A novel surface antigen of relapsing fever spirochetes can discriminate between relapsing fever and Lyme borreliosis

Abstract

In a previous immunoproteome analysis of Borrelia hermsii, candidate antigens that bound IgM antibodies from mice and patients infected with relapsing fever spirochetes were identified. One candidate that was identified is a hypothetical protein with a molecular mass of 57 kDa that we have designated Borrelia immunogenic protein A (BipA). This protein was further investigated as a potential diagnostic antigen for B. hermsii given that it is absent from the Borrelia burgdorferi genome. The bipA locus was amplified and sequenced from 39 isolates of B. hermsii that had been acquired from western North America. bipA was also expressed as a recombinant fusion protein. Serum samples from mice and patients infected with B. hermsii or B. burgdorferi were used to confirm the immunogenicity of the recombinant protein in patients infected with relapsing fever spirochetes. Lastly, in silico and experimental analysis indicated that BipA is a surface-exposed lipoprotein in B. hermsii. These findings enhance the capabilities of diagnosing infection with relapsing fever spirochetes.

FIG. 1.

Phylogram of bipA sequences from GGI and GGII isolates of B. hermsii. The tree was constructed with CLUSTAL V, and the outgroup used was B. turicatae.

FIG. 2.

Amino acid alignments from isolates of B. hermsii that represent the 13 and 3 alleles in GGI and GGII isolates, respectively. Identical amino acids are shaded with a gray background.

FIG. 3.

Immunoblotting to determine antibody reactivity to rBipA and borrelia lysates. The serum samples used included pooled immune sera from three mice (A) and three human patients infected with B. hermsii (B to D) or two human patients infected with B. burgdorferi (E and F). Arrows indicate protein bands of the expected size for rBipA, and the asterisk indicates the expected size of native BipA in B. hermsii DAH lysates. Molecular mass standards are indicated in kilodaltons.

FIG. 4.

Immunoblotting with pooled sera from mice immunized with rBipA. Immune sera against rBipA (A) and preimmunization sera (B) were used to determine seroconversion to BipA in B. hermsii lysates. Five species of Borrelia (C), GGI (D), and GGII (E) isolates of B. hermsii were also probed with anti-rBipA sera (C to E, top panels) and a monoclonal antibody generated against B. hermsii flagellin (C to E, lower panels). Molecular mass standards are indicated on the left of each immunoblot in kilodaltons.

FIG. 5.

Immunoblotting against B. hermsii DAH lysates and DAH rBipA using serum samples from two mice infected with the GGII isolate YOR (A and B) and two mice infected with MTW-4 (D and E). Pooled preinfection serum samples from mice infected with YOR (C) and MTW-4 (F) were also used. Arrows indicate the protein bands of the expected size for rBipA, and the asterisks indicate the expected sizes of native BipA in B. hermsii DAH lysates. Molecular mass standards are indicated in kilodaltons.

FIG. 6.

Analysis of B. hermsii DAH BipA after 0-, 30-, 60-, 90-, and 120-min incubations with proteinase K. The top panel was probed with anti-rBipA immune sera, while the bottom panel was probed with an anti-flagellin monoclonal antibody. Also, (−) PK indicates untreated spirochetes that were incubated at 37°C for the duration of the assay. Molecular mass standards are indicated on the left of the immunoblot in kilodaltons.