Borrelia burgdorferi RevA antigen binds host fibronectin

Abstract

Borrelia burgdorferi, the Lyme disease-causing spirochete, can persistently infect its vertebrate hosts for years. B. burgdorferi is often found associated with host connective tissue, where it interacts with components of the extracellular matrix, including fibronectin. Some years ago, a borrelial surface protein, named BBK32, was identified as a fibronectin-binding protein. However, B. burgdorferi BBK32 mutants are still able to bind fibronectin, indicating that the spirochete possesses additional mechanisms for adherence to fibronectin. We now demonstrate that RevA, an unrelated B. burgdorferi outer surface protein, binds mammalian fibronectin in a saturable manner. Site-directed mutagenesis studies identified the amino terminus of the RevA protein as being required for adhesion to fibronectin. RevA bound to the amino-terminal region of fibronectin. RevA binding to fibronectin was not inhibited by salt or heparin, suggesting that adhesin-ligand interactions are primarily nonionic and occur through the non-heparin-binding regions of the fibronectin amino-terminal domains. revA genes are widely distributed among Lyme disease spirochetes, and the present studies determined that all RevA alleles tested bound fibronectin. In addition, RevB, a paralogous protein found in a subset of B. burgdorferi strains, also bound fibronectin. We also confirmed that RevA is produced during mammalian infection but not during colonization of vector ticks and determined that revA transcription is controlled through a mechanism distinct from that of BBK32.

FIG. 1.

RevA binds human fibronectin. (A) Binding of recombinant RevA_B31 (10 μg/ml) to immobilized human plasma fibronectin or murine laminin was analyzed by ELISA, with bound RevA_B31 detected by using purified specific antibodies. Data represent the means and standard errors of the results from two separate experiments with six replicates per ligand. The asterisk indicates a significantly different value (P = < 0.05, Student's t test assuming unequal variances). (B) Binding of human plasma fibronectin (20 μg/ml) to recombinant RevA, carbonic anhydrase, soybean trypsin inhibitor, or lysozyme as analyzed by ligand affinity blot with bound fibronectin detected by using specific antibodies. Note that all three control proteins exhibited relative affinities for fibronectin below that of even the blocking agent BSA, as demonstrated by the white areas on the blot which correspond to those proteins. (C) Dose-dependent, saturable binding of RevA_B31 to human fibronectin. Binding of recombinant RevA (10 μg/ml) to immobilized human plasma fibronectin was analyzed by ELISA, with bound RevA detected by using purified specific antibodies. Control wells were coated with immobilized laminin, and values represent RevA binding to fibronectin minus background readings for laminin. Data represent the means and standard errors of the results from two separate experiments with six replicates per RevA concentration. Protein binding affinity (K_d) was calculated as the concentration of ligand required for half-maximal binding activity. (D) Fibronectin also binds to RevA in a dose-dependent manner. Binding of human plasma fibronectin (0 to 10 μg/ml) to recombinant RevA or BSA was analyzed by ELISA, with bound fibronectin detected by using specific antibodies. Data represent the means and standard errors of the results from two separate experiments with six replicates per RevA concentration. The asterisks indicate values significantly different from the level of fibronectin binding to BSA (P = < 0.05, Student's t test assuming unequal variances).

FIG. 2.

(A) Predicted amino acid sequences of RevA proteins of B. burgdorferi strains B31, N40, and 297 and B. garinii strain PBi and of RevB of B. burgdorferi strain B31. Identical amino acid residues found in the majority of proteins are boxed and shaded. Arrows above the sequences indicate the truncation ends of the RevA ΔN, ΔC-1, and ΔC-2 mutants of B31 RevA. 297-4, encoded by cp32-4 prophage of strain 297; 297-12, encoded by cp32-4 prophage of strain 297. (B) RevA proteins from B. burgdorferi strains N40 and 297 and from B. garinii all bind human fibronectin. Binding of human plasma fibronectin to immobilized recombinant RevA proteins was analyzed by ELISA, and bound fibronectin was detected by using specific antibodies. Data represent the means and standard errors of the results from two separate experiments with six replicates per RevA protein. Recombinant ErpA of B. burgdorferi strain B31 was also assayed as a negative control protein that does not detectably bind fibronectin, as assessed by signals compared to background in all types of assays (our unpublished results). The asterisks indicate values significantly different from those for ErpA (P < 0.05, Student's t test assuming unequal variances). (C) RevB binds fibronectin. Binding of human fibronectin to immobilized recombinant RevB, as analyzed by ELISA. Data represent the means and standard errors of the results from two separate experiments with six replicates. The asterisk indicates a value significantly different from that with no fibronectin (P < 0.05, Student's t test assuming unequal variances).

FIG. 3.

B. burgdorferi binding to fibronectin is inhibited by soluble recombinant RevA (rRevA) and by antibodies specific for RevA. Glass microscope slides were coated by overnight incubation with 10 μg/ml human plasma fibronectin or control protein BSA in PBS. After being blocked, slides were covered with suspended B. burgdorferi strain B31 MI-16, ML23, or JS315 (BBK32 mutant), incubated, and washed extensively, and then bacteria were visualized by dark-field microscopy. Numbers of adherent bacteria observed at ×200 magnification in 10 fields per slide were counted. Data represent the means and standard errors of the results from three separate experiments.

FIG. 4.

The amino terminus of RevA is essential for ligand binding. Binding of human fibronectin to immobilized wild-type and truncated RevA_B31 recombinant proteins was analyzed by ELISA. Sequences of mutant proteins are indicated in Fig. 2. Data represent the means and standard errors of the results from two separate experiments with six replicates per RevA protein. The asterisk indicates a value significantly different from that for the full-length protein (P = < 0.001, Student's t test assuming unequal variances).

FIG. 5.

RevA-fibronectin interactions. (A) Binding of RevA_B31 to immobilized fibronectin fragments, as analyzed by ELISA. Bound RevA was detected by using purified specific antibody. Data represent the means and standard errors of the results from two separate experiments with six replicates. The asterisks indicate values significantly different from those with binding of RevA to whole fibronectin (P < 0.05, Student's t test assuming unequal variances). Fn, full-length fibronectin; 70kDa, amino-terminal 70-kDa fragment of fibronectin; 30kDa, 30-kDa amino-terminal domain of the 70-kDa fragment; 45kDa, 45-kDa gelatin-binding domain of the 70-kDa fragment. (B) Role of ionic interactions in RevA_B31 binding of fibronectin. Binding of fibronectin to immobilized RevA in the presence of increasing concentrations of NaCl was analyzed by ELISA. Data represent the means and standard errors of the results from three experiments with six replicates per concentration of NaCl. Fn, fibronectin; NS, not statistically significant. (C) Effects of heparin on RevA_B31-fibronectin interactions. Data represent the means and standard errors of the results from three experiments with six replicates per concentration of heparin. Fn, fibronectin; NS, not statistically significant.

FIG. 6.

Temporal analysis of revA expression during mammalian and tick infections. Illustrated are qRT-PCR results from two independently collected and processed pools of 20 to 30 infected, unfed nymphs; three independent pools of infected nymphs that had fed on naïve mice for 72 h; and the ear pinnae (E), hearts (H), and tibiotarsal joints (J) of eight mice that had been infected for 2 weeks following tick bite transmission of B. burgdorferi strain B31 MI-16. Gene expression levels were calculated as nanograms of target gene per nanogram of the constitutively expressed B. burgdorferi flaB gene. Note that at least one tissue from each of the eight mice contained detectable levels of revA mRNA.

FIG. 7.

Expression of revA is dependent upon RpoD (σ70)-containing RNA polymerase. Gene expression levels were calculated as nanograms of target gene per nanogram of the constitutively expressed B. burgdorferi flaB gene. Each experiment was repeated three times, and the error bars represent standard errors.