Acylated cholesteryl galactosides are specific antigens of borrelia causing lyme disease and frequently induce antibodies in late stages of disease

Abstract

Borrelia burgdorferi sensu lato is the causative agent of Lyme disease (LD), an infectious disease occurring in North America, Europe, and Asia in different clinical stages. B. burgdorferi sensu lato encompasses at least 12 species, with B. burgdorferi sensu stricto, B. garinii, and B. afzelii being of highest clinical importance. Immunologic testing for LD as well as recent vaccination strategies exclusively refer to proteinaceous antigens. However, B. burgdorferi sensu stricto exhibits glycolipid antigens, including 6-O-acylated cholesteryl beta-D-galactopyranoside (ACGal), and first the data indicated that this compound may act as an immunogen. Here we investigated whether B. garinii and B. afzelii also possess this antigen, and whether antibodies directed against these compounds are abundant among patients suffering from different stages of LD. Gas-liquid chromatography/mass spectroscopy and NMR spectroscopy showed that both B. garinii and B. afzelii exhibit ACGal in high quantities. In contrast, B. hermsii causing relapsing fever features 6-O-acylated cholesteryl beta-D-glucopyranoside (ACGlc). Sera derived from patients diagnosed for LD contained antibodies against ACGal, with 80% of patients suffering from late stage disease exhibiting this feature. Antibodies reacted with ACGal from all three B. burgdorferi species tested, but not with ACGlc from B. hermsii. These data show that ACGal is present in all clinically important B. burgdorferi species, and that specific antibodies against this compound are frequently found during LD. ACGal may thus be an interesting tool for improving diagnostics as well as for novel vaccination strategies.

FIGURE 1.

TLC analysis of butanol extracted total lipids from the four Borrelia strains examined. 10 μl of the dissolved total lipids of B. afzelii (Baf), B. burgdorferi s.s. (Bbu), B. garinii (Bga), and B. hermsii (Bhe) were applied to silica gel thin-layer plates. The plates were run in CHCl₃/MeOH 85:15 (v/v) (A) and CHCl₃/MeOH/H₂O 65:25:4 (v/v) (B), dried, stained with molybdenum stain, and heated. The visualized fractions were denoted from unpolar to polar as F1 to F5.

FIGURE 2.

Comparative GLC-MS and ¹H NMR analysis of acylated cholesteryl glycosides. A, fractions F2 (acylated cholesteryl glycosides) from B. burgdorferi s.s., B. afzelii, B. garinii, and B. hermsii were purified by flash chromatography and subjected to mild acid methanolysis and subsequent peracetylation. The samples were analyzed by GLC-MS as described under “Experimental Procedures.” The peaks were assigned according to retention time referenced by standards and mass spectra. B, purified fractions F2 of the four strains were dissolved in CHCl₃/MeOD 9:1 (v/v), and ¹H spectra were recorded in 600-MHz NMR spectrometer.

FIGURE 3.

Chemical structures of identified acylated cholesteryl glycosides in LD and RF Borrelia. The structural analysis of fraction F2 in the three LD Borrelia revealed 6-O-acylated cholesteryl-β-d-galactopyranoside (ACGal), whereas the RF-causing B. hermsii contains 6-O-acylated cholesteryl-β-d-glucopyranoside (ACGlc) instead; depicted are the structures with oleic acid (18:1). The only difference in both structures is the configuration of the hydroxyl group of C-4 in the carbohydrate: in d-glucopyranoside equatorial and in d-galactopyranoside axial.

FIGURE 4.

Immunoblots of LD patient sera with separated Borrelia lipids. A, a TLC plate spotted with the total lipids of B. burgdorferi s.s. was run in CHCl₃/MeOH 85:15 (v/v) and transferred to a PVDF membrane using hot iron. Although the TLC was stained with Mostain, the membrane was first incubated with pooled sera from patients diagnosed for erythema migrans (EM), neuroborreliosis (NB), acrodermatitis chronica atrophicans (ACA), or Lyme arthritis (LA), then with anti-human IgG-HRP and eventually with ECL solution. B, 1 μg of sonicate of B. burgdorferi s.s. as positive control, MGalD purified from the same and the acylated cholesteryl glycosides (ACGal and ACGlc) from B. burgdorferi s.s. (Bbu), B. afzelii (Baf), B. garinii (Bga), and B. hermsii (Bhe) were spotted on a PVDF membrane strip. The stripes were incubated with the individual LD patient sera, secondary antibody, and developed with the ECL/film system. Depicted are samples from different LD stages and different recognition patterns.