determined. Each bar represents the mean of four independent determinations ± the standard deviation. The asterisk indicates that incubation of B314/pBBK32 with heparin resulted in significantly less (P < 0.05) spirochetal binding to wells coated with heparin than incubation with chondroitin-6-sulfate.