Complement evasion by Borrelia burgdorferi: serum-resistant strains promote C3b inactivation

Abstract

The most characteristic features of the Lyme disease pathogens, the Borrelia burgdorferi sensu lato (s.l.) group, are their ability to invade tissues and to circumvent the immune defenses of the host for extended periods of time, despite elevated levels of borrelia-specific antibodies in serum and other body fluids. Our aim in the present study was to determine whether B. burgdorferi is able to interfere with complement (C) at the level of C3 by accelerating C3b inactivation and thus to inhibit the amplification of the C cascade. Strains belonging to different genospecies (Borrelia garinii, B. burgdorferi sensu stricto, and Borrelia afzelii) were compared for their sensitivities to normal human serum and abilities to promote factor I-mediated C3b degradation. B. burgdorferi sensu stricto and B. afzelii strains were found to be serum resistant. When the spirochetes were incubated with radiolabeled C3b, factor I-mediated degradation of C3b was observed in the presence of C-resistant B. afzelii (n = 3) and B. burgdorferi sensu stricto (n = 1) strains but not in the presence of C-sensitive B. garinii (n = 7) strains or control bacteria (Escherichia coli, Staphylococcus aureus, and Enterococcus faecalis). Immunoblotting and radioligand binding analyses showed that the C-resistant strains had the capacity to acquire the C inhibitors factor H and factor H-like protein 1 (FHL-1) from growth medium and human serum. A novel surface protein with an apparent molecular mass of 35 kDa was found to preferentially bind to the N terminus region of factor H. Thus, the serum-resistant B. burgdorferi s.l. strains can circumvent C attack by binding the C inhibitors factor H and FHL-1 to their surfaces and promoting factor I-mediated C3b degradation.

FIG. 1

Analysis of serum resistance of B. burgdorferi s.l. strains. The indicated strains were incubated in nonimmune human serum at 37°C for 16 h. The survival of bacteria at specified time points was calculated as the percentage of motile, live bacteria by dark-field microscopy. The different B. burgdorferi strains fell into two distinct categories. While the B. burgdorferi s.s. and B. afzelii strains were resistant to serum killing, all the tested B. garinii strains were classified as serum sensitive.

FIG. 2

SDS-PAGE and autoradiography analysis of C3b cleavage by B. afzelii strain A91. Various amounts of bacteria (lanes 3 to 5 [numbering from the left]: 10⁹, 10⁸, and 10⁷/ml, respectively) grown in BSK-H medium were incubated in a final volume of 40 μl for 1.5 h at 37°C with radiolabeled C3b (1.25 μg/ml; 10⁶ cpm/μg) in the presence of factor I (12.5 μg/ml). Lane 1, negative control with ¹²⁵I-C3b plus I; lane 2, positive control (¹²⁵I-C3b, factor I, and 0.5 μg of factor H/ml). Note the cleavage of the α′ chain of C3b into fragments representing those of iC3B (lane 3). The image was produced using the Fujifilm BAS 2500 instrumentation and MacBAS, version 2.5, and Adobe Photoshop, version 5.5, software.

FIG. 3

Comparison of levels of C3b cleavage by factor I (12.5 μg/ml) in the presence of the three genospecies of B. burgdorferi s.l. grown in BSK-H medium. The C3b inactivation-promoting activity for factor I was tested with approximately 10⁹ bacteria/ml. Lanes 1 and 2 (numbering from the left), cleavage of C3b produced by B. burgdorferi (B. b.) s.s. ia and B. afzelii A91, respectively. Under the conditions used, the cleavage of ¹²⁵I-C3b progressed to a different degree in the presence of factor H (lane 10; 5 μg/ml) and the two strains. Unlike the other strains tested, none of the B. garinii strains (3/96, 5/96, 13/96, 28/97, 40/97, 46/97, and 50/97) promoted ¹²⁵I-C3b cleavage. The image was produced as in Fig. 2.

FIG. 4

Analysis of ¹²⁵I-C3b cleavage by three B. afzelii strains grown in BSK-H. All three strains (A91, 570, and 1082) yielded weak but similar C3b cleavage patterns with factor I (12.5 μg/ml). No cleavage of C3b with B. garinii was observed. The positive control shows ¹²⁵I-C3b cleavage with factors H (5 μg/ml) and I. The image was produced as in Fig. 2.

FIG. 5

Uptake of complement factor H and FHL-1 from the growth medium by the different B. burgdorferi strains. Two batches of B. afzelii A91, two strains of B. garinii, and B. burgdorferi s.s. strain ia were grown in BSK-H medium. After being washed three times with VBS the bacterial preparations were run on SDS-PAGE gel and immunoblotted on nitrocellulose with a polyclonal anti-factor H antibody. Purified factor H (1.0 and 0.1 ng on lanes 6 and 7 [lanes are numbered from the left], respectively) and NHS (lane 8) were run as positive controls. Uptake of 150-kDa factor H occurred with the B. afzelii and B. burgdorferi s.s. strains but not with the B. garinii strains. In addition, B. burgdorferi s.s. bound a protein that corresponds to FHL-1. FHL-1 can be separated from the comigrating FHR-1α because FHL-1 runs as a single band at 43 kDa (lane 5) and FHR-1 runs as a doublet at 43 (FHR-1α) and 37 kDa (FHR-1β; lane 8).

FIG. 6

Ligand blotting analysis of ¹²⁵I-factor H and ¹²⁵I-FHL-1 binding to B. burgdorferi s.l. strains. OM and PC of three strains representing all three genospecies were isolated using ultracentrifugation through sucrose gradients. OM and PC were run on a nonreducing SDS-PAGE gel and electrotransferred onto a nitrocellulose membrane. Factor H binding protein OspE (19 kDa) and a factor H antibody were run as positive controls. The samples were probed with radiolabeled factor H (top) and FHL-1 (bottom). B. burgdorferi (B. b.) s.s. was observed to bind both factor H and FHL-1, whereas B. afzelii A91 bound preferentially FHL-1 and B. garinii bound neither. Factor H and FHL-1 bound to a protein with an approximate molecular mass of 35 kDa which is distinct from the 19-kDa OspE protein.