Integrins alpha(v)beta3 and alpha5beta1 mediate attachment of lyme disease spirochetes to human cells

Abstract

Borrelia burgdorferi (sensu lato), the agent of Lyme disease, is able to cause chronic, multisystemic infections in human and animal hosts. Attachment of the spirochete to host cells is likely to be important for the colonization of diverse tissues. The platelet-specific integrin alpha(IIb)beta3 was previously identified as a receptor for all three species of Lyme disease spirochetes (B. burgdorferi sensu stricto, B. garinii, and B. afzelii). Here we show that B. burgdorferi also recognizes the widely expressed integrins alpha(v)beta3 and alpha5beta1, known as the vitronectin and fibronectin receptors, respectively. Three representatives of each species of Lyme disease spirochete were tested for the ability to bind to purified alpha(v)beta3 and alpha5beta1. All of the strains tested bound to at least one integrin. Binding to one integrin was not always predictive of binding to other integrins, and several different integrin preference profiles were identified. Attachment of the infectious B. burgdorferi strain N40 to purified alpha(v)beta3 and alpha5beta1 was inhibited by RGD peptides and the appropriate receptor-specific antibodies. Binding to alpha(v)beta3 was also shown by using a transfected cell line that expresses this receptor but not alpha(IIb)beta3. Attachment of B. burgdorferi N40 to human erythroleukemia cells and to human saphenous vein endothelial cells was mediated by both alpha5beta1 and alpha(v)beta3. Our results show that multiple integrins mediate attachment of Lyme disease spirochetes to host cells.

FIG. 1

Attachment of B. burgdorferi, B. garinii, and B. afzelii to purified integrins. Bacteria in suspension were added to microtiter wells coated with purified integrins and centrifuged to facilitate bacterial-integrin contact. The plates were incubated for 1 h at room temperature and then washed to remove unbound bacteria. ³⁵S-labeled bacteria were quantitated by liquid scintillation counting. Unlabeled B. burgdorferi, B. garinii, or B. afzelii cells were quantitated by ELISA using a polyclonal rabbit antiserum directed against Lyme disease spirochetes. Binding to uncoated wells was subtracted from that of all others to give the receptor-specific signals displayed. Shown are the means plus the standard deviations of four replicates; the data are representative of multiple determinations. The relative efficiency of binding to each of the three receptors by a given strain was determined in parallel, but the percentage of unlabeled bacteria bound was not quantitated, so comparisons of binding efficiency between strains cannot be made. OD₄₀₅; optical density at 405 nm.

FIG. 2

Specific binding of B. burgdorferi to purified integrins. Purified integrins immobilized in microtiter wells were incubated for 30 min at room temperature with the indicated reagent. Radiolabeled strain N40 or unlabeled strain HB19 was added, and the bacteria were centrifuged, incubated, and quantitated as described in the legend to Fig. 1. Relative binding efficiency is defined as the degree of binding in the presence of each reagent divided by the degree of binding in the absence of any inhibitor. EDTA was used at 10 mM, GRGESP and GRGDSP linear peptides were used at 45 μM, and the cyclic RGD (cRGD) peptide G4120 was used at 150 nM. Purified MAb were used at 10 μg/ml, and ascites was used at a 1:1,000 dilution. See Materials and Methods for the source of each MAb. Shown are the means plus the standard deviations of four replicates. ND, not determined.

FIG. 3

Binding of B. burgdorferi to integrin α_vβ₃ expressed on transfected cells. 835, a cell line that expresses α_vβ₃ as a result of transfection of the genes encoding the α_v and β₃ subunits, was grown to confluence in invasin-coated microtiter wells and incubated with the reagent indicated for 30 min at room temperature. Radiolabeled strain N40 (filled bars) or HB19 (shaded bars) was then added, and the remainder of the assay was performed as described in the legends to Fig. 1 and 2. Platelet factor 4 (PF4) was used at 5 μg/ml. Purified MAb were used at 10 μg/ml and ascites was used at a 1:1,000 dilution. Relative binding efficiency is defined as the degree of binding in the presence of each reagent divided by the degree of binding in the absence of any inhibitor. Shown are the means plus the standard deviations of four replicates.

FIG. 4

Binding of B. burgdorferi to both α_vβ₃ and α₅β₁ on human cells. (A) RGD-Sepharose precipitation of α_vβ₃ from K562 cells. Octyglucoside extracts of K562 cells were incubated with invasin-coated beads, control Sepharose beads, or RGD-Sepharose beads. The beads were washed, and bound proteins were fractionated by SDS-polyacrylamide gel electrophoresis under nonreducing conditions and then transferred to a polyvinylidene difluoride membrane. Lanes containing electrophoresis markers (New England Biolabs) and purified α_vβ₃ were stained with Coomassie blue. Lanes containing proteins precipitated from K562 extracts were probed with anti-α_v or anti-β₃ MAb or with control mouse serum by using standard immunoblot protocols. Bound antibody was revealed by using an anti-mouse immunoglobulin G-horseradish peroxidase conjugate and a chemiluminescent substrate. All primary antibodies were used at a 1:500 dilution, and the enzyme conjugate was diluted 1:5,000. The values to the left are molecular masses in kilodaltons. (B) Inhibition of B. burgdorferi attachment to HSVEC and K562 cells by anti-α₅β₁ and anti-α_vβ₃ MAb. Cells in suspension were incubated with the MAb shown (each at 10 μg/ml) for 30 min at room temperature. Radiolabeled B. burgdorferi N40 was then added, and the incubation was continued for 1 h (see Materials and Methods). After washing, bound spirochetes were quantitated by liquid scintillation counting. Shown are the means plus the standard deviations of four replicates after background subtraction.