Borrelia burgdorferi complement regulator-acquiring surface protein 2 (CspZ) as a serological marker of human Lyme disease

Abstract

Serological diagnosis of Lyme disease may be complicated by antigenic differences between infecting organisms and those used as test references. Accordingly, it would be helpful to include antigens whose sequences are well conserved by a broad range of Lyme disease spirochetes. In the present study, line blot analyses were performed using recombinant complement regulator-acquiring surface protein 2 (BbCRASP-2) from Borrelia burgdorferi sensu stricto strain B31 and serum samples from human Lyme disease patients from throughout the United States and Germany. The results indicated that a large proportion of the patients had produced antibodies recognizing recombinant BbCRASP-2. In addition, Lyme disease spirochetes isolated from across North America and Europe were found to contain genes encoding proteins with high degrees of similarity to the B. burgdorferi type strain B31 BbCRASP-2, consistent with the high percentage of serologically positive patients. These data indicate that BbCRASP-2 may be valuable for use in a widely effective serological assay.

FIG. 1.

Line blot analyses of human patient sera for antibodies recognizing BbCRASP-2. Nitrocellulose membrane strips contained stripes of recombinant BbCRASP-2 in amounts of 32, 16, 8, 4, 2, 1, and 0.1 ng (top of strip to bottom). Many of the patient serum samples from the United States have been used in previous analyses of other borrelial antigens (41, 43), and their designations are presented here for cross-referencing. (A) Paired serum samples from acute and convalescent patients diagnosed with erythema migrans in New York. Numbers above each blot indicate numbers of days between initial diagnosis and collection of serum sample. Pair 1, NY 96-1087 and 96-1088; pair 2, NY 96-1049 and 96-1050; pair 3, NY 96-1077 and 96-1078. A biopsy of the patient who provided serum samples NY 96-1087 and -1088 yielded B. burgdorferi strain 124a, the cspZ gene of which was sequenced, and the predicted BbCRASP-2 amino acid sequence is presented in Fig. 2, below. (B) Acute-phase serum samples from patients diagnosed with erythema migrans at various locations in the United States. The sample designations and reported durations of infection prior to sample collection are as follows: lane 1, NY-2, 30 days; lane 2, NY-3, 30 days; lane 3, NY-5, 30 days; lane 4, NY-6, 30 days; lane 5, NY-7, 30 days; lane 6, NY-8, 30 days; lane 7, CA 92-1682, 1 day; lane 8, WI-93-0206, 86 days; lane 9, WI 93-1414, 5 days. A biopsy of the patient who provided serum sample WI 93-0206 yielded B. burgdorferi strain 93-0117, the cspZ gene of which was sequenced, and the predicted BbCRASP-2 amino acid sequence is presented in Fig. 2, below. (C) Serum samples from German patients diagnosed with erythema migrans with a positive Lyme disease serology. (D) Serum samples from German patients diagnosed with facial palsy, meningitis, or Bannwarth's syndrome (neuroborreliosis, stage II) in the Rhein-Main area and Aschaffenburg, Germany. (E and F) Serum samples from German patients with chronic Lyme disease (ACA and arthritis, respectively). (G) Sera collected from mice 4 weeks following their infestation with ticks infected with B. burgdorferi sensu stricto strain B31. (H to M) Serum samples from non-Lyme disease patients and healthy controls, including patients with a positive syphilis serology and an otherwise-negative borrelia serology (H), leptospirosis patients (I), HIV patients (J), persons with antinuclear antibodies (K), and patients with rheumatoid arthritis (L) and serum samples from blood donors, provided by the blood bank of Frankfurt, Germany (M).

FIG. 2.

Alignment of predicted amino acid sequences of cspZ-encoded proteins of B. burgdorferi sensu lato strains collected from human and tick sources across the United States and Europe (Table 1). Each strain name is prefixed with its genospecies: Bbu, B. burgdorferi sensu stricto; Bbi, B. bissettii; Bg, B. garinii; Bs, B. spielmanii. Identical amino acids present in the majority of proteins are boxed and shaded. Regions of the B. burgdorferi sensu stricto BbCRASP-2 demonstrated to have affinity for factor H and/or FHL-1 are indicated over the alignments (24). All predicted proteins contain an amino-terminal leader polypeptide and a conserved lipobox cleavage sequence/lipid-accepting cysteine residue (56, 61). The additional amino-terminal residues of each B. garinii cspZ-encoded protein have no apparent effect upon ligand binding (53) and likely function as extended “tethers” that give mature proteins extra flexibility (19, 56).