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Lyme disease remains the most prevalent vector-borne infectious disease in North America (17). The spirochete Borrelia burgdorferi was identified as the etiologic agent of Lyme disease in 1982 (1). Since then, at least 10 genospecies representing the complex B. burgdorferi sensu lato have been described (29). Only the genospecies B. burgdorferi sensu stricto, B. garinii, and B. afzelii are well established in causing disease in humans. Recently, however, B. burgdorferi strains resembling the newly described genospecies B. bissettii were isolated from Lyme disease patients in Slovenia (27). Human infections with B. burgdorferi are transmitted by ticks of the Ixodes subgenus. Once transmitted by tick bite, B. burgdorferi establishes a localized infection at the site of tick attachment. The spirochetes migrate in the skin, producing an oval rash termed erythema migrans in 80% of Lyme disease patients (6). Days to weeks later, the spirochetes enter the vasculature and disseminate to multiple tissue and organ sites. At this stage, the patient enters an early disseminated form of Lyme disease and may present with carditis, lymphadenopathy, meningitis, and migratory joint and muscle pain. Despite the presence of a strong host immune response, B. burgdorferi may persist in the host, and spirochetes may be isolated from the patient months to years after transmission. In this late stage of Lyme disease, the patient's clinical manifestations may include cutaneous, musculoskeletal, and neurologic involvement. While early intervention with antibiotics is generally efficacious, this late form of Lyme disease may be more refractory to treatment (26).

The mechanisms by which B. burgdorferi invades and colonizes the host are poorly understood. For many bacterial pathogens, the initial step in host colonization involves the expression of adhesive molecules that mediate bacterial adherence to cells or to the extracellular matrix (20, 30). The capacity of B. burgdorferi to bind a wide variety of cells and extracellular matrix components indicates that these organisms may also express adhesive molecules (24). A common feature of several pathogenic bacteria, most notably Staphylococcus aureus and streptococci, is the expression of adhesins that bind fibronectin (10). Fibronectin is a large, dimeric glycoprotein that is produced by a broad range of cell types (22). It exists as a soluble molecule in body fluids and as an insoluble component of cell membranes and the extracellular matrix. Structurally, fibronectin is a mosaic protein composed of three types of protein modules that are organized into distinct functional domains. Through these functional domains, fibronectin can interact with a variety of macromolecules including fibrin, heparin, collagen, and integrins. Many of these functional domains are also targeted by adhesins expressed by pathogenic microorganisms.

Borrelia species also express adhesins that bind fibronectin (7, 13, 21, 28). We have previously reported on the identification of a gene, bbk32, which encodes a 47-kDa fibronectin-binding adhesin expressed by B. burgdorferi isolate B31 (21). BBK32 was localized to the outer surface of isolate B31, and the adhesin was found to interact specifically with the collagen-binding domain of fibronectin. The ability of recombinant BBK32 to inhibit binding of isolate B31 to immobilized fibronectin was also demonstrated. These results indicated that BBK32 is the primary fibronectin-binding adhesin expressed by B. burgdorferi.

In this study, we extended these earlier observations by mapping the ligand-binding region of BBK32 from B. burgdorferi isolate B31. In addition, we cloned the bbk32 gene from four isolates representing different B. burgdorferi genospecies into Escherichia coli, and determined the degree of sequence conservation and functional activity of the expressed proteins.

Mapping the fibronectin-binding Region of BBK32. The minimal region of BBK32 required to bind fibronectin was localized by creating a bbk32 gene fragment library using the Novatope system (Novagen, Madison, Wis.) and screening the library by ligand blotting. The bbk32 gene was amplified from B. burgdorferi isolate B31 as previously described (21). The amplicon was purified using QIAquick spin columns (Qiagen, Valencia, Calif.), and 5 µg of bbk32 was digested with bovine pancreatic DNase I in 50 mM Tris-HCl (pH 7.5)-0.05 mg of bovine serum albumin/ml-10 mM MnCl₂. The DNA fragments were separated on a 2% NuSieve agarose gel (FMC Bioproducts, Rockland, Maine); the 50- to 150-bp fragments were excised from the gel and purified using QIAquick spin columns. Blunt ends were created by treatment of the DNA fragments with T4 DNA polymerase. A 3' adenosine overhang was then added by Tth polymerase, and the DNA fragments were ligated into the Novagen pScreen T vector, a pET vector derivative possessing a T-cloning site upstream of a T7 gene 10 fusion partner. The bbk32 gene fragment library was constructed by transforming Novablue (DE3) competent E. coli (Novagen) with the ligation mixture and selecting for transformants by plating the bacteria on Luria-Bertani (LB) agar plates containing 50 µg of carbenicillin/ml. Transformants were transferred to nitrocellulose membranes, lysed with chloroform vapors, and denatured with 20 mM Tris (pH 8.0)-6 M urea-0.5 M NaCl. For ligand blotting, the membranes were washed extensively with 25 mM Tris (pH 7.5)-150 mM NaCl-0.05% Tween 20 (TTBS), blocked with 3% bovine serum albumin, and probed for 1 h with a 1:30,000 dilution of alkaline phosphatase-labeled human fibronectin as previously described (21). After the membranes were washed with TTBS, bound fibronectin was detected by immersion of the membranes in a solution of 5-bromo-4-chloro-3-indolylphosphate-nitroblue tetrazolium (Kirkegaard & Perry Laboratories, Gaithersburg, Md.). Following this initial screening of the bbk32 gene fragment library, the fibronectin-binding activity of each positive colony was confirmed by a second round of ligand blotting (data not shown).

View larger version (45K): [in this window] [in a new window]   FIG. 1.   Mapping the ligand-binding region of BBK32. Fragments of the bbk32 gene from B. burgdorferi isolate B31 were cloned into the pScreen T vector and expressed as recombinant fusion proteins in E. coli. Proteins from E. coli lysates were separated on a 12% polyacrylamide gel and stained with Coomassie blue (A) or transferred to a nitrocellulose membrane and probed with alkaline phosphatase-labeled human fibronectin (B). The BBK32 amino acid sequence expressed by each clone is indicated above each lane. A clone expressing the vector-derived peptide of 37 kDa is labeled Vector Only. Sizes of the molecular weight standards (MWS) are provided in kilodaltons.

Functional activity and sequence analysis of BBK32 from different genospecies of B. burgdorferi sensu lato. Having defined the ligand-binding region of BBK32, we sought to establish whether BBK32 is functionally and genetically conserved among isolates of B. burgdorferi sensu lato. To this end, we cloned and expressed bbk32 from four isolates representing different genospecies of B. burgdorferi: isolate B31, B. burgdorferi sensu stricto; isolate IP90, B. garinii; isolate ACA1, B. afzelii; and isolate DN127, B. bissettii. As previously described for isolate B31 (21), the bbk32 gene was amplified from each isolate and cloned into the pMalc2 expression vector (New England Biolabs, Inc., Beverly, Mass.), in which the gene of interest is expressed as a peptide fused to a 42-kDa vector-derived peptide. In each case, the transformation of E. coli with a bbk32 construct derived from either B31, IP90, ACA1, or DN127 resulted in the expression of an 80-kDa recombinant fusion protein that bound fibronectin, as determined by SDS-PAGE and ligand blot analysis (Fig. 2). The appearance of lower-molecular-weight bands having fibronectin-binding activity in Fig. 2 likely represents proteolysis of the overexpressed recombinant proteins. These results demonstrate that each B. burgdorferi isolate possesses a bbk32 gene that was capable of encoding a functional protein upon expression in E. coli. In contrast, when spirochetal lysates of B31, IP90, ACA1, and DN127 were tested by SDS-PAGE and ligand blotting for fibronectin-binding activity, only two of these four B. burgdorferi isolates expressed a fibronectin-binding protein (Fig. 3). As observed previously (21), isolate B31 expressed relatively high levels of a 47-kDa fibronectin-binding protein (BBK32), whereas ACA1 fibronectin-binding activity was visible as a faint band of 45 kDa. No fibronectin-binding activity was detected for the IP90 and DN127 lysates by ligand blotting. The lack of fibronectin-binding activity observed for isolates IP90 and DN127, despite the presence in both cases of a bbk32 gene capable of encoding a functional protein, suggest that bbk32 expression may be tightly regulated during in vitro cultivation of these isolates.

View larger version (36K): [in this window] [in a new window]   FIG. 2.   Fibronectin-binding activities of E. coli clones expressing the bbk32 gene from isolates representing different genospecies of B. burgdorferi. The bbk32 gene was amplified from each isolate and cloned into the pMalc2 vector. Expression of the cloned gene results in a recombinant fusion protein of 80 kDa. Lysates from each E. coli clone were subjected to SDS-PAGE on a 12% polyacrylamide gel and stained with Coomassie blue (A) or transferred to nitrocellulose for ligand blotting with alkaline phosphatase-labeled human fibronectin (B). The isolate from which bbk32 was derived is indicated above each lane. The genospecies designation for each isolate is as follows: B31, B. burgdorferi sensu stricto; IP90, B. garinii; ACA1, B. afzelii; and DN127, B. bissettii. Expression of the pMalc2 vector in E. coli produces a fusion protein of 52 kDa (Vector Only). Sizes of the molecular weight markers (MWS) are provided in kilodaltons.

View larger version (42K): [in this window] [in a new window]   FIG. 3.   Fibronectin-binding activities of isolates representing different B. burgdorferi genospecies. Proteins from the spirochete lysates were separated by SDS-PAGE on a 12% polyacrylamide gel. The proteins were detected by staining with Coomassie blue (A) or transferred to a nitrocellulose membrane for ligand blotting with alkaline phosphatase-labeled human fibronectin (B). Isolates B31, IP90, ACA1, and DN127 have been genotyped as B. burgdorferi sensu stricto, B. garinii, B. afzelii, and B. bissettii, respectively. The weak fibronectin-binding activity of strain ACA1 was evident in the original ligand blot but may not be clearly visible in photograph (B). Sizes of the molecular weight markers (MWS) are shown in kilodaltons.

View larger version (11K): [in this window] [in a new window]   FIG. 4.   Alignment of BBK32 amino acid sequences from isolates representing different genospecies of B. burgdorferi with the ligand-binding region (amino acids 131 to 162) of BBK32 from isolate B31. The genospecies designations for the isolates are as follows: B31, B. burgdorferi sensu stricto; IP90, B. garinii; ACA1, B. afzelii; and DN127, B. bissettii. Sequence dissimilarity is indicated with a single-letter amino acid code. Sequence identity is shown as a period. Shaded amino acids residues represent sequence identity between the ligand-binding regions of BBK32 from isolate B31 and the UR region of protein F1 from S. pyogenes.

Nucleotide sequence accession numbers. The GenBank accession numbers for the bbk32 sequence from B. burgdorferi isolates B31, IP90, ACA1, DN127, and N40 are AE000788, AF213178, AF213179, AF213180, and U82107, respectively.