Complement Factor H-Related Proteins CFHR2 and CFHR5 Represent Novel Ligands for the Infection-Associated CRASP Proteins of Borrelia burgdorferi

Background: One virulence property of Borrelia burgdorferi is its resistance to innate immunity, in particular to complement-mediated killing. Serum-resistant B. burgdorferi express up to five distinct complement regulator-acquiring surface proteins (CRASP) which interact with complement regulator factor H (CFH) and factor H-like protein 1 (FHL1) or factor H-related protein 1 (CFHR1). In the present study we elucidate the role of the infection-associated CRASP-3 and CRASP-5 protein to serve as ligands for additional complement regulatory proteins as well as for complement resistance of B. burgdorferi. Methodology/Principal Findings: To elucidate whether CRASP-5 and CRASP-3 interact with various human proteins, both borrelial proteins were immobilized on magnetic beads. Following incubation with human serum, bound proteins were eluted and separated by Glycine-SDS-PAGE. In addition to CFH and CFHR1, complement regulators CFHR2 and CFHR5 were identified as novel ligands for both borrelial proteins by employing MALDI-TOF. To further assess the contributions of CRASP-3 and CRASP-5 to complement resistance, a serum-sensitive B. garinii strain G1 which lacks all CFH-binding proteins was used as a valuable model for functional analyses. Both CRASPs expressed on the B. garinii outer surface bound CFH as well as CFHR1 and CFHR2 in ELISA. In contrast, live B. garinii bound CFHR1, CFHR2, and CFHR5 and only miniscute amounts of CFH as demonstrated by serum adsorption assays and FACS analyses. Further functional analysis revealed that upon NHS incubation, CRASP-3 or CRASP-5 expressing borreliae were killed by complement. Conclusions/Significance: In the absence of CFH and the presence of CFHR1, CFHR2 and CFHR5, assembly and integration of the membrane attack complex was not efficiently inhibited indicating that CFH in co-operation with CFHR1, CFHR2 and CFHR5 supports complement evasion of B. burgdorferi

CRASP-3 and CRASP-5 bind diverse complement proteins.

<p>Binding of equimolar amounts of CFH, CFHR1, CFHR2, CFHR5 (33 µM) to immobilized CRASP-3, CRASP-5, ErpX, and ErpQ (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 the three CFHRs. Data represent the means and standard errors from three separate experiments.</p

Detection of C3b inactivation products after incubation of CFH with <i>B. garinii</i> transformants.

<p>Factor I-mediated conversion of C3b to iC3b was analyzed by detection of C3b cleavage fragments after incubation of spirochetes with purified CFH. <i>B. burgdorferi</i> s.s. LW2 producing all five CRASP proteins (control strain), <i>B. garinii</i> G1, G1/pKFSS1, G1/pCRASP-3, and G1/pCRASP-5 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. For control purposes all reactions were also performed in the absence of CFH. Subsequently, the probes were boiled for 5 min, subjected to 12.5% Glycine-SDS-PAGE and transferred onto a nitrocellulose membrane. The various C3b degradation products (α'46- and α'43-kDa bands) were visualized by Western blotting using a polyclonal goat anti-human C3 antiserum (Calbiochem). As additional controls, reaction mixtures containing C3b and factor I were incubated with or without purified CFH (lanes 11 and 12), respectively.</p

Deposition of complement components C3, C6 and C5b-9 on the surface of <i>B. garinii</i> G1 producing CRASP-3 or CRASP-5.

<p>Complement components deposited on <i>B. burgdorferi</i> s.s. LW2 (control strain), transformants G1/pKFSS1, G1/pCRASP-3, and G1/pCRASP-5 were detected by indirect immunofluorescence microscopy. Spirochetes were incubated with either 25% normal human serum (NHS) or heat-inactivated NHS (hiNHS). Bound C3, C6, or C5b-9 were 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 ×1000 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.</p

Characterization of <i>B. garinii</i> G1 transformants producing CRASP-3 or CRASP-5.

<p>(A and B) <i>B. garinii</i> G1 and transformed G1 strains were characterized by PCR amplification using <i>flaB</i>, <i>aadA</i>, <i>erpP</i>, and <i>erpA</i> gene-specific primers, as listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013519#pone-0013519-t001" target="_blank">Table 1</a>. Both panels, left to right: wild-type <i>B. garinii</i> G1, <i>B. garinii</i> G1 transformed with the empty cloning vector pKFSS1, <i>B. garinii</i> G1 transformed with either pCRASP-3 or pCRASP-5, and purified pCRASP-3 or pCRASP-5 alone. (C) Demonstration of surface expression of CRASP-3 and CRASP-5 by transformed <i>B. garinii</i> G1, by indirect immunofluoresecence microscopy of intact borrelial cells. Spirochetes were incubated with rabbit polyclonal anti-CRASP-3 or anti-CRASP-5 antisera 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 were used to identify all bacteria. Slides were visualized at a magnification of ×1000 using a Olympus CX40 fluorescence microscope mounted with a DS-5Mc charge-coupled device camera (Nikon). (D) Surface localization of CRASP-3 and CRASP-5 in transformed G1/pCRASP-3 and G1/pCRASP-5. Spirochetes were incubated with or without proteinase K, then lysed by sonication and total proteins separated by Tricine-SDS-PAGE. CRASP-3 and CRASP-5 were identified using NHS and MAb VIG8 specific for the C-terminus of CFH by ligand affinity analysis. Flagellin (FlaB) was detected with MAb L41 1C11 (dilution 1/1000) by Western blotting. (E and F) Synthesis of CRASP-3 (panel D) and CRASP-5 (panel E) by transformed G1 as assessed by ligand-affinity blotting. Whole cell lysates were separated by Tricine-SDS-PAGE and transferred to nitrocellulose. The membranes were incubated with NHS and binding of CFH to borrelial proteins was detected with mAb VIG8. Monoclonal antibody, L41 1C11, specific for the flagellin protein FlaB, was used to show equal loading of bacterial lysates.</p

Serum susceptibility of transformed <i>B. garinii</i> G1.

<p>A growth inhibition assay was used to investigate susceptibility to human serum of <i>B. burgdorferi</i> s.s. strain LW2 (A), and <i>B. garinii</i> strains G1 (B), G1/pKFSS1 (C), G1/pCRASP-3 (D), and G1/pCRASP-5 (E). Spirochetes were incubated in either 50% NHS (filled diamonds) or 50% heat-inactivated NHS (open diamonds) over a cultivation period of 8 days at 33°C, respectively. Color changes were monitored by measurement of the absorbance at 562/630 nm. All experiments were performed three times in which each test was done at least threefold with very similar results. For clarity only data from representative experiments are shown. Error bars represent ± SD.</p

Oligonucleotides used in this study.

a<p>sequences of specific restriction endonuclease recognition sites are underlined.</p