Borreliacidal activity of early Lyme disease sera against complement-resistant Borrelia afzelii FEM1 wild-type and an OspC-lacking FEM1 variant

Abstract

Sera obtained from 14 Lyme borreliosis patients at early stages (stages I and II) of the disease were examined for their borreliacidal properties against Borrelia afzelii isolate FEM1 by use of a growth inhibition assay. Five of 14 immune sera exhibited borreliacidal activity against isolate FEM1. Heat-inactivated immune sera failed to kill the spirochaetes. Immunoblotting experiments with outer-membrane preparations showed that OspC and 11 additional proteins of 14.0, 16.0, 17.7, 19.3, 21.7, 27.5, 32.7, 40.7, 48.9, 51.3 and 53.6 kDa were recognised by borreliacidal immune sera. To analyse the borreliacidal properties of anti-OspC antibodies, two sera (EM4 and EM5), which beside antibodies against a 51.3-kDa protein contained exclusively anti-OspC antibodies, were further investigated by comparative analysis with a FEM1 wild-type and a FEM1 variant lacking OspC in a growth inhibition assay. Only FEM1 wild-type and not variant FEM1OspC(2) was killed by immune sera EM4 and EM5. Complementdependent killing of FEM1 wild-type was mediated by formation of the terminal complement complex that was found to be attached directly to the outer membrane as con®rmed by immuno-electron microscopy. No complement deposition was observed on the surface of variant FEM1OspC(2) after incubation with immune sera EM4 and EM5, thereby suggesting that only anti-OspC antibodies in these two immune sera were responsible for borreliacidal activity. These results provide direct evidence that antiOspC antibodies, once developed during the immune response, are of critical importance for ef®cient killing of borreliae in the early phase of infection.

Differential Binding of Host Complement Inhibitor Factor H byBorrelia burgdorferiErp Surface Proteins: a Possible Mechanism Underlying the Expansive Host Range of Lyme Disease Spirochetes

“…It has been noted that while some strains of B. burgdorferi are resistant to complement-mediated killing, mutants of the same strains may be sensitive to killing (39,61). Since B. burgdorferi regulates synthesis of Erp proteins (73), it is possible that such mutants lack the ability to produce Erp proteins under the conditions tested.…”

“…Since B. burgdorferi regulates synthesis of Erp proteins (73), it is possible that such mutants lack the ability to produce Erp proteins under the conditions tested. Indeed, many such mutants are defective in gene regulation and exhibit protein profiles different from those of their parent strains (39,61). Additionally, B. burgdorferi is known to lose plasmids during cultivation, including the plasmids that encode Erp proteins (14), so it is also possible that the mutants lost plasmids encoding the Erp proteins that bind factor H for the type of serum being tested.…”

Further Characterization of Complement Regulator-Acquiring Surface Proteins ofBorrelia burgdorferi

“…After centrifugation, the pellets were resuspended in 0.1 ml of PBS and whole-cell lysates were obtained by sonication of the cells using a Branson B-12 Sonifier (Heinemann, Schwäbisch Gmünd, Germany). The lysates (15 g) were separated by Tricinesodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) via 4% stacking and 10% separating gels as described previously (21). Wide-range molecular mass markers were obtained from Sigma-Aldrich (Deisenhofen, Ger- Tween 20) for 6 h at room temperature.…”

“…5). HSP70 and flagellin served as controls for temperature-independent proteins, and OspC served as a control for a temperature-dependent protein (21,36,39). SDS-PAGE followed by Coomassie blue staining confirmed expression of HSP70 and flagellin independently of temperature and passage time.…”

C-Terminal Region of Outer Surface Protein C Binds Borreliacidal Antibodies in Sera from Patients with Lyme Disease

“…The antibodies opsonize the spirochetes to facilitate ingestion by phagocytic cells (25) or kill the spirochetes with or without activation of complement (20,21,22,28). The killing (borreliacidal) antibodies appear to be ineffective at eliminating an existing infection, presumably because the spirochetes undergo antigenic variation (32,37), suppress the immune response (5), or localize in immune privileged sites (27).…”