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INTRODUCTION |
Spirochete bacteria of the Borrelia burgdorferi sensu
lato complex are the causative agents of Lyme borreliosis. The most common manifestation of borreliosis is erythema migrans near the initial site of a tick bite. This can be followed by a complex of
cardiac, neurological, or arthritic disorders (1-3). The three main
genospecies of B. burgdorferi can present with somewhat
different clinical manifestations: B. burgdorferi sensu
stricto often causes arthritis, Borrelia garinii
neuroborreliosis, and Borrelia afzelii has been
preferentially associated with acrodermatitis chronica atrophicans (3,
4).
A striking feature of B. burgdorferi is that it persists for
long periods in its mammal host as well as in several species of ticks
of the genus Ixodes, which transmit the bacteria between the
different mammals. The prolonged survival indicates that the spirochetes are capable of effectively evading the host immune mechanisms. During the different stages of its life cycle,
Borrelia shows dramatic changes in the expression of its
surface proteins (5, 6). It has been shown that many of the of
borrelial surface proteins raise an antibody response in the human host and in animal models (7-9). However, only a few of the antigens have
proven useful as vaccine candidates. Recently developed Lyme disease
vaccines use either the outer surface protein A (OspA) (10, 11) or OspC
(12) as an immunogen. OspA is mainly expressed while the spirochetes
persist in the tick and the expression of OspA is already
down-regulated when the pathogen is transmitted to the human host. The
OspA vaccine leads to a 70-90% protection (10, 11, 13). On the other
hand, OspC is preferentially expressed in the mammalian host. In an
optimal situation, antibodies against vaccine proteins would neutralize
important virulence factors on the surface of the spirochetes. The
physiological roles of most of the borrelial antigens are yet unknown.
However, the recently deciphered genome sequence of B. burgdorferi sensu stricto serves as a basis also for functional
studies (14, 15).
The complement (C)1 system is
an important effector system of the innate immunity in the first line
of defense against invading microbes. C activation is a major mechanism
whereby the spirochetes become coated by opsonizing molecules (C1q,
C3b, iC3b) after entering the human host. This may be initiated by
antibodies or antibody-independently via direct activation of the
classical or the alternative C pathway. Assembly of the C membrane
attack complex usually leads to lysis of Gram-negative bacteria, unless
the microbes have a means to escape the attack.
Several factors may be involved in the interplay between
Borrelia bacteria and their different hosts. Recent studies
support the importance of the C system before and during the infection. The tick Ixodes scapularis expresses a complement-regulatory
protein in its saliva, which down-regulates C activation at an early
stage in the midgut of the tick (16). The spirochetes may benefit from
the protective effect of this protein, first, after the tick's blood
meal when the spirochetes are still in the tick but already in contact
with human plasma, and second, when the pathogens in the tick saliva
enter the human body. However, at least after the transmission of the
pathogen to the human host and dissemination of Borreliae
from the site of infection, the spirochetes must cope with the powerful
effects of the human C system. The three main subspecies of the
B. burgdorferi sensu lato complex have been tested for their
sensitivity to serum complement-mediated bacteriolysis. Although
B. garinii strains were considered serum-sensitive, the
B. afzelii and B. burgdorferi sensu stricto
strains were resistant or partially resistant with no effective
deposition of the terminal complement complexes on the pathogen surface
(17, 18). Analogously, differences in complement-mediated
opsonophagocytosis of the different Borrelia subspecies have
been observed. In an assay dependent on an interaction between iC3b and
the integrin CD11b/CD18 (CR3, Mac-1), neutrophils showed a strong
response to serum-opsonized B. burgdorferi sensu stricto
(19).
An important mechanism of complement activation is the alternative
pathway (AP). The AP is unique in its spontaneous initiation and the
random nature of C3b deposition on target structures. The latter leads
to an attack against all particles, membranes, and cells that are not
specifically protected against AP activation (20). In addition to
direct activation, the AP can enhance C activation initiated by the
classical or the lectin pathway. Complement activation via AP is
strictly controlled by a number of membrane-bound proteins on host cell
surfaces and by the fluid phase regulators factor H and its truncated
form factor H-like protein 1 (FHL-1) (21, 22). Factor H is composed of
20 and FHL-1 of seven short consensus repeat (SCR) domains. Factor H
and FHL-1 control AP activation in three ways. They (i) act as
cofactors for the serine protease factor I in the cleavage of C3b; (ii)
accelerate the decay of the AP C3 convertase, C3bBb, which promotes the
amplification of complement activation by cleaving C3 into its active
form C3b; and (iii) compete with factor B for binding to C3b and thus
prevent formation of the AP C3 convertase (23-26). Recently,
increasing interest has been focused on the interactions between
microbial surface proteins and human complement regulators.
Exploitation of C inhibitors of the host could be an efficient
mechanism whereby the microbes could avoid both direct C attack and
C-mediated enhancement of the adaptive immune response (27, 28).
The aim of the present study was to investigate whether B. burgdorferi spirochetes can protect themselves against complement attack by direct binding of the complement regulator factor H to a
ligand on their outer surface. We analyzed the binding of factor H to
different subspecies of the B. burgdorferi sensu lato complex. After observing factor H binding to the outer membrane of
B. burgdorferi sensu stricto, we identified the outer
surface protein E (OspE) as a specific ligand for factor H. The
addition of soluble OspE inhibited the binding of factor H to
Borrelia and promoted killing of Borrelia in
human serum. The binding site of factor H for Borrelia was
located on the C-terminal part of the molecule. Binding of factor H to
OspE may help Borreliae to evade C-mediated
opsonophagocytosis and direct killing.
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EXPERIMENTAL PROCEDURES |
Expression and Purification of Complement Components--
Plasma
proteins factor H and C3b were purified from human plasma as described
previously (29, 30). Human C5 was purchased from Calbiochem (La Jolla,
CA). Recombinant proteins FHL-1 (representing the N-terminal seven SCRs
of factor H plus four unique additional amino acids), SCRs 8-20 and
SCR 15-20 of factor H, factor H-related proteins FHR-3 and FHR-4, and
a construct SCR 1-4 of FHR-3 were expressed with the baculovirus
expression system as described previously (31-34). The recombinant
proteins were purified by Ni2+-chelate chromatography as
described (35). Purity of the proteins was analyzed by SDS-PAGE and
silver staining.
Bacterial Strains--
Borrelial strains B. burgdorferi sensu stricto (Bbia), isolated from the cerebrospinal
fluid of a Finnish neuroborreliosis patient, B. afzelii
(BaA91, 1082) and B. garinii (Bg13, -28, -40, -46, and -50),
isolated from skin biopsies of Finnish patients with erythema migrans,
were kindly provided by Matti Viljanen (National Public Health
Institute, Turku, Finland). The B. afzelii strain 570 was
isolated from a tick in the Helsinki park area. The strains were
identified to the species level by sequencing the PCR-amplified
fragment of the flagellin gene of B. burgdorferi (36).
Briefly, a 277-base pair segment of the flagellin gene was obtained by
PCR using primers FL7 (biotinylated) and FL59 (37). The biotinylated
PCR products were rendered single-stranded using streptavidin-coated
Dynabeads according to the instructions of the manufacturer (Dynabeads
M-280 streptavidin; Dynal AS, Oslo, Norway). Manual sequencing was
performed by using Sanger's dideoxynucleotide chain termination method
and Sequenase 2.0 (U. S. Biochemical Corp., Cleveland, OH) as
described previously (38). The obtained sequences were compared with
the flagellin gene sequences of the type strains of B. afzelii Bo23 and B. garinii 387. All isolates were
grown in BSK-H medium (Sigma) at 33 °C in 5% CO2
atmosphere until the cultures reached the late exponential phase. The
cultures were harvested by centrifugation, washed, and diluted to a
final bacterium concentration of 2 × 107/ml in
veronal-buffered saline (VBS) or 1/3 GVB (1/3× VBS with 0.1%
gelatin), and subsequently used in complement component binding experiments.
Expression and Purification of Recombinant Borrelia Surface
Proteins--
The recombinant OspE protein was generated by PCR
amplification of the DNA encoding the OspE protein from the American
B. burgdorferi sensu stricto strain N40 (39). The OspE gene
was cloned into a pGEX-2T vector (Amersham Pharmacia Biotech) in
Michael Norgard's laboratory (Dallas, TX). The recombinant GST-OspE
fusion protein was expressed in Escherichia coli DH5-
host cells. Briefly, a primary culture was started by inoculating a
single colony from a fresh transformant plate to 50 ml of Luria-Bertani
(LB) broth containing 100 µg/ml ampicillin. The culture was incubated
at 37 °C with shaking overnight. The culture was diluted 1:50 to 1500 ml of LB broth containing 100 µg/ml ampicillin and incubated at
37 °C for 3 h (until the growth reached the mid-log phase with an optical density of ~0.6 at 600 nm).
Isopropyl-
-D-thiogalactoside was added to a final
concentration of 0.7 mM. After an additional incubation of
3 h, the cells were harvested, washed, and sonicated. The GST-OspE
fusion protein was purified by affinity chromatography on
glutathione-Sepharose matrix according to the instructions of the
manufacturer (Amersham Pharmacia Biotech). The recombinant OspE was
cleaved from GST with bovine thrombin (Sigma) in thrombin cleavage
buffer (150 mM NaCl, 100 mM KCl, 2.5 mM CaCl2, 1 mM dithiothreitol, 20 mM Hepes, pH 7.6) for 3 h and then eluted with
thrombin cleavage buffer. The cleaved OspE was dialyzed against 10 mM diaminopropane, pH 8.6, for 20 h. The dialyzed OspE
was applied on a MonoQ (Amersham Pharmacia Biotech) anion exchange
column in the same buffer and eluted by adding a gradient of NaCl to
the starting buffer. The purity of the OspE protein after MonoQ
purification was confirmed by SDS-PAGE.
OspA, OspD, and decorin-binding protein A (DbpA) were from the American
B. burgdorferi sensu stricto strains 297, N40, and N40,
respectively. They were cloned originally in Michael Norgard's laboratory. The GST-OspD and GST-OspA fusion proteins were expressed in
E. coli XL1Blue host cells. Their expression and
purification were done as described for OspE. The recombinant DbpA was
a hexa-His fusion protein construct expressed in E. coli
DH5-
host cells, and expression was done as with OspE. DbpA, OspC,
and P35/BBK32 recombinant proteins were generated by PCR amplification
of the DNA encoding the predicted mature portion of these proteins from the Finnish B. burgdorferi sensu stricto strain ia (Bbia).
DbpA, OspC, and P35/BBK32 coding sequences were cloned into pQE-30
vectors according to the manufacturer's instructions (Qiagen,
Valencia, CA). These hexa-His fusion proteins were expressed in
E. coli M15 host cells. They were expressed similar to OspE.
Fusion proteins were purified by affinity chromatography on a chelating
Sepharose Fast Flow matrix containing Ni2+ ions. The
proteins were eluted from the column by increasing the amount of
imidazole. The purity of the proteins was confirmed by SDS-PAGE.
Binding of Factor H and FHL-1 to Whole Bacteria--
The
Borrelia spirochetes were cultured as described above.
B. burgdorferi sensu stricto, B. afzelii, B. garinii, or Staphylococcus aureus (used as a negative
control) were used at a concentration of 2 × 107
cells/ml in 1/3 × VBS, pH 7.4, containing 0.1% gelatin (GVB). In
the binding assay, the indicated amounts of bacteria were incubated (20 min at 37 °C) with the radiolabeled factor H or FHL-1 (60,000 cpm)
in a total volume of 100 µl of 1/3 GVB (33). Particle-associated factor H/FHL-1 was separated from unbound ligand by centrifuging the
mixtures through 20% sucrose in 1/3 GVB. The tube was then frozen at
80 °C to avoid mixing the supernatant with the pellet. After
cutting off the pellets, the binding of the factor H/FHL-1 proteins was
calculated as a percentage of the total radioactivity input. All
experiments were performed a least in duplicate and repeated twice. For
the heparin inhibition assay, the indicated amounts of low molecular
weight heparin (Sigma) were added to the reaction mixture
simultaneously with factor H/FHL-1.
The inhibitory effects of increasing amounts of OspE (ranging from 0 to
300 µg/ml) or 300 µg of OspA (control protein) on the binding of
factor H to whole B. burgdorferi sensu stricto was assayed
similarly by adding the competing proteins together with
125I-labeled factor H.
Effect of Soluble OspE on the Survival of B. burgdorferi in Human
Serum--
The B. burgdorferi sensu stricto (strain ia),
B. afzelii (A91), and B. garinii (46) were
cultured as described above. Freshly harvested or frozen bacteria
(6.4 × 107) were washed with GVB and incubated with
serum for 2 h at 37 °C in heat-inactivated serum (control); NHS
(nonimmune serum, no IgG or IgM antibodies against B. burgdorferi detectable by enzyme-linked immunosorbent assay; Ref.
40); or NHS supplemented with 0, 20, or 200 µg/ml (final
concentration) of soluble OspE protein. Serum was added after 30 and 60 min to avoid depletion of complement components caused by blocking
factor H. The final serum concentration shifted from 20% to 33%
during the experiment. The surviving bacteria were counted
microscopically after 2 h, and the percentage of live spirochetes
was calculated.
Immunofluorescence Analysis of C3 and Factor H Binding to
Borrelia Spirochetes--
B. burgdorferi sensu stricto
(strain ia), B. afzelii (strain 1082), and B. garinii (strain 46) were cultured as described above. Freshly
harvested (sensu stricto ia (6.4 × 107)) or frozen
bacteria (B. garinii (5.4 × 109) and B. afzelii (6.4 × 109)) were washed with GVB and incubated 1:2 with nonimmune
NHS or EDTA-plasma from the same donor for 60 min at 37 °C. Bacteria were washed three times with GVB and incubated with a monoclonal antibody against the iC3b neoepitope (Quidel Corp., La Jolla, CA). For
detection of factor H, the monoclonal antibody VIG8 (kindly provided by
Dr. Wolfgang Prodinger, University of Innsbruck, Austria; Refs. 32 and
41) for specific detection of the C terminus (SCRs 19-20) of factor H
was used. An unrelated B-cell lymphoma idiotype-specific AF1 antibody
served as a control. Samples were incubated for 30 min at 37 °C with
primary antibodies (10 µg/ml) and washed three times with GVB before
adding a polyclonal fluorescein isothiocyanate-conjugated goat
anti-mouse IgG (Alexa 488, Molecular Probes, Eugene, OR) used at a
1:200 dilution. The stained samples were mounted with Mowiol (42) and
examined on an Olympus BX50 standard microscope equipped with a filter
specific for fluorescein isothiocyanate fluorescence. The samples were
photographed with a Spot RT Slider digital camera and processed with
Spot RT Software 3.0 (Diagnostic Instruments, Sterling Heights, MI).
Surface Plasmon Resonance Assays--
Protein-protein
interactions were analyzed in real time by the surface plasmon
resonance technique using the Biacore 2000 instrument as described
recently (33, 34). Briefly, factor H or the recombinant borrelial
surface proteins OspA, OspC, OspD, OspE, P35/BBK32, and DbpA were
immobilized via a standard amine-coupling procedure to flow cells of a
sensor chip (carboxylated dextran chip CM5; Biacore AB, Uppsala,
Sweden). Two flow cells were activated with 35 µl of a mixture of 0.2 M
N-ethyl-N'-(dimethylaminopropyl)carbodiimide and 0.05 M N-hydroxysuccinimide (Biacore AB).
The protein to be immobilized was dialyzed against 10 mM
acetate buffer (pH 4.8-5.5) and 20 µg portions (>150 µg/ml) of
the proteins were injected into one of the flow cells until an
appropriate level of coupling for the binding experiments (>4,000
resonance units) was reached. The other flow cell without any protein
was used as a control. Unreacted groups in both flow cells were
inactivated by a standard ethanolamine-HCl injection (35 µl). After
the coupling procedure, the flow cells were washed thoroughly with
sequential injection of 1/3 VBS, pH 7.4, and 3 M NaCl in 10 mM acetate buffer, pH 4.6.
First, the binding of a panel of Borrelia surface proteins
(OspA, OspC, OspD, OspE, P35/BBK32, DbpA) to factor H was tested. The
proteins were dialyzed against 1/3 VBS, and protein concentrations were
measured using the BCA protein assay (Pierce). Each ligand was injected
separately to a blank control flow cell and the flow cell with factor H
using a flow rate of 5 µl/min at 22 °C. The final concentrations
of the fluid-phase ligands in the binding assay ranged from 125 to 200 µg/ml. In a second set of experiments, a reverse setting was used.
The Borrelia surface proteins were immobilized to the sensor
chip surface as described above. Factor H, recombinant deletion
constructs of factor H, or factor H-related proteins (proteins which
are similar but not identical to factor H; Ref. 43) were injected to a
flow cell coated with a borrelial surface protein and to a blank
channel. Binding was assayed at least in duplicate using independently
prepared sensor chips.
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RESULTS |
Binding of Factor H and FHL-1 to Different Strains of B. burgdorferi--
The binding of human complement regulatory proteins
as one mechanism to evade complement attack and phagocytosis has been reported recently for some pathogenic organisms, like
Streptococcus pyogenes (44-46), Streptococcus
pneumoniae (47), and Neisseria gonorrhoeae (48, 49).
Our first aim was to investigate whether the 150-kDa factor H protein
and/or its truncated 42-kDa form FHL-1, the main soluble inhibitors of
the alternative pathway of complement, bind to the surfaces of the
spirochete bacteria of the B. burgdorferi sensu lato complex.
Immunofluorescence Microscopy Analysis of C3 and Factor H Binding
to Borrelia--
To examine complement deposition and regulation on
the borrelial surface, we incubated spirochetes of the three subspecies with nonimmune human serum or EDTA plasma. Complement C3 activation and
inactivation to iC3b and factor H binding were analyzed by immunofluorescence microscopy using specific antibodies (Fig. 1). With an antibody directed against an
iC3b neoepitope, strong complement deposition was detected on all the
three strains. This indicated that incubation of Borrelia in
NHS led to complement activation on the borrelial surface. An antibody
specific for the C-terminal domain SCR 19-20 of factor H (VIG8) showed
strong signals for the B. burgdorferi sensu stricto and for
B. afzelii strains but a weaker staining for the B. garinii strain. Factor H from serum may bind to C3b deposited on
the borrelial surface. In addition, the binding might have been
enhanced by a specific ligand on the spirochete. After incubation of
Borrelia in NHS-EDTA, where complement activation is
inhibited, factor H binding was detected only to the serum-resistant
B. burgdorferi sensu stricto and B. afzelii
strains. The deposition on B. garinii was weaker. A
noncomplement antibody, serving as a negative control, showed no
positive staining at all (Fig. 1, last row).

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Fig. 1.
Immunofluorescence analysis of serum iC3b
deposition and factor H/FHL-1 binding to different strains of B. burgdorferi. The strains B. burgdorferi sensu stricto
ia, B. afzelii 1082, and B. garinii 46 were
treated with undiluted nonimmune NHS or EDTA-plasma (30 min, 37 °C),
washed, and immunostained with specific monoclonal antibodies against
iC3b (row 1) or factor H. The antibody
VIG8 (rows 2 and 3) detects the
C-terminal SCR 19-20 domain of factor H. In the control
(row 4), the serum-treated bacteria were
incubated with an unrelated antibody (AF-1) against an idiotypic B cell
surface marker. Bound antibodies were detected with a fluorescein
isothiocyanate-conjugated goat anti-mouse IgG antibody. Controls were
negative for all strains.
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With the next experiment we wanted to analyze direct binding of factor
H and FHL-1 to Borrelia in the absence of any possibly interfering serum component. Radiolabeled factor H and FHL-1 proteins were incubated (30 min at 37 °C) with six different strains of the
three subspecies of B. burgdorferi: B. garinii strains 13, 28, and 50; B. afzelii 570 and 1082; and B. burgdorferi sensu stricto ia (each at 2 × 107).
The bound protein was separated by centrifugation through a 20%
sucrose solution and the binding was quantified with a
counter. Sedimentation of radioactivity in the absence of bacteria was taken as
background (always below 0.3% of total offered radioactivity). Both
factor H and FHL-1 bound to B. burgdorferi sensu stricto ia
strain (Fig. 2). The values were 10 times
higher for factor H and 5 times higher for FHL-1 compared with the
B. garinii and B. afzelii strains. The latter
strains showed a binding of ~1% of the offered protein with no
significant differences between the strains (Fig. 2). To test the
specificity of this interaction for the borrelial surface, the
experiment was repeated using 11 strains of Staphylococcus
aureus or S. epidermidis. Neither factor H nor FHL-1
bound to these bacteria (data not shown). Factor H and FHL-1 thus seem
to interact specifically with a ligand on the surface of
Borrelia, preferentially with that of the sensu stricto ia
strain.

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Fig. 2.
Binding of factor H and FHL-1 to different
B. burgdorferi strains. Radiolabeled complement
regulators factor H and FHL-1 were tested for binding to six different
strains of the B. burgdorferi sensu lato complex: B. garinii strains 13, 28, and 50; B. afzelii 570 and
1082; and B. burgdorferi sensu stricto strain ia.
Radiolabeled 125I-factor H or 125I-FHL-1 were
incubated with the bacteria for 30 min at 37 °C, and bound protein
on the bacteria was separated by centrifugation through a 20% sucrose
solution. Radioactivity was quantified with a counter. A parallel
batch without addition of bacteria served as a negative control
(control).
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Screening of Borrelial Surface Proteins for Binding of Factor H and
FHL-1--
Given the interaction between factor H and the whole
Borrelia bacteria, we wanted to identify potential ligands
on the Borrelia surface. To this end we expressed and
screened a set of six surface proteins of Borrelia: the
outer surface proteins OspA, OspC, OspD, and OspE; the decorin-binding
protein DbpA; and the protein P35/BBK32 (Fig.
3). This set represents a selection of
prominent borrelial outer surface proteins, selected by their potential
importance for diagnostic analysis. For most of these proteins, immune
responses in the human host have been reported.

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Fig. 3.
SDS-PAGE analysis of borrelial surface
proteins. Approximately 2 µg of each recombinant protein was
loaded on a 12.5% gel under reducing conditions. After separation the
proteins were visualized by silver staining. The mobility of the size
markers is indicated.
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To analyze the putative protein-protein interactions, we used the
Biacore biosensor technique. This method allows detection of binding in
real time without a need for labeling of the binding partners. We have
recently shown that the method is useful for analyzing the interaction
between proteins of the complement system (33, 34). In the first set of
experiments, factor H was immobilized to the surface of the sensor chip
by standard amine coupling. The various borrelial surface proteins were
injected to the flow cell containing factor H and to a blank flow cell
used as an internal control. One of the outer surface proteins, OspE,
bound to factor H while OspA, OspC, OspD, and DbpA did not bind. The
P35/BBK32 protein showed some binding to factor H but interacted also
with the dextran matrix of the control blank channel (data not shown). Examples of the binding analyses for OspA, OspD, and DbpA are shown in
Fig. 4 (A, B, and
C, respectively).

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Fig. 4.
Analysis of Borrelia surface
proteins for binding of factor H by the surface plasmon resonance
technique. Binding of proteins to the immobilized ligand is
detected as an increase of resonance units per time. Factor H in the
fluid phase (125 µg/ml in 1/3 VBS, 10 µl) was injected into a flow
cell precoupled with borrelial surface proteins (solid
lines) and into the control flow cell (blank channel,
plotted as dotted lines), where only the bulk
effect is seen. The left part of the panel shows
the binding results for OspA (A), OspD (B), and
DbpA (C) to immobilized factor H. The right
part shows binding analysis of factor H to immobilized OspC
(D), P35/BBK32 (E), and OspE (F). All
recombinant fragments were tested on two different chip surfaces, and
representative figures are shown in panels A-F.
Note the strong binding of factor H to OspE, but not to the other
Borrelia surface proteins.
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To confirm the result for OspE and to simulate the in vivo
situation (borrelial protein on the surface and factor H in the fluid
phase), we analyzed binding of the proteins in a reverse setting. The
Borrelia surface proteins were immobilized onto the chip
surface, and factor H was used as an analyte in the fluid phase. In
this approach the binding of OspE to factor H was confirmed, whereas no
binding could be observed between factor H and OspC or P35/BBK32 (Fig.
4, D-F) or with any of the other proteins. Thus, factor H interacts
with OspE regardless of which of the binding partners is immobilized.
Influence of Soluble OspE on the Interaction between Factor H and
Borrelia--
To test the specificity of factor H interaction with the
surface protein OspE, we measured the effect of soluble OspE on the binding of factor H to Borrelia. As shown in Fig.
5, OspE derived from B. burgdorferi sensu stricto, inhibited the binding of factor H
in a dose-dependent manner. At a concentration of 300 µg/ml OspE, the binding was completely abrogated. The protein OspA, used as a control, did not show a significant effect. OspE thus seems
to be a specific target structure for factor H on the surface of
B. burgdorferi sensu stricto.

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Fig. 5.
Inhibition of interaction between factor H
and Borrelia by soluble OspE protein. Binding of
factor H to the borrelial surface was measured as described for Fig. 2.
The influence of increasing amounts of OspE (0-300 µg/ml) or OspA
(control protein) on the binding of factor H to whole B. burgdorferi sensu stricto was assayed.
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Binding of OspE to Other Complement Components--
To further
assess the specificity of factor H binding to OspE, we examined binding
of OspE to two other complement components, C3b and C5. For this assay,
the binding of C3b or C5 in the fluid phase or the reverse setting with
immobilized C3b or C5 and soluble recombinant OspE and P35/BBK32
proteins was used. As shown in Fig. 6, no
direct interaction between C3b or C5 and OspE was observed. The reverse
setting with immobilized C3b or C5 and fluid-phase OspE gave the same
negative results. In contrast, however, the P35/BBK32 protein bound to
both C3b and C5, when the latter proteins were coupled to the chip
(data not shown). Thus, the interaction between P35/BBK32 and factor
H/C3b/C5 seems to be more due to a general "stickiness" of the
protein rather than to a specific interaction. In conclusion, from the
panel of surface proteins that were tested, OspE was identified as a
specific ligand for the complement regulator factor H.

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Fig. 6.
Binding of complement components C3b and C5
to OspE. The ability of OspE to bind to the complement components
C3b and C5 was tested using a biosensor assay similarly as described
for Fig. 4. OspE was immobilized on the chip surface, and C3b and C5
were injected as fluid-phase ligands. In the control flow cell, no
protein was immobilized.
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Effect of Soluble OspE Protein on the Survival of Different B. burgdorferi Strains in Human Serum--
The B. burgdorferi
sensu stricto, B. burgdorferi afzelii, and
B. burgdorferi garinii strains were incubated
either in heat-inactivated serum (control), normal human serum without
adding OspE or in normal human serum supplemented with 20 or 200 µg/ml soluble OspE (Fig. 7). The
mixture was replenished twice with serum to compensate for potential
depletion of the AP in the fluid phase because of blockade of the
regulator factor H. After 2 h, the number of surviving bacteria
was counted microscopically. In normal human serum, the survival rate
was reduced to 30% as compared with 90% in heat-inactivated serum.
When OspE was added at a concentration of 20 or 200 µg/ml, only 10%
or 5% of bacteria survived, respectively. B. burgdorferi afzelii showed a much higher serum resistance and was not affected by the addition of human normal serum. Only at the high OspE
concentration the survival rate was reduced from 80% to 70%. The
B. burgdorferi garinii strain, known to be serum-sensitive,
showed 20% survival in the absence of OspE and only 1% of the
spirochetes survived when OspE was added. Thus, OspE seems to be an
important factor for the survival of B. burgdorferi in human
serum, in particular for the sensu stricto strain.

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Fig. 7.
Effect of soluble OspE protein on the
survival of different B. burgdorferi strains in human
serum. The indicated strains were incubated in heat-inactivated
serum (control), in normal human serum without adding OspE, or in
normal human serum supplemented with 20 or 200 µg/ml (final
concentrations) of soluble OspE protein. Serum was added stepwise to
avoid depletion of complement components. The percentage of surviving
bacteria after 2 h was counted microscopically.
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Mapping of the OspE Binding Site on Factor H--
As we had
identified OspE as a specific ligand for factor H, we wanted to
localize the binding site on factor H responsible for the interaction.
To this end, we tested a set of recombinant constructs of factor H and
factor H-related proteins for binding to OspE. The binding was again
analyzed with the Biacore method. OspE was immobilized on the chip
surface, and six different recombinant constructs were tested. The
recombinant proteins were expressed in the baculovirus system, and
their functional activity has been shown recently (e.g.
binding to the complement component C3b) (33, 34). We tested the
following constructs of factor H: SCR 1-7 (FHL-1), SCR 8-20, and SCR
15-20 (Fig. 8). Surprisingly, the
construct SCR 1-7 did not bind to OspE. This construct contains the
domain responsible for the basic complement regulatory functions of
factor H in SCRs 1-4 and a domain that has been shown to interact with
heparin, streptococcal M protein, and the C-reactive protein. A
construct consisting of the SCRs 8-20 clearly bound to OspE. The
construct SCR 15-20 of factor H also bound to OspE, suggesting that
the interacting site is located in the C-terminal region of the
protein.

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Fig. 8.
Mapping of the binding site for OspE on
factor H. Binding was measured as described in Fig. 4. The
binding site for OspE on factor H was localized in a biosensor assay
with OspE immobilized on the sensor chip surface and the following
recombinant constructs, expressed in the baculovirus system, as
fluid-phase ligands: SCR 1-7 (FHL-1) (A), SCR 8-20
(B) and 15-20 (C) of factor H, and the factor
H-related proteins FHR-3 (E), SCR 1-4 of FHR-3
(F) and FHR-4 (D). The binding curves are shown
as solid lines and controls (no protein
immobilized) as dotted lines.
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To confirm the specificity of these results, we used constructs of two
factor H-related (FHR) proteins. The FHR proteins are encoded by
distinct genes and are similar but not identical to factor H (43). Both
FHR-3 and FHR-4 are composed of five SCR units. We used the proteins as
"natural mutants" to compare the binding results with those
obtained for factor H. The SCR 2 of FHR-3 is homologous to SCR 7 of
factor H, and it contains a binding site both for heparin and
streptococcal M protein. This binding domain is not present in FHR-4.
Fig. 8 shows the result of the binding analysis. Neither the entire
FHR-3 or FHR-4 nor a construct of SCRs 1-4 of FHR-3 bound to OspE. As
the construct 15-20 binds to OspE but the FHRs do not, the interaction
seems to be mediated by a region that differs between the SCR 15-20
and the FHRs proteins. Thus, we have localized a domain for interaction
with OspE to the SCRs 15-20 of factor H, a site that is distinct from
the recently described binding site for M proteins of streptococci.
Influence of Heparin on the Interaction between Factor H and
OspE--
To test whether the binding of factor H to the borrelial
surface is dependent on charge, the effect of heparin on the
interaction between factor H/FHL-1 and Borrelia was
investigated. Radiolabeled factor H and FHL-1 were incubated with whole
B. burgdorferi sensu stricto bacteria, and the influence of
heparin on the binding was analyzed (Fig.
9). At heparin concentrations below 30 µg/ml, a small enhancing effect on factor H binding to the
spirochetes was observed. At higher heparin concentrations, the binding
of factor H to Borrelia decreased. In contrast, the binding
of FHL-1 showed only a small reduction when the heparin concentration
increased. Taken together, the results suggest that the C-terminal
heparin/sialic acid binding site of factor H is involved in the
interaction with B. burgdorferi and the binding site seems
to be located on SCR domains 15-20 of factor H.

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Fig. 9.
Inhibition of the interaction between factor
H/FHL-1 and B. burgdorferi sensu stricto by
heparin. Binding of factor H and FHL-1 to the borrelial surface
was measured as described in Fig. 2. The influence of increasing
amounts of heparin (0-300 µg/ml) on the binding was assayed.
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DISCUSSION |
In this study we discovered an interaction between the outer
surface protein OspE of the human pathogen B. burgdorferi
sensu stricto and factor H, the main regulatory protein of the
alternative pathway of complement activation. Additional evidence
suggests that this may not be the only interaction between factor H and Borrelia. The consequence of factor H binding is that the
pathogen can avoid or suppress ongoing complement activation on its
surface by inhibiting C3/C5 convertases and promoting the degradation of C3b. As shown by our experiments, OspE is one surface component of
Borrelia that can interfere with the human complement
system. OspE binds to the C terminus of factor H, thus leaving the
N-terminal regulatory domains of the complement inhibitor free to exert
their regulatory activities.
Borrelia spirochetes are able to persist for a long time in
the human tissues and in circulation. As the complement system is a
powerful effector system of the innate immunity, it is advantageous for
pathogens to evade complement activation. The direct binding of
complement regulators like factor H or its truncated form FHL-1 has
recently been reported for several pathogens (27, 28). The best studied
example is the interaction with the M protein of group A streptococci
(44-46). Both factor H and FHL-1 bind to certain M proteins. FHL-1
seems to be the preferred ligand of the hypervariable region of M
proteins, and the binding of the protein does not inhibit its
complement regulatory effects. Recently, it has been proposed that
factor H also binds to S. pneumoniae and contributes to
their resistance to phagocytosis (47). Interactions of factor H with
bacterial surfaces have also been reported to contribute to the serum
resistance of further pathogens. Factor H binds to sialylated
lipo-oligosaccharides of N. gonorrhoeae by an interaction
via SCRs 16-20 (48). In addition, factor H also binds to nonsialylated
Neisseria strains by an interaction with porin proteins, the
main outer membrane proteins of the pathogen (49). However, the
interacting region on factor H responsible for the interaction could
not be localized in the latter example.
Borrelia is very active in changing its surface properties.
The expression of the outer surface proteins, which are in direct contact with the environment, varies according to the alternating hosts, temperature, culture conditions, and several other factors (50-52). The outer surface proteins are thus candidates for a possible interaction with the immune system of the host. We expressed and tested
a set of six borrelial surface proteins for their potential binding to
factor H/FHL-1: the outer surface lipoproteins OspA, OspC, OspD, and
OspE; the fibronectin-binding protein P35/BBK32; and the
decorin-binding protein DbpA. OspA is the best studied protein of
Borrelia. It is the immunogen in a recently developed Lyme
disease vaccine (10, 11). OspA is expressed at a high level during the
life stage of the spirochete within the tick, but the expression
decreases after the tick feeds on blood. On the contrary, the OspC
protein is up-regulated after transmission of the bacteria to the
mammalian host. When the temperature shifts from 23 °C in the tick
to 35 °C in the mammalian host, the expression of the lipoproteins
OspE and OspF and of some other antigens is up-regulated among other
antigens, an effect that also occurs with Borrelia cultures
in vitro (50-52). In light of the current results, this
phenomenon is beneficial for the spirochete in its ability to resist
complement attack. Like many bacterial virulence factors, the 19.2-kDa
lipoprotein OspE is encoded by a circular plasmid and coexpressed with
OspF in one operon (39). No functions have yet been assigned to OspE.
In this study, we describe that OspE binds the plasma complement
inhibitor factor H. The interacting region was localized to the
C-terminal part of factor H. Binding of factor H to the pathogen
surface with this region leaves the complement regulatory domains,
which reside in SCRs 1-4 of factor H, free for an efficient control of
C3b deposition on the bacterial surface similarly as demonstrated
earlier for streptococci (45). Direct evidence for the functional
relevance of the factor H-OspE interaction came from serum sensitivity
tests. Blocking of serum factor H binding to Borrelia
burgdorferi sensu stricto by soluble OspE increased the lytic
activity of serum toward the bacteria. Our result suggests that OspE
could have a potential as a vaccine. However, in mice vaccinated with
OspE, no protective immunity against the spirochetes was achieved (53).
The situation may, however, be different in humans, as the complement
system is species-specific and it is not known whether murine factor H
binds to OspE. If antibodies in humans block the complement inhibitory
activity of OspE, they would neutralize an important virulence factor
and thereby promote destruction of the pathogen.
We also tested the decorin-binding protein DbpA, which has been seen to
raise an antibody response in the human host. Although this protein has
adhesive properties and may mediate the adherence of spirochetes to
collagen fibers in the tissue (54), no interaction with factor H was
observed. Another protein, first called P35 and later BBK32, bound to
factor H but also to the control channel of our assay. P35/BBK32 has
been described to raise a protective antibody response in mice after
vaccination (9). Later, this protein was characterized as a
fibronectin-binding protein (55, 56). The importance of P35/BBK32
binding to factor H requires further studies because when P35/BBK32 was
immobilized on the sensor chip, it did not bind factor H anymore. As
P35/BBK32 bound also to C3b and C5 to some extent, the interactions of
P35/BBK32 could just indicate a general adhesive property of this protein.
When pathogens evade complement activation, inhibition of the
deposition of C3b on the pathogen surface or a late intervention in
preventing the formation of membrane attack complexes are possible mechanisms. It has been reported that Borreliae can prevent
efficient deposition of membrane attack complex on their surfaces (15). Thus, it is possible that C activation is controlled at the level of
C3. In our immunofluorescence stainings, we found deposition of iC3b
after serum incubation of all strains of the three subspecies of the
B. burgdorferi sensu lato complex. By providing C3-related ligands on B. burgdorferi, the serum inactivation also
resulted in a deposition of factor H on the surface of all tested
strains. When complement activation in plasma was inhibited by EDTA,
binding of factor H was observed mainly to B. burgdorferi
sensu stricto and B. afzelii, which are strains known to be
complement-resistant. The fact that under plasma-free conditions
radiolabeled factor H bound strongly only to the B. burgdorferi sensu stricto strain may indicate that other plasma
factors are needed for the binding to the B. afzelii strain.
The binding of factor H to Borrelia was influenced by
heparin. Upon increasing the heparin concentration, factor H first
showed an increase and then a decrease in binding, while the binding of
FHL-1 was reduced to a lesser extent. The reason for this different effect on factor H and FHL-1 binding could be explained by the presence
of at least two binding sites for glycosaminoglycans on the longer
factor H molecule of which only one is used for binding to
Borrelia. Heparin probably dimerizes factor H at low concentrations, thereby increasing the binding activity, whereas at
higher concentrations binding is inhibited. Thus, the effect of heparin
on the interaction of factor H with Borrelia seems to
indicate both oligomerization of factor H molecules and direct inhibition of factor H binding to Borrelia bacteria.
An interaction of Borrelia OspE with factor H is one
possible mechanism for the serum resistance of the sensu stricto
strain. However, it has been shown that in most cases more than one
factor contributes to the resistance. Although we saw binding of FHL-1 to the whole bacteria, on the basis of our experiments OspE does not
seem to be a ligand for this complement regulator.
Since FHL-1, nevertheless, bound to B. burgdorferi sensu
stricto (Fig. 2) and the immunofluorescence microscopy experiment (Fig.
1) suggested binding of factor H/FHL-1 also to the B. afzelii strain, it is likely that additional ligands for factor H
and FHL-1 exist. A study reported at the recent complement workshop investigated the interaction of Borrelia with factor H and
FHL-1 (57). Two still unidentified Borrelia proteins were
detected by ligand blotting with molecular masses of ~20/21 and 27.5 kDa. The 20/21-kDa protein may be identical with the OspE protein. In
conclusion, we have identified a new putative virulence factor of the
human pathogen B. burgdorferi sensu stricto, which binds a
human complement regulator to evade complement attack. The results bring up OspE as an important functional protein and a potential vaccine candidate for studies in human beings.