A Surface-Exposed Region of a Novel Outer Membrane Protein (P66) ofBorreliaspp. Is Variable in Size and Sequence

Abstract

A model of the 66-kDa outer membrane protein (P66) of Lyme disease Borrelia spp. predicts a surfaceexposed 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.

Borrelia burgdorferi HtrA: evidence for twofold proteolysis of outer membrane protein p66

“…A clear preference for the hydrophobic amino acids isoleucine and leucine was noted, followed by threonine, alanine, valine and serine. P66 has a surface exposed loop structure containing a trypsin-sensitive lysine residue (Bunikis et al, 1998;Kenedy et al, 2014). The surface-exposed loop sequence was cleaved at five different positions, including on either side of trypsin-sensitive residue K487.…”

“…Recombinant full-length p66 (BB0603) protein fused to maltose binding protein (MBP-p66) was used as the target substrate for most degradation experiments was the gift of Dr. Jenifer Coburn (the inherent insolubility of p66 necessitated its use in experiments as an MBP fusion protein) (Bunikis et al, 1998;Coburn et al, 1999). Purified MBP (Novus Biologicals, Littleton, CO) and recombinant B. burgdorferi OspB (Katona et al, 2000;Coleman et al, 2013) were used as control substrates.…”

Access of Antibody or Trypsin to an Integral Outer Membrane Protein (P66) ofBorrelia burgdorferiIs Hindered by Osp Lipoproteins

“…Proteolytic products were evaluated by Western blot analysis with monoclonal antibodies against OspA (H5332), OspB (H6831), flagellin (H9724), BmpA, and P66 (H914). H914 binds to an epitope of P66 that is not susceptible to proteinase K treatment (12). Figure 1 shows that the bulk of P66 and OspB of intact cells were proteolyzed by low levels of proteinase K; i.e., 10 and 12.5 g/ml cleaved 50% of P66 and 50% of OspB, respectively.…”

Non‐heritable change of a spirochaete's phenotype by decoration of the cell surface with exogenous lipoproteins

“…The relapsing fever Borrelia spp., such as B. hermsii and B. turicatae, have on their surface Vsp and Vlp lipoproteins, which are involved with antigenic variation and are associated with different tissue localizations during mammalian infections (Barbour et al, 1982;Cadavid et al, 1993;Cadavid et al, 1997;Pennington et al, 1997;Schwan and Hinnebusch, 1998;Pennington et al, 1999). The outer membrane of spirochaetes also includes integral membrane proteins, such as the P66 protein (Probert et al, 1995;Skare et al, 1997;Bunikis et al, 1998). Perhaps because of scarcity of integral membrane proteins (Radolf, 1994) and the absence of lipopolysaccharides (Takayama et al, 1987), the borrelial outer membrane forms blebs or exhibits patching or aggregation of outer membrane antigens when surface-exposed lipoproteins are bound by antibodies (Barbour et al, 1983;Escudero et al, 1997).…”