Tricine

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 (link)).
Borrelia were grown at 33 °C for 5–6 days up to cell
densities of 1 × 10⁷ ml^-1 in modified
Barbour-Stoenner-Kelly (BSK) medium as described previously
(19 (link)). Escherichia
coli JM109 was used as host for cloning and purification of recombinant
proteins.
Site-directed Mutagenesis, Sequence Analysis, and Generation of
Recombinant Proteins—To introduce single or double amino acid
substitutions into the BbCRASP-2-encoding cspZ gene, a site-directed
mutagenesis approach was conducted as described by the QuikChange™
protocol (Stratagene). The mutagenic high-performance liquid
chromatography-purified primers employed in this study were purchased from
Sigma-Aldrich (Steinheim, Germany) and listed in supplemental Table S1.
Briefly, PCR reactions were carried out for 18 cycles (95 °C for 30 s, 55
°C for 30 s, and 68 °C for 13 min) using 100 ng of expression vector
pGEX CSPZ, 125 ng of each mutagenic primer, and 2.5 units of Pfu DNA
polymerase (Stratagene) in a final volume of 50 μl. For the introduction of
site-specific mutations into shuttle vector pCSPZ (previously referred as
pCRASP-2 (31 (link))), thermal
cycling was performed for 18 cycles at 95 °C for 30 s, 55 °C for 60 s,
and 68 °C for 16 min. Before transformation of E. coli, the
reactions were treated with 10 units of DpnI (New England Biolabs, Frankfurt,
Germany) for 1 h at 37 °C. All mutations introduced into the cspZ gene were verified by DNA sequencing of both DNA strands. Proteins were
purified using glutathione-Sepharose columns, with conditions as recommended
by the manufacturer (GE Healthcare).
Binding and Inhibition Assay—Purified GST-BbCRASP-2, mutated
GST-BbCRASP-2 proteins, and GST (2.5 ng/μl each) were immobilized overnight
at 4 °C using Immobilizier™ glutathione microtiter plates (Nunc,
Wiesbaden, Germany). Non-specific binding sites were blocked with 0.2% gelatin
in PBS for 6 h at 4 °C. CFH (Calbiochem, Darmstadt, Germany) or purified
CFHL1 (16 (link)) (5 μg/ml each)
was added to the wells and left overnight at 4 °C. After addition of
polyclonal anti-CFH antibodies (Calbiochem) for 2 h at room temperature,
protein complexes were identified using a secondary peroxidase-conjugated
anti-goat IgG antibody. Reactions were developed with 1,2-phenylenediamine
dihydrochloride (Sigma-Aldrich).
Influences of heparin and salt on CFH binding were analyzed by ELISA.
Purified GST-BbCRASP-2 (15 ng/μl) was immobilized onto wells of a
microtiter plate (MaxiSorb, Nunc) overnight at 4 °C. Following three
washing steps with PBS containing 0.05% Tween 20 (PBST), nonspecific binding
sites were blocked with 0.2% gelatin in PBS for 6 h at 4 °C. Plates
covered with BbCRASP-2 fusion protein were incubated overnight at 4 °C
with CFH (0.25 ng/well) in PBST containing increasing concentrations of either
low molecular weight heparin (Sigma-Aldrich, 0.5–16 μg/ml) or NaCl
(29 mm to 1.16 m). After washing three times in PBS,
binding of CFH was detected using mAb VIG8 antibody
(35 ). The experiments were
conducted at least three times, and the means ± S.D. were
calculated.
SDS-PAGE, Ligand Affinity Blot, and Western Blot
Analysis—Borrelial cell extracts (15 μg) were subjected to 10%
Tris/Tricine-SDS-PAGE under reducing conditions and transferred to
nitrocellulose. Binding of CFH and CFHL1 to borrelial proteins was assessed by
ligand affinity blotting as previously described
(19 (link)).
For Western blot analysis, membranes were incubated for 60 min at room
temperature with either mAb L41 1C11 (FlaB)
(36 (link)) or polyclonal anti-GST
antibody (GE Healthcare, Germany) as described elsewhere
(19 (link)).
Transformation of Serum-sensitive B. garinii and Characterization of
Transformants—High passage, non-infectious B. garinii strain G1 was grown in 100 ml of BSK medium and harvested at mid-exponential
phase (5 × 10⁷ to 1 × 10⁸ cells/ml).
Electrocompetent cells were prepared as described previously
(37 ) with slight
modifications. Briefly, 50-μl aliquots of competent B. garinii strain G1 cells were electroporated at 12.5 kV/cm in 2-mm cuvettes with 10
μg of plasmid DNA. For control purpose B. garinii strain G1 cells
also were transformed with pKFSS1 vector
(38 (link)) alone. Cells were
immediately diluted into 10 ml of BSK medium and incubated with antibiotic
selection at 33 °C for 48–72 h. Bacteria were then diluted into 100
ml of BSK medium containing streptomycin (25 μg/ml), and 200-μl aliquots
were plated into 96-well cell culture plates (Corning) for selection of
transformants. Several clones selected were expanded in 1 ml of fresh BSK
medium without antibiotic selection for 7 days, and then transferred into 10
ml of fresh BSK medium containing streptomycin (50 μg/ml).
The cspZ genes of transformed B. garinii G1 strains were
detected by PCR with specific primers (Table S1). Spirochetes (100 μl) were
sedimented by centrifugation, washed with PBS, and suspended in 50 μl of
water. Five microliters of suspension was amplified by PCR using
oligonucleotide primers at final concentrations of 100 nm each,
plus 200 μm dNTPs. PCR was carried out for 25 cycles using the
following parameters: denaturation at 94 °C for 1 min, annealing at 50
°C for 1 min, and extension at 72 °C for 1 min.
Plasmid DNA was prepared from the presumptive E. coli transformants with the Wizard® Plus SV Miniprep DNA purification system
(Promega, Mannheim, Germany), and DNA inserts were sequenced by a commercial
provider (GATC, Konstanz, Germany).
Immunofluorescence Assay for Detection of Surface-exposed
BbCRASP-2—For indirect immunofluorescence assays, spirochetes (1
× 10⁷) were incubated with 1:50 dilution of mouse polyclonal
anti-BbCRASP-2 for 1 h at room temperature with gentle agitation. Following
three washes with PBS containing 0.2% BSA, cells were collected by
centrifugation at 14,000 × g for 10 min and resuspended in 100
μl of PBS containing 0.2% BSA. Aliquots of 10 μl were then spotted on
coverslips and allowed to air dry overnight. After fixation with methanol,
samples were dried for 90 min at room temperature and incubated for 90 min in
a humidified chamber with a 1:500 dilution of Alexa 488-conjugated goat
anti-mouse IgG (Molecular Probes). Slides were then washes four times with
0.2% BSA in PBS and mounted in ProLong® Gold Antifade reagent (Molecular
Probes) before being sealed with glass slides. 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).
Immunofluorescence Assay for Detection of Complement
Proteins—Spirochetes (6 × 10⁶) were incubated with
25% NHS for 30 min at 37 °C with gentle agitation, washed three times with
PBS containing 1% BSA (PBS-BSA). Aliquots of 10 μl were then spotted on
microscope slides and allowed to air dry overnight. After fixation, slides
were incubated for 1 h in a humidified chamber with antibodies against
complement components C3 (dilution of 1:1000), C6 (dilution of 1:200), and
C5b-9 (dilution of 1:50). 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). Slides were then washed, sealed, and visualized as described
above.
Serum Adsorption Experiments—Spirochetes harvested by
centrifugation were resuspended in 500 μl veronal-buffered saline
(supplemented with 1 mm Mg²⁺, 0.15 mm Ca²⁺, 0.1% gelatin, pH 7.4) and a portion of 1 ×
10⁹ organisms were sedimented by centrifugation. The cell sediment
was then resuspended in 750 μl of NHS supplemented with 34 mm EDTA and incubated for 1 h at room temperature with gentle agitation. After
three washes with PBSA (0.15 m NaCl, 0.03 m phosphate,
0.02% sodium azide, pH 7.2) containing 0.05% Tween 20, the proteins bound to
the spirochetes were eluted by incubation with 0.1 m glycine-HCl,
pH 2.0, for 15 min. The bacterial cells were sedimented by centrifugation
(14,000 × g, 20 min, 4 °C), and the proteins in the
supernatant were analyzed by SDS-PAGE and Western blotting.
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 (link)). Each
experiment was conducted at least three times, and means ± S.D. were
calculated.
Human Sera, CFH, CFHL1, and Monoclonal and Polyclonal
Antibodies—NHS obtained from 20 healthy human blood donors without
known history of spirochetal infections was used as the source for CFH.
Purified human CFH was purchased from Calbiochem. CFHL1 were expressed in
Spodoptera frugiperda Sf9 insect cells infected with recombinant
baculovirus (16 (link),
39 (link)).
Generation of mAb L41 1C11 against FlaB was described elsewhere
(35 ). To detect GST fusion
proteins a goat anti-GST antibody (GE Healthcare, Germany) was used.
Polyclonal rabbit αSCR1–4 antiserum was used for detection of
CFHL1 (16 (link)), and the mAb VIG8
was applied to specifically detect CFH
(35 ). For detection of both
complement regulators a goat anti-human CFH antiserum (Merck Biosciences, Bad
Soden, Germany) was used. The goat anti-human C3 and C6 antibodies were
purchased from Calbiochem, and the monoclonal anti-human C5b-9 antibody was
from Quidel (San Diego, CA). Polyclonal mouse anti-BbCRASP-2 sera were
generated by injection (intraperitoneal) of recombinant BbCRASP-2 into Balb/c
mice (31 (link)).