BORRELIABAKTEERI PAKENEE IMMUUNIPUOLUSTUSTA

[soijuv]

Borreliabakteerien on useissa tutkimuksissa todettu selviytyvän elimistössä antibioottihoidoista ja immuunipuolustuksen hyökkäyksistä huolimatta käyttämällä useita eri mekanismeja.

Uusi suomalainen tutkimus: Pietikäinen, Meria, Blomb; Meria 2010

"Borreliabakteerin tiedetään aiheuttavan kroonisia infektioita eri puolilla elimistöä. Selviytyäkseen elimistössä bakteerin täytyy kyetä pakenemaan immuunipuolustuksen hyökkäyksiltä.

Borreliabakteereiden todettiin sitoutuvan C4bp proteiiniin. Sen avulla bakteeri selviytyy elimistössä vasta-aineiden muodostumisesta huolimatta. Erityisen voimakkaasti bakteerien alajajeista sitoutui B. garinii."


GModel
MIMM-3360; No. of Pages7
Molecular Immunology xxx (2009) xxx?xxx
Contents lists available at ScienceDirect
Molecular Immunology
journal homepage: www.elsevier.com/locate/molimm

Binding of the complement inhibitor C4b-binding protein to Lyme
disease borreliae

Johanna Pietikäinena,1, Taru Meria,∗,1, Anna M. Blomb, Seppo Meria
a Infection Biology Research Program, Haartman Institute, Department of Bacteriology and Immunology,
University of Helsinki and HUSLAB, Helsinki, Finland
b Department of Clinical Chemistry, University Hospital Malmö, University of Lund, Malmö, Sweden

a r t i c l e i n f o
Article history:
Received 8 October 2009
Received in revised form
19 November 2009
Accepted 21 November 2009
Available online xxx
Keywords:
Innate immunity
Infection
Immune evasion

a b s t r a c t

The Lyme disease spirochetes, Borrelia burgdorferi sensu stricto, Borrelia afzelii and Borrelia garinii, are tickborne
pathogens that can cause chronic disseminated infections. To survive in the human host borreliae
need to evade the immune system. It is already well known that B. burgdorferi ss. and B. afzelii bind
the complement (C) alternative pathway inhibitor factor H from serum using OspE and CRASP-1/Bba68
proteins to escape C attack. In the presence of natural antibodies and in chronic infections, when specific
antibodies develop, borreliae have to protect themselves from antibody-induced classical pathway C
attack.

In this study we demonstrate binding of the classical pathway inhibitor, C4b-binding protein (C4bp),
to three genospecies of B. burgdorferi sensu lato. Binding was strongest to B. garinii, which has
been found to be the weakest factorHbinder. The bacteria bound both purified 125I-labeled C4bp and C4bp
from serum. Unlabeled C4bp competed for binding with 125I-C4bp, whereas BSA had no effect. Binding
was salt-sensitive and inhibited by C4b and partially by heparin. C4bp maintained its cofactor activity for
factor I in cleaving C4b when bound to the bacterial surface. Ligand blotting analysis of whole cell lysates
and fractionated outer cell membranes of the bacteria suggested one major receptor of approximately
43 kDa (P43) for C4bp in B. garinii and B. burgdorferi sensu stricto. Binding of C4bp may thus allow Lyme
disease borreliae to escape activation of the classical C pathway and allow chronic infections in humans
even in the presence of specific antibodies.
© 2009 Elsevier Ltd. All rights reserved.

1. Introduction

Lyme borreliosis is a tick-borne multisystemic infection. It is
caused by three genospecies of the Borrelia burgdorferi sensu lato
complex: B. burgdorferi sensu stricto, Borrelia garinii or Borrelia
afzelii (Steere, 1989). The spirochetes are transmitted to humans
by the bites of the Ixodes group ticks. Erythema migrans occurring
at the initial site of a tick bite is the first and the most common
symptom of borreliosis occurring in about 50?70% of cases. Clinical
manifestations of the disseminated disease differ depending on
the causative genospecies. B. burgdorferi sensu stricto causes mainly
Abbreviations: C4bp, C4b-binding protein; C, complement; CP, classical pathway;
AP, alternative pathway; MAC, membrane attack complex; FHL-1, factor H-like
protein 1; CNS, central nervous system; SCR, short consensus repeat; BSA, bovine
serum albumin; FI, factor I; NHS, normal human serum; HI-NHS, heat-inactivated
NHS; ECL, enhanced chemiluminescence.
∗ Corresponding author at: Haartman Institute, Department of Bacteriology and
Immunology, P.O. Box 21, Haartmaninkatu 3, FIN-00014 University of Helsinki,
Helsinki, Finland. Tel.: +358 9 1912 6395; fax: +358 9 191 26382.
E-mail address: taru.meri@helsinki.fi (T. Meri).

1 These authors contributed equally.
arthritis, while B. garinii is associated with neurological symptoms
and B. afzelii with skin manifestations.
Complement (C) is an important part of the innate immunity
as it protects the host from microbial infections by directly killing
targets, marking them for phagocytosis and by enhancing inflammatory
reactions. The C system can be activated via the classical
(CP), alternative (AP) or the lectin pathway. Activation of C leads to
C3-amplification that promotes formation of the membrane attack
complexes and direct lysis of the targets. During prolonged infections
antibodies against borreliae develop and could activate the
CP. As borreliae do not have capsules or a peptidoglycan layer for
their defense against C, evasion of the C system by specific membrane
proteins has been considered to be especially important for
the virulence of borreliae.
B. burgdorferi sensu stricto and B. afzelii gain protection against
the AP by binding the AP inhibitors factor H (FH) (Hellwage et al.,
2001) and factor H-like protein (FHL-1) to their surfaces (Alitalo et
al., 2001). The resistance of borreliae to lysis in nonimmune serum
has been shown to correlate with the binding of FH and FHL-1. The
receptors for FH belong to two distinct classes of proteins: cp32 circular
plasmid-encoded OspE-family (Hellwage et al., 2001) and the
lp54 linear plasmid-encoded CRASP-1/Bba68 proteins (Kraiczy et
al., 2004). In addition, a third type of a putative FH-binding protein

2. Materials and methods

2.1. Bacterial strains
B. burgdorferi sensu stricto strains (B31, 297, N40) and a tick
isolate B. afzelii (600) were kind gifts from docent Ilkka Seppälä
from our department. B. afzelii (BaA91, 600, 1082) and B. garinii
strains (Bg40, Bg46, Bg50) were isolated from skin biopsies of
Finnish patients and they were kind gifts from Prof. Matti Viljanen
(Department of Microbiology, University of Turku, Finland). All
strains were grown at +33 ◦C in BSK-H complete medium (Sigma,
St. Louis, MO). Whole cell lysates were prepared by washing the
bacteria five times with VBS before diluting them in VBS (approx.
1×109 bacteria/500l). Complete EDTA-free protease inhibitor
coctail was added to the suspension prior to sonication, which was
done using a Soniprep 150 ultrasonic disintegrator (MSE, London,
UK) five times/strain with an amplitude of 5m for 5 s.

2.2. Proteins, antibodies and buffers
Bovine serum albumin (BSA) and heparin were purchased
from Sigma. C4bp was purified from human plasma as described
(Dahlback, 1983). C4b and factor I were from Quidel (San Diego,
CA, USA). C4bp and C4b were labeled with 125I using the Iodogen
method (Pierce Chemical Corp., Rockford, IL) (Salacinski et al.,
1981). Sheep polyclonal anti-C4bp antibody was from The Binding
Site (Birmingham, UK). HRP-conjugated donkey anti-sheep
IgG antibody was from the Jackson Immunoresearch Laboratories
(Cambridgeshire, UK). Veronal-buffered saline (VBS) contained
145mM NaCl and 5mM barbiturate, pH 7.4. Phosphate-buffered
saline (PBS) contained 120mMNaCl and 30mMphosphate, pH 7.3.
Normal human serum (NHS) was collected from healthy laboratory
personnel, pooled and stored at −70 ◦C prior usage. To inactivate C
serum was incubated at +55 ◦C for 30 min (heat-inactivated NHS,
HI-NHS). IgG was depleted from NHS (containing 0.5M EDTA, pH
7.7) using Protein G Sepharose 4 Fast Flow colum (Amersham
Biosciences, Uppsala, Sweden) according to the manufacturer?s
instructions. The serum was passed through the column three
times, with elutions in between 20mM sodium phosphate, pH
7.0, was used as the binding buffer. Complete EDTA-free protease
inhibitor cocktail was from Roche Diagnostics GmbH, Mannheim,
Germany.

2.3. Absorption of C4bp from serum
The bacteria (approx. 5×108 assay) were washed five times
with VBS and incubated with HI-NHS (at a 1:2 dilution) for 60 min
at 37 ◦C on a shaker (750 rpm). The bacteria were washed five
times with VBS, and the last wash fractions were collected. Proteins
bound to the surfaces of the bacteria were eluted with 0.1M
glycine?HCl, pH 2 (15 min at RT). After centrifugation (8000×g,
4min) the supernatants were collected. Wash and elute fractions
were subjected to a non-reducing SDS-PAGE and transferred to
nitrocellulose membranes. Nonspecific binding was blocked by
incubating the membranes in 5% fat-free milk in PBS (1?12 h, RT
or +4 ◦C). Thereafter, the membranes were incubated with a polyclonal
sheep anti-C4bp antibody (at a dilution of 1:5000) for 12 h
at 4 ◦C or 1h at +37◦C. After three washes with PBS/0.05%Tween
20, and three washes with PBS, a HRP-conjugated donkey antisheep
IgG antibody was added at a 1:5000 dilution. The membranes
were incubated for 3 h at RT. After one wash with PBS/0.05%Tween
20 and three washes with PBS, the antibodies were detected by
an enhanced chemiluminescence (ECL) method: the immunoblot
was incubated for 5?15 min at 22 ◦C with a substrate solution
containing nitroblue tetrazolium (Sigma) and 5-bromo-4-chloro-
3-indolyl phosphate (Boehringer Mannheim GmbH, Mannheim,
Germany), each dissolved in dimethyl formamide (Merck, Darmstadt,
Germany) and finally diluted into 0.1M NaHCO3 buffer (pH
9.7) containing 1mM MgCl2.

2.4. Direct 125I-C4bp-binding assays
The spirochetes (1.25×108 bacteria/assay) were washed three
times with VBS and incubated in 1/3 VBS containing 0.1% gelatin
(1/3 GVBS) for 30 min at 37 ◦C on a shaker (750 rpm) with
125I-C4bp (approx. 20,000 cpm/assay). In the inhibition assays unlabeled
C4bp, BSA, C4b (0.1?10g/ml) or heparin (10?200 IU/ml)
were added to the reaction mixture. In the salt inhibition assay
the bacteria were incubated with 125I-C4bp in increasing NaCl
concentrations (50?450 mM). After incubation the bacteria were
centrifuged (10,600×g, 4 min) through 20% sucrose gradients in
1/3 GVBS. Radioactivity was measured from the pellets and the
supernatants with a gammacounter. The ratios of the bound (pellet)
to the total (pellet + supernatant) radioactivities were calculated.

2.5. Analysis of cofactor activity for C4b cleavage
The functional activity of borrelia-bound C4bp was tested as
its ability to promote inactivation of C4b by factor I. The bacteria
(approx. 1×109 assay−1) were washed three times with VBS
and incubated with HI-NHS (at a dilution 1:2) or purified C4BP
(75g/ml) for 60 min at 37 ◦C on a shaker (750 rpm). As a negative
control the bacteria were incubated with VBS. The bacteria
were washed three times with VBS and incubated further with
125I-C4b (approx. 50,000 cpm/assay) and factor I (1g/reaction) at
37 ◦C for 60 min. The mixtures were centrifuged (8000×g, 5 min),
and the supernatants were subjected to reducing SDS-PAGE elec
trophoresis. The gel was fixed with 5% acetic acid for 30 min, dried
and subjected to autoradiography. Cleavage of C4b was analyzed by
the appearance of a 43 kDa cleavage fragment. As a positive control
125I-C4b was incubated with purified C4bp (7.5#g) and factor
I (1#g). As a negative control 125I-C4b was incubated with FI alone
in 100#l of VBS.

2.6. Sucrose density gradient fractionation of borreliae outer
membranes
Borrelial outer cell membranes were separated from protoplasmic
cylinders by ultracentrifugation as described (Radolf
et al., 1995). Approximately 1×109 borrelia spirochetes
were washed three times with VBS and suspended in 0.5 ml
ice-cold outer membrane buffer consisting of 10mM Hepes
(N-hydroxyethylpiperazine-N#-2-ethanesulphonic acid), 150mM
NaCl, 1mM MgCl2 (pH 7.4), protease inhibitors and 20% (wt/vol)
sucrose. The bacteria were incubated for 1 h after which 0.5 ml
aliquots were added on tops of linear gradients containing 20?60%
sucrose and a protease inhibitor cocktail. The tubes were centrifuged
at 30,000rpm for 18 h. After warming the tubes to room
temperature 0.6 ml fractions were collected starting from the top.

2.7. Ligand blotting test for C4bp binding
Solubilizates of spirochetes or sucrose density gradient
fractions were subjected to non-reducing SDS-PAGE
and transferred to nitrocellulose membranes. After incubating
the membranes in 5% milk in PBS (1.5 h, RT), 30% HI-NHS
depleted of IgG in 5% milk PBS was added (12 h, +4 ◦C). After three
washes with PBS/0.05% Tween 20 and three washes with PBS a
polyclonal sheep anti-C4bp was added (1:5000) and the Western
blotting was continued as described above.

3. Results

3.1. B. burgdorferi sensu stricto, B. afzelii and B. garinii bind C4bp
from normal human serum
To study whether the three major genospecies of Borrelia bind
the CP inhibitor C4bp from serum representative strains of each
type were incubated in HI-NHS. After extensive washing bound
proteins were eluted. The wash and elute fractions were subjected
to a non-reducing SDS-PAGE and Western blotting with an anti-
C4bp antibody. B. burgdorferi sensu stricto (B31, N40, 297), B. afzelii
(A91, 600, 1082) and B. garinii (40, 46, 50) strains acquired C4bp to
their surfaces as observed by the presence of C4bp in elute fractions
(Fig. 1). The intensities of the C4bp bands in the eluate fractions
showed strain-specific variation, which did not clearly correlate
with the different genospecies.

3.2. Binding of purified C4bp
To test the binding of purified C4bp to borrelia strains, C4bp was
radiolabeled with iodine and incubated with the bacteria (approx.
1.25×108 assay) under hypotonic circumstances (1/3GVBS). To
separate unbound 125I-C4bp from the bacteria-bound 125I-C4bp,
mixtures were centrifuged through 20% sucrose. The 125I-C4bp
bound clearly to all the tested strains (Fig. 2A). B. garinii (46, 50), B.
afzelii (A91, 600, 1082) and B. burgdorferi sensu stricto (B31, N40) all
bound a large proportion of the offered 125I-C4bp (range: 23?67%).
B. garinii strains (strain 50: 65.3%±0.6, n = 3) bound 125I-C4bp more
strongly than B. afzelii (strain 600: 45.2%±1.6, n = 3) and B. burgdorferi
sensu stricto (strain B31: 26.6%±3.9, n = 3). To verify that the
binding of C4bp was specific we performed a competition assay
where purified non-labeled C4bp was incubated together with the
Fig. 1. Binding of C4bp from human serum to various Borrelia strains. (A) B. burgdorferi
sensu stricto (strains B31, 297, N40), (B) B. garinii (strains g50, g40, g46) and (C)
B. afzelii (strains A91, 600, 1082) were incubated in heat-inactivated NHS or VBS.
After incubation the bacteria were washed extensively and possibly bound C4bp
was eluted. Proteins in the last wash and the elute fractions were detected with
a polyclonal anti-C4bp antibody. Binding was seen to all strains but with variable
intensities. No nonspecific binding of the detection antibodies to any secreted bacterial
components in the VBS-controls was seen. Only one representative VBS control
of each genospecies is shown.
spirochetes using two representative strains of B. burgdorferi sensu
stricto (B31, N40), B. garinii (g46, g50), three strains of B. afzelii (A91,
1082, 600) and 125I-C4bp. As shown in Fig. 2B?D, non-labeled C4bp
reduced binding of 125I-C4bp to bacteria in a dose-dependent manner
indicating a specific interaction. BSA, tested as a control, had no
effect on the binding of 125I-C4bp.

3.3. The effect of salt and heparin and C4b on binding
To study whether binding of C4bp to borreliae was of ionic
nature we tested the effect of salt on binding of C4bp to B.
burgdorferi sensu stricto strain B31. The binding of 125I-C4bp was
significantly reduced under hypertonic circumstances at 450mM
NaCl (7.5%±1.8 at 150mM vs. 21.8%±1.9 at 450mM) (Fig. 3A).
Furthermore, a reduction of 80% in binding was seen under isotonic
vs. hypotonic (50mMNaCl) conditions. The result thus showed that
the interaction of C4bp with borrelia spirochetes is ionic in nature.
We obtained similar results using another strain of B. burgdorferi
sensu stricto (N40) as well as B. garinii strains g46, g50 (results not
shown). Importantly, however, considerable binding still occurs
under physiological conditions.

C4bp has a heparin and a C4b-binding site in its #-chains at
the SCR domains 1?2. To study if the heparin site is involved in
binding of C4bp to borreliae we tested the effect of increasing
heparin concentrations on the interaction using B. burgdorferi sensu
stricto strain B31. Increasing amounts of heparin were added to
the reaction mixture prior incubation with 125I-C4bp. As shown
in Fig. 3B heparin inhibited binding of 125I-C4bp to B. burgdorferi
sensu stricto strain B31 (65% of binding was inhibited at the
highest heparin concentration tested). C4b added to the reaction
mixture (0.1, 1 and 10g/ml) also inhibited the binding of 125IC4bp
in a dose-dependent manner (Fig. 3C). Heparin and C4b also
inhibited binding of C4bp with two other borreliae strains tested (B.
burgdorferi s.s. strain N40 and B. garinii strain g40; data not shown).
Taken together these results suggest that the binding sites for hep-
Fig. 3. The effects of increasing salt concentration, heparin and C4b on the binding
of 125I-C4bp to B. burgdorferi sensu stricto strain B31. B. burgdorferi sensu stricto
(B31) was incubated with 125I-C4bp in the presence of varying amounts of NaCl (A),
heparin (B) or C4b (C). Cell-bound C4bp was determined as in Fig. 2 and binding in
the absence of inhibitor was set as 100%.
arin and the borrelial receptor(s) on C4bp are at least partially
overlapping.

3.4. Bound C4bp maintains cofactor activity for C4b cleavage
We next studied if C4bp bound to the surface of bacteria
maintained cofactor activity for factor I in the cleavage of C4b.
Spirochetes were incubated in HI-NHS or with purified C4bp where
after they were washed and incubated further with 125I-C4b and
factor I. Supernatants were subjected SDS-PAGE analysis under
reducing conditions and cleavage of the 125I-C4b was detected by
autoradiography. As shown in Fig. 4, bacteria preincubated in HINHS
or with purified C4bp showed cofactor activity for the cleavage
of C4b as seen by the reduction in the -chain and the appearance
of the 45 kDa C4d and other cleavage fragments of the C4b-chain.
The cleavage fragments were similar to those produced by purified
C4bp with factor I. Radiolabeled C4b was not cleaved when factor
I was not added to the reaction mixture or when the bacteria were
preincubated with VBS alone, indicating the lack of endogenous
C4b cleaving activity in the tested borrelia strains.

3.5. The receptor for C4bp in B. garinii and B. burgdorferi sensu
stricto
To detect a possible receptor for C4bp we next solubilized and
fractionated bacteria and studied C4bp binding to membrane fractions.
The solubilized outer membranes of B. garinii spirochetes
were fractionated by sucrose density gradient centrifugation. The
fractions were subjected to a non-reducing SDS-PAGE, transferred
to nitrocellulose membranes and incubated with HI-NHS depleted
of IgG to prevent nonspecific interactions by serum antibodies. The
binding of C4bp to the outer membrane proteins was analyzed
using a polyclonal anti-C4bp antibody. As seen in Fig. 5A, B. Garinii
(strain g50) solubilizate and its fractions 13 and 14 showed a prominent
band of approximately 43 kDa suggesting that it corresponds
to a potential receptor for C4bp in B. garinii. A major putative receptor
of 43 kDa was seen also with B. burgdorferi sensu stricto strain
B31 (Fig. 5B). Control blots incubated without HI-NHS or without
the first antibody were clear of bands (not shown).
The major C4bp-binding band of approximately 43 kDa was
shared by B. garinii g50, B. garinii g46, B. burgdorferi sensu stricto
B31 and weakly by B. burgdorferi sensu stricto N40 (Fig. 6). When
compared to B. garinii g50 sugar density gradient fractions 13 and
14, the major band was of similar size and apparently represents
the same protein.

4. Discussion

In the present report, we describe the binding of the classical
pathway inhibitor C4bp to the surface of Borrelia spirochetes, which
is a novel interaction between borrelia and the C system. The borreliae
bound C4bp from human serum with a putative receptor of
43 kDa, tentatively labeled as P43. When tested with purified C4bp
it was seen that binding was specific and sensitive to salt and heparin
indicating that it is primarily of ionic nature. C4bp maintained
its cofactor activity for factor I in the cleavage of C4b while bound
to the borrelial surface. These results thus suggest that in addition
to the previously well defined utilization of FH and FHL-1, binding
of C4bp is a novel C evasion mechanism of B. burgdorferi. Binding
of C4bp would particularly control activation of the classical and
lectin pathways of complement.

In the many disease manifestations of borreliosis, symptoms are
thought to result from the presence of spirochetes in various tissues
(Bolz and Weis, 2004). The bacteria invade joints, heart and the CNS
after hematogenous dissemination. Even in the absence of specific
antibodies, as in the early infections, the CP of C can be activated
via direct C1q binding to the target, by natural antibodies (IgM) or
by CRP during an acute phase reaction. Activation of the mannanbinding
lectin pathway has not been reported and may be unlikely
because the borrelial outer membrane does not contain mannan.
On the other hand, this pathway cold become activated by ficolins
if they bind to borrelial surfaces. Although antibody formation
against borreliae is slow, IgG antibodies are formed after bacterial
dissemination. New IgM and IgG antibodies continue to appear
after months to years in chronically ill patients and can reach relatively
high levels. Kochi and Johnson (1988) demonstrated a role
for specific IgG in classical C pathway-mediated killing of serum
resistant borrelial strains by enhanced MAC formation. However,
Brade et al. (1992) did not find differences in the bactericidal effects
of immune and nonimmune sera. Many studies, however, point
towards a protective role of immune sera against borrelial reinfection.
IgG antibodies could prevent relapses of infection by initiating
a fully bactericidal C activation. In most cases, however, activation
is controlled at the C3-amplification loop level by FH and FHL-1. The
efficiency of antibodies in activating C depends on the nature and
stability of the target antigens. The target antigens could include
also borrelial inhibitors of C, whereby killing of the bacteria would
be enhanced if the inhibitory activities are neutralized.
Relapsing fever spirochetes are able to vary their outer surface
proteins to a much greater extent than Lyme disease spirochetes.
It has been thought that because relapsing fever borreliae evade
CP by varying their variable outer surface proteins, they are able
to cause more severe infections. Multiple fever episodes and reappearances
in blood are characteristic to the disease caused by these
spirochetes until they have used all possible combinations of their
variable proteins. As we have shown recently, it is, however, possible
that relapsing fever spirochetes are unduly resistant to C
because they are able to bind also C4bp from human serum (Meri
et al., 2006).

Serum sensitivity of the different genospecies of B. burgdorferi
varies. B. burgdorferi sensu stricto and B. afzelii have in earlier studies
been found to be serum resistant, while B. garinii has in these
assays been serum sensitive (Alitalo et al., 2001; Breitner-Ruddock
et al., 1997; vanDamet al., 1997). Serum sensitivity has been shown
to correlate with the ability of the bacteria to bind the AP inhibitor
FH. B. burgdorferi sensu stricto and B. afzelii express the FH-binding
proteins OspE and Bba68/CRASP-1 (Hellwage et al., 2001; Kraiczy
et al., 2003), whereas B. garinii has in most studies been shown not
to bind FH or FHL-1. In our recent report, however, neurovirulent
B. garinii strains were found to express FH-binding proteins, but
their overall FH-binding capacity was weaker when compared to B.
afzelii and B. burgdorferi sensu stricto strains (Alitalo et al., 2005).
Depending on growth conditions, e.g. in vivo vs. in vitro, Borrelia
spirochetes seem to vary considerably the expression of their outer
surface proteins.

In this study B. garinii was found to bind more C4bp than B.
afzelii or B. burgdorferi sensu stricto (Fig. 2), although all genospecies
bound a fairly high proportion of the offered 125I-C4bp under the
conditions used. Under similar conditions, the binding of FH has
been much weaker. The difference in C4bp-binding between B.
garinii and B. afzelii genospecies was not great, but the fact that B.
garinii is a weak binder of FH/FHL-1 makes it remarkable. Limited
expression of OspE and CRASP-1/Csp-1 makes B. garinii more susceptible
to lysis by the alternative C pathway. However, later in the
course of infection at least anti-OspE antibodies develop (Panelius
et al., 2008). Analogously, antibodies may develop against putative
C4bp-binding proteins, which would limit the spread of bacteria
in the body. B. garinii causes mainly neuroborreliosis. The central
nervous system is considered to be an immunoprivileged site. It is
a good place to escape host defenses because of nonlymphatic circulation
of fluids and lesser degree of C activation. We and others
have speculated earlier that this genospecies favors CNS because
there is less AP activation and a better possibility to hide in the
cerebrospinal space. Thus, preferential binding of C4bp to B. garinii
(versus B. afzelii and B. burgdorferi sensu stricto) could compensate
for the relative lack of FH-binding proteins in B. garinii. Notably, also
N. meningitidis, another gram-negative bacterium with preference
for CNS infections, binds C4bp (Jarva et al., 2005).

B. burgdorferi sensu stricto, B. garinii and B. afzelii bound C4bp
from whole human serum (Fig. 1). C4bp bound to bacterial surface
maintained its cofactor activity for fI in cleaving C4b (Fig. 5),
suggesting that enough active sites on C4bp are available for effector
functions. The most obvious function for bound C4bp would
be down regulation of CP activation in the bacterial microenvironment.

Gram-negative bacteria, including Borreliae, are generally
lysed by MAC unless C is specifically inhibited. On the other
hand, efficient phagocytosis under nonimmune conditions requires
opsonization by C components (Underhill and Ozinsky, 2002). By
interfering with C deposition borrelia can thus evade immune
defenses more efficiently. Studies with S. pyogenes have shown
that deposition of C occurs almost exclusively via CP but C4bp
bound to bacteria limits CP activation under nonimmune conditions
(Carlsson et al., 2003). C4bp-binding is thus associated with
phagocytosis resistance by streptococci. Other possible functions
for bacteria-bound C4bp, as Johnsson et al. (1996) proposed, could
be modification of coagulation using protein S bound to the -
chain of C4bp (Dahlback, 1991) and mediation of adhesion between
bacteria and a putative receptor on human cells.

The hypervariable N-terminal region on the M-protein has
been shown to be the main receptor site for C4bp on S. pyogenes
(Johnsson et al., 1996). It binds to the SCR1-2 domains of the-chain
of C4bp in a hydrophobic manner, which also involves the C4bbinding
site (Accardo et al., 1996). S. pyogenes uses M-protein to
bind not only C4bp but also immunoglobulins, C inhibitors FH and
FHL-1, fibrinogen, fibronectin and albumin (Kotarsky et al., 1998;
Thern et al., 1995). The FH-binding proteins of B. burgdorferi, OspE
and Bba68/CRASP-1 are 15?17 kDa and ∼27 kDa, respectively. The
putative C4bp-binding protein in borrelia was found to be∼43 kDa.
This C4bp-binding receptor P43 is thus different from the known
FH-binding proteins in borrelia

In contrast to the interaction between M-protein of S. pyogenes
and C4bp, the interaction between C4bp and borrelia was found to
be sensitive to salt. Salt sensitivity points to an interaction based
on ionic forces. This seems to be the case for a number of other
bacterium?C4bp interactions as well. For example, B. pertussis, the
agent causing whooping cough, binds C4bp through filamentous
hemagglutinin using charged amino acids at the junction of the -
chain SCR1-2 domains (Berggard et al., 2001). N. gonorrhoeae uses
two porin molecules to bind C4bp. The C4bp?Por1B interaction is
sensitive to both salt and heparin, whereas the C4bp?Por1A interaction
is hydrophobic and therefore insensitive to increasing salt
concentrations (Ngampasutadol et al., 2005). We observed that the
borrelia?C4bp interaction was sensitive to heparin, which binds to
SCR1-3, SCR2 being the most important domain (Blom et al., 2001).
Therefore, the binding sites on C4bp for a putative borrelial receptor
and heparin seem to overlap to some extent. Also, C4b competed
out 125I-C4bp in binding to C4bp pointing towards a shared binding
site between the putative receptor and C4b on C4bp. C4b has
been shown to bind to SCR1-3 (Blom et al., 2001). A cluster of positively
charged amino acids at the interface between SCR1 and SCR2was
found to be most important region for this highly salt-sensitive
interaction. It should be noted that multiple -chains in C4bp allow
the use of similar binding sites simultaneously as ligand sites for
bacteria and C4b. Experimentally, this was verified as the ability of
bacteria-bound C4bp to act as cofactor for C4b cleavage.
In summary, we have demonstrated, for the first time, binding
of the C classical pathway inhibitor C4bp to all genospecies of

Lyme disease causing borreliosis and identified a putative 43 kDa
receptor (P43) for C4bp. There are only few examples of microbes,
which utilize both AP and CP inhibitors. S. pyogenes as well as N.
gonorrhoeae binds both C4bp and FH. Binding of C4bp may help
B. burgdorferi to cause chronic infections even in the presence of
specific antibodies.

Acknowledgements

We thank Marjatta Ahonen for excellent technical assistance.
This researchwassupported by theAcademyof Finland in the frame
of the ERA-NET PathoGenoMics, Sigrid Juselius Foundation, Finnish
Cultural Foundation, Maud Kuistila Foundation, Helsinki University
Central Hospital Funds (EVO) and Finska Läkaresällskapet.
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