Figure 1.
Serum susceptibility testing of B. valaisiana.
A colorimetric growth survival assay was used to investigate susceptibility to human serum of B. valaisiana Bv9, VS116, ZWU3 Ny3, B. garinii G1, and B. burgdorferi LW2. Spirochetes were incubated in either 50% NHS (diamonds) or 50% hiNHS (rectangles) over an incubation period of 9 days at 33°C. Color changes were monitored by measurement of the absorbance at 562/630 nm. All experiments were performed three times with at least three replicates, obtaining very similar results. For clarity, data of a representative experiment are shown. Error bars represent ± SD.
Figure 2.
Determination of activated complement components on the surface of B. valaisiana.
Complement components C3, C4, C6 and MAC deposited on the surface of B. valaisiana isolates Bv9, VS116, ZWU3 Ny3, B. garinii G1, B. burgdorferi LW2 were visualized by indirect immunofluorescence microscopy. Spirochetes were incubated with 25% NHS for 30 min at 37°C with gentle agitation and deposited C3, C4, C6, and MAC were analyzed with specific antibodies against each component and appropriate Alexa 488-conjugated secondary antibodies. For visualization of the spirochetes in a given microscopic field, the DNA-binding dye DAPI was used. The spirochetes were observed at a magnification of 100× objective. The data were recorded with a DS-5Mc CCD camera (Nikon) mounted on an Olympus CX40 fluorescence microscope. Each panel is representative of at least 20 microscope fields.
Figure 3.
Identification of CRASPs among B. valaisiana.
Cell lysates (30 µg each) obtained from control strains B. burgdorferi LW2 and B. garinii G1 as well as from B. valaisiana isolates Bv9, VS116, and ZWU3 Ny3 were subjected to 10% tris/tricine SDS-PAGE and transferred to nitrocellulose. The membranes were then incubated with either NHS as source for CFH and CFHR1 or with cell culture supernatant containing recombinant FHL1. Potential CFH/FHL1/CFHR1-binding proteins were detected using a polyclonal anti-CFH serum, a polyclonal anti-CFHR1 serum or a polyclonal anti-SCR1-4 antiserum. Monoclonal antibody L41 1C11 recognizing the FlaB protein was used to show equal loading of bacterial lysates. The identified CRASP proteins, CspA, CspZ, ErpP, and ErpA of B. burgdorferi LW2, the CFH-binding protein of B. valaisiana Bv9 and VS116, and the CFHR1-binding protein of B. valaisiana Bv9 are indicated on the right and the mobility of the marker proteins is indicated on the left.
Figure 4.
Binding of complement regulators to intact spirochetes.
(A) Ligand affinity blotting was performed to detect binding of complement regulators. B. burgdorferi LW2, B. garinii G1, B. valaisiana isolates Bv9, VS116, and ZWU3 Ny3 as well as B. duttonii LA1 were incubated in NHS-EDTA to prevent complement activation, washed extensively, and bound proteins were eluted using 100 mM glycine-HCl (pH 2.0). Both the last wash (W) and the eluate (E) fractions obtained from each strain were separated by 12.5% Laemmli-SDS-PAGE (detection of CFH, FHL1, FHR1, FHR2, and C1-Inh) or by 10% tris/tricine SDS-PAGE (C4Bp detection) and transferred to nitrocellulose. Membranes were probed either with a polyclonal anti-SCR1-4 antiserum for the detection of both CFH and FHL1, a polyclonal anti-CFHR1 antiserum for the detection of CFHR1 and CFHR2, a polyclonal anti-C4Bp antiserum, or with a polyclonal anti-C1-Inh antiserum. Binding of CFH (B) or C4Bp (C) to spirochetes (5×107 cells) was assessed by ELISA. Bound CFH was detected with a polyclonal goat anti-CFH antiserum and C4Bp was detected with a polyclonal sheep anti-C4Bp antiserum. Data represent means and SD from three separate experiments, each performed at least in triplicate. ***p<0.001; **p<0.01. Raw data were analyzed by one-way ANOVA with post hoc Bonferroni correction.
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.
Figure 6.
Determination of the C4b proteolytic activity of B. valaisiana.
(A) Schematic representation of the α-, β-, and the γ-chain of C4b and the cleavage fragments of the α-chain generated by C4Bp and Factor I. (B) Degradation of C4b by an intrinsic proteolytic activity of borrelial cells (5×108) was analyzed by detection of characteristic cleavage fragments after incubation of spirochetes with (+) or without (−) purified C4Bp. B. burgdorferi LW2, B. garinii G1, B. valaisiana isolates Bv9, VS116, and ZWU3 Ny3 were incubated with C4Bp for 60 min at room temperature. After extensive washing with GVB++, C4b (1 µg/ml) and Factor I (1 µg/ml) were added and the mixture was incubated for 120 min at 37°C. Subsequently, the samples were heated to 95°C for 5 min, subjected to 10% tris/tricine SDS-PAGE and transferred onto a nitrocellulose membrane. The C4b cleavage products were visualized by Western blotting using a polyclonal goat anti-human C4 antiserum. As a positive control, purified C4b (1 µg) was incubated with C4Bp and Factor I, and as a negative control complement proteins were incubated in the absence of complement regulator C4Bp. The mobility of the α’-, β’-, and γ’-chain and the α’4 fragment is indicated. (+) incubation with all complement proteins; (−) incubation without C4Bp.
Figure 7.
Determination of the C5 proteolytic activity of B. valaisiana.
(A) Schematic representation of the α-, and β-chain of C5. (B) Degradation of C5 by an intrinsic proteolytic activity of spirochetes (5×108) was analyzed by detection of potential cleavage products after incubation of cells with purified C5. B. burgdorferi LW2, B. garinii G1, B. valaisiana isolates Bv9, VS116, ZWU3 Ny3, and B. duttonii LA1 were incubated with 1 µg C5 for 60 min and 120 min at 37°C. After centrifugation, supernatants were subjected to 10% tris/tricine SDS-PAGE and transferred onto a nitrocellulose membrane. The C5 fragments were visualized by Western blotting using a polyclonal goat anti-human C5 antiserum. As a negative control, purified C5 (1 µg) was incubated under the same conditions. The mobility of the 118 kDa α-chain and the 74 kDa β-chain is indicated.
Figure 8.
Determination of the C3a generation by B. valaisiana.
Spirochetes (6×106) were incubated with 25% of NHS for 5 min at 21°C and activation of C3 was then analyzed by the MicroVue C3a Plus ELISA. Generated C3a was detected using a monoclonal anti-C3a capture antibody and a HRP-conjugated polyclonal anti-C3 antiserum. All experiments were performed three times with at least three replicates, obtaining very similar results. For clarity, data of a representative experiment are shown. Error bars represent ± SD. Raw data were analyzed by one-way ANOVA with post hoc Bonferroni correction. ***p<0.001; *p<0.05.