Contribution of the infection-associated complement regulator-acquiring surface protein 4 (ErpC) to complement resistance of Borrelia burgdorferi

Abstract

Borrelia burgdorferi evades complement-mediated killing by interacting with complement regulators through distinct complement regulator-acquiring surface proteins (CRASPs). Here, we extend our analyses to the contribution of CRASP-4 in mediating complement resistance of B. burgdorferi and its interaction with human complement regulators. CRASP-4 (also known as ErpC) was immobilized onto magnetic beads and used to capture proteins from human serum. Following Western blotting, factor H (CFH), CFH-related protein 1 (CFHR1), CFHR2, and CFHR5 were identified as ligands of CRASP-4. To analyze the impact of native CRASP-4 on mediating survival of serum-sensitive cells in human serum, a B. garinii strain was generated that ectopically expresses CRASP-4. CRASP-4-producing bacteria bound CFHR1, CFHR2, and CFHR5 but not CFH. In addition, transformed spirochetes deposited significant amounts of lethal complement components on their surface and were susceptible to human serum, thus indicating that CRASP-4 plays a subordinate role in complement resistance of B. burgdorferi.

Figure 1

Identification of serum proteins that bind to recombinant CRASP-4. Recombinant, polyhistidine-tagged CRASP-4 was immobilized onto magnetic beads and incubated with NHS. Uncoated beads were also treated under the same conditions and used as a control to identify nonspecific binding of serum proteins. After extensive washing, bound proteins were eluted with 100mM glycine-HCl (pH 2.0) and the eluate fractions were separated by SDS-PAGE under nonreducing conditions. (a) Silver stain of a gel loaded with purified polyhistidine-tagged CRASP-4 (1 μg), eluate fraction of the uncoated beads, and the final wash and eluate fraction of CRASP-4-coated beads. (b) Western blot analysis of the eluate fraction of CRASP-4-coated beads using a polyclonal anti-CFH or a polyclonal anti-CFHR1 antiserum. Mobilities of molecular mass standards are indicated to the left.

Figure 2

CRASP-4 binds distinct complement proteins. Binding of equimolar amounts of CFH, CFHR1, CFHR2, and CFHR5 (33 μM) to immobilized CRASP-4 (5 μg/mL) was analyzed by ELISA. Bound CFH or CFHR proteins were detected with either goat CFH polyclonal antiserum or mouse CFHR1 monoclonal antiserum (JHD 7.10), which reacts with all three CFHRs. Data represent the means and standard errors from three separate experiments.

Figure 3

Characterization of B. garinii G1 producing CRASP-4. (a) B. garinii G1 and transformed strains G1/pKFSS1 and G1/pCRASP-4 were characterized by PCR amplification using flaB-, aadA-, and erpC-specific primers, as listed in Table 1. (b) Synthesis of CRASP-4 by transformed G1 was assessed using ligand affinity blotting. Whole cell lysates (15 μg each) of G1, G1/pKFSS1 and G1/pCRASP-4 were separated by SDS-PAGE, and transferred to nitrocellulose. After incubation with NHS, binding of CFH to CRASP-4 was identified using a polyclonal antiserum. A monoclonal antibody, L41 1C11, specific for the flagellin protein FlaB, was applied to show equal loading of borrelial lysates. (c) Surface localization of CRASP-4 in transformed G1 cells. Spirochetes were incubated with or without proteinase K or trypsin, respectively, then lysed by sonication, and total proteins were separated by SDS-PAGE. CRASP-4 was identified by ligand affinity analysis as described above. Flagellin (FlaB) was detected with MAb L41 1C11 (dilution 1/1000) by Western blotting. (d) Demonstration of surface expression of CRASP-4 by transformed B. garinii G1, by indirect immunofluoresecence microscopy of intact borrelial cells. Spirochetes were incubated with rabbit polyclonal anti-ErpA/ErpC antiserum before fixation. Periplasmic FlaB, used as control, was detected by mAb L41 1C11 using fixed and unfixed cells. For counterstaining, the DNA-binding dye DAPI was used to identify all bacteria. Slides were visualized at a magnification of ×1,000 using an Olympus CX40 fluorescence microscope mounted with a DS-5Mc charge-coupled device camera (Nikon).

Figure 4

Binding of serum molecules by B. garinii transformants. B. garinii strains G1, G1/pKFSS1, and G1/pCRASP-4 and B. burgdorferi strain LW2 (used as control) were incubated in NHS plus EDTA to prevent complement activation and washed extensively, and bound proteins were eluted using 0.1 M glycine (pH 2.0). Both the last wash (w) and the eluate (e) fractions obtained from each strain were separated by SDS-PAGE and transferred to nitrocellulose. As an additional control purified CFH (1 μg) was also applied. Membranes were probed with a polyclonal anti-FHR1 antiserum which recognizes CFH, CFHR1, CFHR2, and CFHR5. Mobilities of molecular mass standards are shown to the left of the panels.

Figure 5

Serum susceptibility of transformed B. garinii G1. A growth inhibition assay was used to investigate susceptibility to human serum of B. burgdorferi strain LW2 and B. garinii strains G1 and G1/pCRASP-4. Spirochetes were incubated in either 50% NHS (open triangles) or 50% heat-inactivated NHS (filled triangles) over a cultivation period of 9 days at 33°C, respectively. Color changes were monitored by measurement of the absorbance at 562/630 nm. All experiments were performed three times during which each test was done at least in triplicate with very similar results. For clarity only data from a representative experiment are shown. Error bars represent ± SD.

Figure 6

Deposition of complement components C3 and C6, and MAC on the surface of borrelial strains. Deposition of complement components on B. burgdorferi LW2 (control strain), B. garinii G1 and transformant G1/pCRASP-4 were detected by indirect immunofluorescence microscopy. Spirochetes were incubated with 25% NHS. Bound C3, C6, or MAC was detected using specific antibodies against each component plus appropriate Alexa-488-conjugated secondary antibodies. For visualization of intact spirochetes, the DNA-binding dye DAPI was used. Slides were visualized at a magnification of ×1,000 and the data were recorded via a DS-5Mc CCD camera (Nikon) mounted on an Olympus CX40 fluorescence microscope. Panels shown are representative of at least 20 microscope fields.