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A surface-exposed region of a novel outer membrane protein (P66) of Borrelia spp. is variable in size and sequence
J Bunikis1, C J Luke, E Bunikiene
1Department of Microbiology, University of California Irvine, 92697-4025, USA.
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.
Insights
The outer membrane protein P66 in Lyme disease bacteria has a variable surface loop. This loop region is targeted by antibodies and shows signs of evolutionary selection across different Borrelia species.
Area of Science:
- Microbiology
- Immunology
- Genetics
Background:
- The 66-kilodalton outer membrane protein (P66) of Lyme disease Borrelia species possesses a surface-exposed loop.
- This loop region contains an antigen frequently recognized by sera from Lyme disease patients.
Purpose of the Study:
- To investigate the surface-exposed loop of P66 and homologous proteins in other Borrelia species.
- To characterize the epitope recognized by monoclonal antibodies and analyze sequence variability.
Main Methods:
- Monoclonal antibody binding and epitope mapping of Borrelia burgdorferi P66.
- Cloning and sequencing of homologous P66 genes from various Borrelia species (e.g., B. hermsii, B. turicatae).
- Polymerase Chain Reaction (PCR) using conserved primers.
Main Results:
- A monoclonal antibody bound to a proteolysis-accessible region of B. burgdorferi P66, mapping to a variable surface loop.
- Homologous P66 proteins from other Borrelia species showed conserved hydrophobic regions flanking a variable loop.
- The surface loop sequences exhibited significant variation in size and composition across different Borrelia species.
Conclusions:
- The surface-exposed loop of P66 in Borrelia species is a highly variable region.
- This variability suggests that the P66 surface loop is under selective evolutionary pressure.
- The functional significance of P66 remains to be elucidated, but its variable surface region is a key finding.