A surface-exposed region of a novel outer membrane protein (P66) of Borrelia spp. is variable in size and sequence

Abstract

A model of the 66-kDa outer membrane protein (P66) of Lyme disease Borrelia spp. predicts a surface-exposed loop near the C terminus. This region contains an antigen commonly recognized by sera from Lyme disease patients. In the present study, this region of P66 and homologous proteins of other Borrelia spp. were further investigated by using monoclonal antibodies, epitope mapping of P66 of Borrelia burgdorferi, and DNA sequencing. A monoclonal antibody specific for B. burgdorferi bound to the portion of P66 that was accessible to proteolysis in situ. The linear epitope for the antibody was mapped within a variable segment of the surface-exposed region. To further study this protein, the complete gene of Borrelia hermsii for a protein homologous to P66 was cloned. The deduced protein was 589 amino acids in length and 58% identical to P66 of B. burgdorferi. The B. hermsii P66 protein was predicted to have a surface-exposed region in the same location as that of B. burgdorferi's P66 protein. With primers designed on the basis of conserved sequences and PCR, we identified and cloned the same regions of P66 proteins of Borrelia turicatae, Borrelia parkeri, Borrelia coriaceae, and Borrelia anserina. The deduced protein sequences from all species demonstrated two conserved hydrophobic regions flanking a surface-exposed loop. The loop sequences were highly variable between different Borrelia spp. in both sequence and size, varying between 35 and 45 amino acids. Although the actual function of P66 of Borrelia spp. is unknown, the results suggest that its surface-exposed region is subject to selective pressure.

FIG. 1

Western blot analysis of reactivity of P66-specific monoclonal antibodies against protease-treated B. burgdorferi B31. Spirochetes were treated with buffer alone (N), trypsin (T), or proteinase K (P). After PAGE and transfer to membrane, the cell components were reacted with murine monoclonal antibody H914 or H1337. Molecular weight standards (MWS) in thousands are shown to the left.

FIG. 2

Monoclonal antibody H1337 epitope mapping. Shown are sequences of overlapping recombinant peptides (fragments 1 to 5) representing amino acids 440 to 528 of P66 of B. burgdorferi. Origins of fragments (Frag.) 1 and 5 of P66 and their Western blot reactivities with serum specimens from patients with Lyme disease have been described elsewhere (11). The amino acids of predicted transmembrane regions flanking the putative surface-exposed loop of P66 are underlined. Lysine and arginine residues at predicted trypsin cleavage sites are indicated by double underlines. Amino acids are numbered according to the processed P66 sequence of B. burgdorferi B31 (10).

FIG. 3

Western blot reactivity of monoclonal antibody H1337 to fragments 1 to 5. The E. coli lysates containing pGEX-encoded glutathione S-transferase or a product from pTOPE bearing an irrelevant insert were used for negative controls in lanes Ec1 and Ec2, respectively. Fragments 1 to 5 are described in the legend to Fig. 2. Reactivity of H1337 to P66 of B. burgdorferi B31 is shown in the rightmost lane (Bb). MWS, molecular weight standards (in thousands).

FIG. 4

Sequence comparison of predicted surface-exposed region and flanking transmembrane regions of P66 from Lyme disease Borrelia spp., relapsing fever Borrelia spp., B. coriaceae, and B. anserina. The surface-exposed segment is delimited from the transmembrane regions by spaces. Gaps are indicated by hyphens. Consensus (Cons) amino acids at each position were those that occurred in at least six of the eight sequences. Amino acids are numbered according to the processed P66 sequence of B. burgdorferi B31. Bb, B. burgdorferi B31; Baf, B. afzelii ACAI; Bg, B. garinii Ip90; Bh, B. hermsii; Bp, B. parkeri; Bt, B. turicatae; Bc, B. coriaceae; Ban, B. anserina.