Determination of the C3a generation by <i>B. valaisiana</i>.

<p>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.</p

Determination of activated complement components on the surface of <i>B. valaisiana.</i>

<p>Complement components C3, C4, C6 and MAC deposited on the surface of <i>B. valaisiana</i> isolates Bv9, VS116, ZWU3 Ny3, <i>B. garinii</i> G1, <i>B. burgdorferi</i> 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.</p

Determination of the C4b proteolytic activity of <i>B. valaisiana</i>.

<p>(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. <i>B. burgdorferi</i> LW2, <i>B. garinii</i> G1, <i>B. valaisiana</i> 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.</p

Serum susceptibility testing of <i>B. valaisiana</i>.

<p>A colorimetric growth survival assay was used to investigate susceptibility to human serum of <i>B. valaisiana</i> Bv9, VS116, ZWU3 Ny3, <i>B. garinii</i> G1, and <i>B. burgdorferi</i> 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.</p

Determination of the C5 proteolytic activity of <i>B. valaisiana</i>.

<p>(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. <i>B. burgdorferi</i> LW2, <i>B. garinii</i> G1, <i>B. valaisiana</i> isolates Bv9, VS116, ZWU3 Ny3, and <i>B. duttonii</i> 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.</p