Mapping the ligand-binding region of Borrelia burgdorferi fibronectin-binding protein BBK32

Abstract

The cellular attachment and entry of pathogenic microorganisms can be facilitated by the expression of microbial adhesins that bind fibronectin. We have previously described a Borrelia burgdorferi gene, bbk32, that encodes a 47-kDa fibronectin-binding protein. In this study, the ligand-binding region of BBK32 from B. burgdorferi isolate B31 was localized to 32 amino acids. The bbk32 gene was cloned and sequenced from three additional B. burgdorferi isolates representing different genospecies of B. burgdorferi sensu lato. All four bbk32 genes encoded proteins having fibronectin-binding activity when expressed in Escherichia coli, and the deduced proteins shared 81 to 91% amino acid sequence identity within the ligand-binding domain. In addition, the ligand-binding region of BBK32 was found to share sequence homology with a fibronectin-binding peptide defined for protein F1 of Streptococcus pyogenes. The structural and functional similarity between the ligand-binding region of BBK32 and the UR region of protein F1 suggests a common mechanism of cellular adhesion and entry for B. burgdorferi and S. pyogenes.

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.

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.

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.

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.