Deciphering the Ligand-binding Sites in the Borrelia burgdorferi Complement Regulator-acquiring Surface Protein 2 Required for Interactions with the Human Immune Regulators Factor H and Factor H-like Protein 1

Corinna Siegel,Johanna Schreiber,Katrin Haupt,Christine Skerka,Volker Brade,Markus M Simon,Brian Stevenson,Reinhard Wallich,Peter F Zipfel,Peter Kraiczy

doi:10.1074/jbc.m805844200

Abstract

Borrelia burgdorferi, the etiologic agent of Lyme disease, employs sophisticated means to evade killing by its mammalian hosts. One important immune escape mechanism is the inhibition of complement activation mediated by interactions of the host-derived immune regulators factor H (CFH) and factor H-like protein 1 (CFHL1) with borrelial complement regulator-acquiring surface proteins (BbCRASPs). BbCRASP-2 is a distinctive CFH- and CFHL1-binding protein that is produced by serum-resistant B. burgdorferi strains. Here we show that binding of CFH by BbCRASP-2 is due to electrostatic as well as hydrophobic forces. In addition, 14 individual amino acid residues of BbCRASP-2 were identified as being involved in CFH and CFHL1 binding. Alanine substitutions of most of those residues significantly inhibited binding of CFH and/or CFHL1 by recombinant BbCRASP-2 proteins. To conclusively define the effects of BbCRASP-2 residue substitutions on serum sensitivity in the bacterial context, a serum-sensitive Borrelia garinii strain was transformed with plasmids that directed production of either wild-type or mutated BbCRASP-2 proteins. Critical amino acid residues within BbCRASP-2 were identified, with bacteria producing distinct mutant proteins being unable to bind either CFH or CFHL1, showing high levels of complement components C3, C6, and C5b-9 deposited on their surfaces and being highly sensitive to killing by normal serum. Collectively, we mapped a structurally sensitive CFH/CFHL1 binding site within borrelial BbCRASP-2 and identified single amino acid residues potentially involved in the interaction with both complement regulators.

Highlights

The ability of Borrelia burgdorferi sensu lato, the etiologic agent of Lyme disease, to infect immunocompetent mammalian hosts requires complex environmental sensing mechanisms and coordinated expression of proteins essential to overcome host defenses, in particular the innate and adaptive immune responses [1,2,3,4]
Identification of Amino Acid Residues within BbCRASP-2 Region 2 Required for CFH and CFHL1 Binding—To identify amino acid residues potentially involved in the interaction of BbCRASP-2 with complement regulators CFH and CFHL1, a comprehensive alanine-scanning approach was conducted (Fig. 1)
These results indicate that several amino acid residues within region 2 mediate BbCRASP-2 binding to CFH and CFHL1 and suggest somewhat different interactions with each host protein

Summary

EXPERIMENTAL PROCEDURES

Bacterial Strains and Culture Condition—B. garinii strain G1 was originally isolated from the cerebrospinal fluid of a Lyme disease patient and is highly susceptible to complement-mediated killing in vitro [7]. Following four washes with PBS, the slides were incubated for 1 h at room temperature with 1:2000 dilutions of appropriate Alexa 488-conjugated secondary antibodies (Molecular Probes, Leiden, The Netherlands). Serum Susceptibility Testing of Borrelia Strains—Serum susceptibility of B. garinii strains G1, G1/pKFSS1, G1/pCSPZ, and G1 containing shuttle vector pCSPZ that harbor point mutations in the cspZ gene was assessed by using a growth inhibition assay as described previously [7]. Polyclonal rabbit ␣SCR1– 4 antiserum was used for detection of CFHL1 [16], and the mAb VIG8 was applied to detect CFH [35] For detection of both complement regulators a goat anti-human CFH antiserum (Merck Biosciences, Bad Soden, Germany) was used. Polyclonal mouse anti-BbCRASP-2 sera were generated by injection (intraperitoneal) of recombinant BbCRASP-2 into Balb/c mice [31]

RESULTS

To further characterize the nature of interactions between

DISCUSSION