Borrelia valaisiana Resist Complement-Mediated Killing Independently of the Recruitment of Immune Regulators and Inactivation of Complement Components
Figure 5
Determination of the C3b proteolytic activity of B. valaisiana.
(A) Schematic representation of the α- and the β-chain of C3b and the cleavage fragments of the α-chain generated by CFH and Factor I. (B) Degradation of C3b by an intrinsic proteolytic activity of borrelial cells (4×107) was analyzed by detection of characteristic cleavage fragments after incubation of spirochetes with (+) or without (−) purified CFH. B. burgdorferi LW2, B. garinii G1, B. valaisiana isolates Bv9, VS116, and ZWU3 Ny3 were incubated with CFH for 60 min at room temperature. After extensive washing with PBS, C3b (10 ng/ml) and Factor I (20 ng/ml) were added and the mixture was incubated for 30 min at 37°C. Subsequently, samples were heated to 95°C for 5 min, subjected to 12.5% Laemmli-SDS-PAGE and transferred onto a nitrocellulose membrane. The C3b degradation products were visualized by Western blotting using a polyclonal goat anti-human C3 antiserum. As a positive control, purified CFH (50 ng) was incubated with C3b and Factor I, and as a negative control complement proteins were incubated in the absence of CFH. The mobility of the α’- and the β-chain of C3b and the cleavage products of the α’-chain (α’-68 and α’-43) is indicated. (+) incubation with all complement proteins; (−) incubation without CFH.