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*
Department of Clinical Chemistry, Lund University, University Hospital Malmö, Malmö, Sweden;
Laboratory for Bacteriology, Microbiology and Tumor Biology Center, Karolinska Institute, Stockholm, Sweden; and
Department of Laboratory Medicine, Lund University, Lund, Sweden
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-chains and one 
-chain
linked together with disulfide bridges. We found that pili bind to the
-chain of C4BP, which is composed of eight homologous complement
control protein (CCP) domains. From the results of an inhibition assay
with C4b and a competition assay in which we tested mutants of C4BP
lacking individual CCPs, we concluded that the binding area for pili is
localized to CCP1 and CCP2 of the -chain. The binding between pili
and C4BP was abolished at 0.25 M NaCl, implying that it is based mostly
on ionic interactions, similarly to what have been observed for
C4b-C4BP binding. Furthermore, the N-terminal part of PilC, a
structural component of pili, appeared to be responsible for binding of
C4BP. Membrane cofactor protein, previously shown to be a receptor for
pathogenic N. gonorrhoeae on the surface of epithelial
cells, competed with C4BP for binding to pili only at high
concentrations, suggesting that different parts of pili are involved in
these two interactions. Accordingly, high concentrations of C4BP were
required to inhibit binding of N. gonorrhoeae to Chang
conjunctiva cells, and no inhibition of binding was observed with
cervical epithelial cells. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The cellular pilus receptor, MCP, is widely distributed on the surface
of various human cells with the exception of erythrocytes and is
involved in the protection of tissues from the attack that could result
from activation of the complement system (14). MCP
consists of four complement control protein (CCP) domains followed by
an O-glycosylated Ser-Thr-Pro rich region, a transmembrane
domain and a cytoplasmic tail (15). CCP domains are
typically composed of 
60 aa forming five or more strands
surrounding a compact hydrophobic core (16). They are
present mainly, but not exclusively, in proteins involved in the
activation and regulation of the complement system. One of these
regulators is C4BP, a large plasma protein consisting of seven
identical -chains and one -chain held together by disulfide bonds
(17, 18). The - and -chains consist of eight and
three CCP domains, respectively, and have C-terminal domains involved
in the polymerization of the molecule (19). C4BP prevents
the formation of the C3-convertase of the classical pathway of
complement, accelerates the decay of this convertase (20),
and serves as a cofactor to factor I in the degradation of C4b
(21, 22). Interestingly, C4BP binds to two major human
pathogens, the Gram-positive bacterium Streptococcus
pyogenes (23) and the Gram-negative Bordetella
pertussis (24), which may result in local
down-regulation of the complement system, thereby increasing the
pathogenicity of these bacteria. Both S. pyogenes and
B. pertussis bind to a region of C4BP partially overlapping
with the C4b binding site (24, 25, 26). Recently, it has been
reported that C4BP also binds to certain nonpiliated strains of
N. gonorrhoeae and that the binding is mediated by porins
(13). Studies of these interactions between C4BP and
bacterial pathogens are of general interest, because many pathogenic
micro-organisms are now known to interact with human complement
regulators (27). In the present study we describe and
characterize a binding between type IV pili of N.
gonorrhoeae and human C4BP.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human C4BP purified from plasma (28), C4
(29), and streptococcal IgA/C4BP-binding M protein (Arp4;
equal to Emm4 in an alternative nomenclature (30)).
Concentrations of C4BP and C4 were determined by measurement of
absorbance at 280 nm; the extinction coefficients (1%, 1 cm) used were
14.1 for C4BP and 8.3 for C4. The concentration of Arp4 was determined
after 24-h hydrolysis in 6 M HCl and analysis of amino acid content. C4
met (C4b-like molecules) were prepared by incubation of human plasma
purified C4 with 100 mM methylamine, pH 7.6, for 1 h at 37°C and
subsequent dialysis against TBS. Recombinant wild-type C4BP and mutant
R39Q/R64Q/R66Q were expressed in human kidney cells after selection of
stable clones (293, no. 1573-CRL, American Type Culture Collection,
Manassas, VA) and purified by affinity chromatography using mAb104
directed against the -chain of C4BP. The expression and
characterization of the two proteins were described previously
(31). C4BP mutants lacking single CCP domains were
constructed by overlapping extension PCR, expressed in 293 cells, and
purified by affinity chromatography (13). Proteins were
labeled with 125I using the chloramine T method.
The sp. act. was 20–25 MBq/µg of protein.
Recombinant MCP was obtained as described previously (1). Briefly, MCP was expressed in bacteria as a fusion protein together with maltose-binding protein (MBP). Bacterial lysates containing MBP or MBP-MCP were applied to an amylose column, and the proteins were eluted with 10 mM maltose. Similarly, two fusion proteins containing fragments of MS11 PilC1 were constructed and purified. PilC.A contains the N-terminal part of the PilC1 protein (aa 1–257), whereas PilC.C is composed of the C-terminal part of the PilC1 (aa 501-1016), both followed by the MBP fusion partner (32). Concentrations of fusion proteins were measured using the Bio-Rad protein assay (Hercules, CA). A polyclonal anti-PilC Ab was generated against a fusion protein containing the C-terminal half of MS11 PilC1 (32).
Binding of [125I]C4BP to bacteria
Bacteria were added to wells of microtiter plates (1.25 x 108 organisms/well) and incubated for 10 min at 37°C. They were then fixed with 0.3% glutaraldehyde for 15 min at room temperature and washed four times with PBS containing 0.05% Tween 20. [125I]C4BP was added (50 kcpm/well) in a volume of 50 µl and incubated for 60 min at 37°C. The wells were washed four times with PBS/0.05% Tween 20, and the amount of radioactivity was associated with immobilized bacteria was measured in a gamma counter.
Direct ligand binding assay
Microtiter plates were incubated overnight at 4°C with 50 µl
of a solution containing 10 µg/ml of isolated pili in 75 mM sodium
carbonate, pH 9.6. The wells were washed three times with washing
buffer (50 mM Tris-HCl, 0.15 M NaCl, and 0.1% Tween 20, pH 7.5) and
then incubated at room temperature with 200 µl of quenching solution
(washing buffer supplemented with 3% fish gelatin). After another
three washes, 50 µl of C4BP diluted in 100 mM HEPES-NaOH, 0.1% Tween
20, and 0.1% BSA, pH 7.2, was added to the wells. The samples were
incubated overnight at 4°C and washed three times, and biotinylated
mAb 67, directed against CCP4 of the C4BP -chain, was added. After
2-h incubation at 4°C, the wells were washed three times and
incubated with biotin-avidin-conjugated HRP. After another 2 h at
4°C and washing, the enzymatic activity was detected with a substrate
according to a manufacturer’s instructions (Dakopatts, Glostrup,
Denmark).
When binding of C4BP to PilC was tested, microtiter plates were incubated overnight at 4°C with 200 µl of solution containing 60 µg/ml of PilC.A or PilC.C in 75 mM sodium carbonate, pH 9.6. The wells were washed three times with PBS/0.05% Tween 20 and then incubated with incubation buffer (PBS, 0.1% BSA, and 0.05% Tween 20) for 1 h. Fifty microliters of C4BP (40 µg/ml) in incubation buffer was added for 1 h, and the plates were washed and incubated with C4BP Ab (1/5000) in incubation buffer for 1 h at 37°C. After another set of washing, HRP-conjugated anti-rabbit IgG in incubation buffer was added for 1 h at 37°C. The plates were washed for the last time, and enzymatic activity was detected by adding HRP substrate (Roche).
Competition assay
Microtiter plates were incubated overnight at 4°C with 50 µl
of a solution containing 10 µg/ml of isolated pili in 75 mM sodium
carbonate, pH 9.6. The wells were washed three times with the washing
buffer and then incubated at room temperature with 200 µl of
quenching solution. After another three washes, the
125I-labeled C4BP purified from plasma was added
(50 kcpm/well, 0.1 nM) together with various unlabeled proteins
diluted in 100 mM HEPES-NaOH, 0.1% Tween 20, and 0.1% BSA, pH 7.2.
The samples were incubated for 16–20 h at 4°C and washed four times,
and the amount of radioactivity associated with each well was measured
in a gamma counter. About 15–30% of the added
125I-labeled C4BP tracer bound to the immobilized
pili, and the binding of radiolabeled tracer could be competed out by
unlabeled C4BP.
C4BP overlay assay
Ten micrograms of purified pili were loaded in each well and subjected to electrophoresis in the presence of SDS in a 12% polyacrylamide gel. The gel was cut into three pieces. One piece was stained with Coomassie brilliant blue, and the remaining lanes were blotted onto a polyvinylidene difluoride membrane and blocked overnight with 5% milk powder. One membrane was incubated with PilC antiserum (1/5000). Another membrane was incubated in 10 ml of TBS supplemented with 0.2% Tween containing 100 µg of C4BP for 1 h and then with polyclonal anti-C4BP Ab (1/5000). A control membrane was incubated with C4BP Ab only. All filters were finally incubated with HRP-conjugated goat anti-rabbit Ab (1/10000; Sigma) and developed with an ECL kit (NEN Life Science Products, Boston, MA).
Plasmon surface resonance
The interaction between purified pili of N.
gonorrhoeae and C4BP was analyzed using plasmon surface resonance
(Biacore 2000, Biacore, Uppsala, Sweden). Two flow cells of a
CM5 sensor chip were activated, each with 20 µl of a mixture of 0.2 M
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 0.05 M
N-hydroxysulfosuccinimide at a flow rate of 5 µl/min,
after which C4BP purified from plasma (0.01 mg/ml in 10 mM sodium
acetate buffer, pH 4.5) was injected over one flow cell to reach
3000 resonance units. Unreacted groups were blocked with 20 µl of
1 M ethanolamine, pH 8.5. A negative control was prepared by activating
and subsequently blocking the surface of the second flow cell. The
association kinetics were studied for various concentrations (12.5–200
µg/ml) of purified pili. The flow buffer was 10 mM HEPES-KOH, pH 7.4,
supplemented with 150 mM NaCl, 0.005% Tween 20, and 3.4 mM EDTA.
Aliquots of pili stock solution (0.7 mg/ml in PBS) were diluted in the
flow buffer, and 90 µl were injected during the association phase at
a constant flow rate of 30 µl/min. The sample was injected first over
the negative control surface and then over immobilized C4BP. Signal
from the control surface was subtracted. The dissociation was followed
for 15 min at the same flow rate. In all experiments 20 µl of 100 mM
HCl was used to remove bound ligands during a regeneration step.
BiaEvaluation 3.0 software (Biacore) was used to analyze
obtained sensograms and to calculate rate affinity constants.
Bacterial strains and growth conditions.
N. gonorrhoeae MS11 (P+) and MS11 (P-n) were gifts from M. Koomey (33). Piliated and nonpiliated variants were distinguished by colony morphology under a binocular microscope. Bacteria were grown on GCB-agar plates (Difco, Detroit, MI) containing Kellogg’s supplement at 37°C in 5% CO2 and passaged daily.
Wild-type pili were purified from N. gonorrhoeae strain MS11 by several cycles of crystallization and solubilization as previously described (3). The pili preparations used in this study contained <1% of contaminating proteins as judged from Coomassie Blue staining.
Cell lines and growth conditions
ME 180 (no. HTB33; American Type Culture Collection), an epithelial-like human cell line derived from a cervical carcinoma was cultured in McCoy’s 5A medium (Life Technologies, Grand Island, NY) supplemented with 10% FBS and 2 mM glutamine. The Wong-Kilbourne derivative of Chang conjunctiva (American Type Culture Collection, no. CCL 20.2) was grown in medium 199 with Earle’s salts supplemented with 10% FBS and glutamine. Cells were grown at 37°C in 5% CO2. Binding experiments were performed in a medium free of serum, antibiotics, and glutamine.
Binding of bacteria to epithelial cells
The cells were cultured in 24-well tissue culture plates for 2–3 days until they were 60–80% confluent. The monolayers were then washed with serum-free medium, and bacteria, previously grown for 18–20 h and suspended in serum-free culture medium, were mixed with different amounts of C4BP and incubated at 37°C in 5% CO2 for 30 min. The suspensions of bacteria preincubated with C4BP were added to the wells (1.25 x 107 bacteria/well), and additional cell culture medium was added. After incubation for 60 min at 37°C in 5% CO2, infected cell layers were washed three times for 5 min each time, treated with 1% saponin for 5 min, serially diluted, and spread on GCB plates. The bacteria were grown at 37°C in 5% CO2 overnight, and CFU were counted.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C4BP interacts with several bacterial pathogens, and it has been
recently reported that some strains of N. gonorrhoeae bind
human C4BP via outer membrane porins (13). Because of this
observation and the fact that pili are responsible for attachment of
N. gonorrhoeae to MCP, a complement regulator similar in
structure and function to C4BP, we decided to test whether the
interaction between N. gonorrhoeae and C4BP could also be
mediated by pili. We found a significant difference
(p < 0.05 in Student’s t test) in
the binding of 125I-labeled human C4BP purified
from plasma to piliated and nonpiliated strain MS11 of N.
gonorrhoeae (Fig. 1
A).
There was a substantial binding of C4BP to a nonpiliated strain of
Neisseria, probably mediated by porins (13). As
a control, immobilized bacteria were detected with an Ab whose affinity
for N. gonorrhoeae is not affected by the presence of pili
(data not shown). By doing so we have ascertained that piliated
bacteria were immobilized at the same level as the bacteria
lacking pili.
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B, 
). As a
control, C4BP was added to wells that were only blocked with fish
gelatin and did not contain pili proteins (Fig. 1B, •).
Binding of C4BP was dose dependent and saturable at 80 nM C4BP,
while no binding was seen in control wells. The polymeric nature of
C4BP did not allow calculation of affinity constants, but half-maximal
binding of C4BP to pili was obtained at 20 nM protein.
Binding between pili and C4BP was also confirmed by an experiment using
surface plasmon resonance technology (Biacore 2000). C4BP purified from
plasma was covalently immobilized on the surface of the CM5 chip, and
increasing concentrations of pili isolated from gonococcal strain MS11
were added. The flow buffer used was 10 mM HEPES-KOH, pH 7.4,
supplemented with 150 mM NaCl, 0.005% Tween 20, and 3.4 mM EDTA. The
association and dissociation reactions were recorded and confirmed a
dose-dependent binding between pili and C4BP (Fig. 2). We found that binding of pili to C4BP
was limited by a mass transfer at all flow rates tested, which is
consistent with the fact that pili are very large structures (5000
kDa). This together with a fact that polymeric C4BP contains multiple
binding sites for most of its ligand, made it difficult to calculate
rate affinity constants. However, the dissociation rate affinity
constant (Kd,
Koff) could be estimated as 4.8
x 10-4 s-1.
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An overlay assay was used to identify the C4BP-binding subunit of
pili, which are complex assemblies of several proteins. First, we found
that Abs raised against gel-purified full-length PilC recognized a band
with an apparent Mr of 110 kDa in the
preparation of purified pili (Fig. 3A, lane 2). This
Mr is consistent with properties of PilC.
A protein showing a similar migration pattern upon SDS-PAGE also bound
C4BP (Fig. 3A, lane 3), which suggested that C4BP
interacts with the PilC subunit. Furthermore, we tested direct binding
of C4BP to immobilized fusion proteins consisting of MBP and fragments
of PilC. Using a microtiter plate-based binding assay, we found that
C4BP bound to an N-terminal fragment of PilC encompassing aa 1–257
(PilC.A), whereas binding to the C-terminal part of PilC comprising aa
501-1016 was negligible (PilC.C; Fig. 3B). Furthermore, we
tested whether PilC.A was able to inhibit binding between
[125I]C4BP and immobilized pili. We found that
2 µM recombinant protein was needed to block the interaction by 50%
(Fig. 3C), which further implies that binding for C4BP is
localized to the N-terminal part of PilC. As a negative control we used
recombinant MBP, which did not affect the interaction.
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-chain
Human C4BP consists of two types of subunits, seven identical
-chains involved in the regulation of complement activation, and a
single -chain that interacts with anticoagulant, vitamin K-dependent
protein S (18, 34). To assess which subunit is responsible
for interaction with pili we tested whether C4BP purified from plasma
(with -chain) and recombinant C4BP (polymerized -chains, without
-chain) have the same abilities to compete with
125I-labeled C4BP purified from plasma for
binding to immobilized pili (Fig. 4A). We found that in both
cases 2 nM C4BP was needed to block binding by 50%
(EC50), which suggests that binding is localized
within the -chain and that the -chain does not contribute to the
interaction. The same result was obtained with C4BP carrying protein S
bound to -chain (data not shown).
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A shows that the R39Q/R64Q/R66Q mutant had lower apparent
affinity for pili, suggesting that the binding site for pili overlaps
with the cluster of positively charged amino acids on the interface
between CCP1 and CCP2. To further test this hypothesis, we tested
whether C4 met and streptococcal protein Arp4, which bind to
overlapping, but nonidentical, sites on C4BP (26), would
compete for binding of C4BP to pili. Fig. 4B shows that
0.8 nM C4 met was needed to decrease the interaction between C4BP
and pili by 50%. About 5-fold (3.5 nM) more Arp4 was needed to obtain
a similar effect, which suggests that the area of C4BP interacting with
pili is more similar to the C4 met- than to the Arp4-binding site. We
have further confirmed these results by using a competition assay in
which we tested a series of recombinant C4BP molecules lacking single
CCP domains. We found that the removal of CCP2 had a slight effect on
the binding of C4BP to pili and that the mutant lacking both CCP1 and
CCP2 entirely lost its ability to bind immobilized pili (Fig. 5). This finding strengthened the
hypothesis that the binding site for pili is localized to CCP1 and CCP2
of the -chain and therefore overlaps with a C4b-binding site. We
have also observed that the mutant lacking CCP7 showed decreased
apparent affinity for pili. This finding seems at first surprising, but
there is some evidence implying that CCP7 or CCP8 may contain a
secondary binding site for C3b/C4b (35).
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To further characterize the binding site for pili we tested
whether this interaction is inhibited in the presence of increasing
salt concentrations. 125I-labeled recombinant
wild-type C4BP was incubated with pili immobilized in wells of a
microtiter plate. The buffer (50 mM HEPES-NaOH, pH 7.2) was
supplemented with 0.1% BSA, 0.1% Tween 20, and various NaCl
concentrations ranging from 5 mM to 1.0 M. After 16-h incubation at
4°C and washing with 50 mM Tris-HCl, pH 7.5, supplemented with 0.1%
Tween 20, bound C4BP was measured in a gamma counter. Fig. 6 shows that binding between C4BP and
pili was sensitive to the salt concentration, because it was abolished
at 0.25 M NaCl. These results are similar to those previously obtained
for the C4b-C4BP interaction (26) and suggest that
mechanisms of both interactions are based upon ionic interactions
between amino acids.
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Because MCP is a cellular receptor for pili and is similar in
function and structure to C4BP, we tested whether recombinant MCP would
block binding between C4BP and pili. MCP was expressed as a fusion
protein with MBP and purified. We found that MCP-MBP was able to
compete with the C4BP-pili interaction when MCP-MBP was present in
sufficiently high concentration. About 300 nM MBP-MCP was needed to
block binding by 50% (Fig. 7A). MBP alone could not
inhibit binding of 125I-labeled C4BP to
immobilized pili even at the highest concentration tested.
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B shows that C4BP even at a
concentration of 900 nM did not inhibit binding of N.
gonorrhoeae to the epithelial cervical ME 180 cell line. However,
100 nM C4BP was required to block binding of N.
gonorrhoeae to Chang conjunctiva cells by 50% when the cells were
60–70% confluent. No inhibition was observed when the cells reached
higher density. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Pili present on the surface of pathogenic N. gonorrhoeae are
important virulence factors responsible for the adhesion of bacteria to
epithelial cells. MCP, a membrane protein regulating activation of the
complement system, was shown to be a receptor for pili
(1). Therefore, it was interesting to investigate whether
there is a detectable interaction between pili and C4BP, which is
similar in structure and function to MCP. Indeed, using a microtiter
plate-based assay and surface plasmon resonance technique (Biacore) we
could demonstrate a direct, dose-dependent, and saturable binding
between the two molecules. Half-maximal binding of C4BP to immobilized
pili occurred at 20 nM. Taking into consideration that the serum
concentration of C4BP is 0.35 µM, the interaction is highly
physiologically relevant in the blood environment.
Gonococcal pili undergo both phase and antigenic variations. Pilin, the major protein constituent of pili, is expressed from one active PilE locus, and there are also several silent loci of this gene. Structural variation in pili results mainly from recombination between silent and expressed pili gene sequences (37). We found that four pili variants containing different PilE sequences (38) bound C4BP with similar apparent affinities (not shown). PilC is a 110-kDa pilus biogenesis protein that seems to be involved in the interaction between pili and MCP, because it was possible to select or genetically construct adherence negative mutants that expressed pili without detectable amounts of PilC (39, 40). The results of an overlay assay suggested that C4BP interacts with the PilC subunit of pili. We then used fusion proteins containing N- and C-terminal fragments of PilC to further identify the binding site for C4BP, and we found that the N-terminal fragment of PilC is apparently required for the interaction.
We then attempted to localize the binding site for pili on the
molecular surface of C4BP. First, we tested whether C4BP purified from
plasma consisting of seven -chains and one -chain will bind pili
with the same apparent affinity as the recombinant C4BP that is
composed exclusively of -chains. -Chains are known to bind C4b
(17), heparin (41), Bordetella
pertussis (24), Streptococcus pyogenes
(23), and serum amyloid P component (42),
whereas -chain binds anticoagulant protein S (18). We
found that both forms of C4BP bound to pili with similar affinities,
implying that -chains confer the ability to bind gonococcal pili. To
further localize the binding site on C4BP we tested whether C4 met and
streptococcal surface M-protein Arp4 were able to block the interaction
between C4BP and pili. We found that C4 met in particular was an
efficient inhibitor; 0.8 nM of the protein was needed to block
binding by 50%. We also tested whether the R39Q/R64Q/R66Q mutant of
C4BP, where three positively charged amino acids were replaced by Gln,
would bind pili. That mutant was previously shown to have impaired
binding ability for both C4 met and streptococcal Arp4 and Sir22
proteins (26, 31). We found that the R39Q/R64Q/R66Q mutant
bound to pili with lower apparent affinity compared with the wild type.
However, the effect of the mutation was not as dramatic as in the case
of binding to C4 met (26, 31). We also tested a panel of
mAbs directed against the -chain of C4BP (26, 43). We
found that several of these strongly inhibited binding of C4BP to pili.
These Abs have previously been analyzed for their ability to block
binding between C4BP and C4 met or Arp4 (26). The results
obtained for pili were similar to those previously observed for the
C4b-C4BP interaction. Thus, the interaction between C4BP and pili was
inhibited in the presence of Abs directed against CCP1 and CCP2 of the
-chain (data not shown). Furthermore, we found that C4BP lacking
both CCP1 and CCP2 was not able to compete with
125I-labeled wild-type C4BP for binding to pili.
The influence of NaCl on the pili-C4BP interaction was also tested, and
we found that the binding was entirely abolished at 0.25 M NaCl.
Similar results were previously obtained for the C4 met-C4BP
interaction and were in sharp contrast to the Arp4-C4BP interaction,
which is almost insensitive to the presence of salt and governed mostly
by hydrophobic forces (26). Taken together, these results
suggest that binding site for pili overlaps with the interaction site
for C4b/C4 met on CCP1-CCP2 and that the mechanism of pili-C4BP
interaction resembles that of C4b-C4BP binding. Surprisingly, we found
that a mutant lacking CCP7 showed decreased binding to pili. The
surface of CCP7, predicted by homology-based computer modeling,
displays a large patch of positively charged amino acids, similar to
that present on the interface of CCP1 and CCP2 (19). It is
unclear at this point whether this cluster of amino acids could be
involved in binding of pili in a specific manner as a second binding
site. The same mutant, but lacking CCP7, displayed decreased affinity
for C4 met (not shown).
We tested whether recombinant MCP could inhibit binding between C4BP and pili, and we found that rather high concentrations of the protein (300 nM) were needed to reach 50% inhibition. This suggests that there are different binding sites for C4BP and MCP on pili. This suggestion is further strengthened by the fact that MCP interacts with the C-terminal part of PilC (our manuscript in preparation), whereas C4BP seems to bind the N terminus of the protein. These observations are consistent with the finding that binding of Neisseria to epithelial cells was inhibited only by high concentrations of C4BP or not at all. This together with the rest of our results suggest that the pili-C4BP interaction more likely plays a role in the later stages of the infection and may not be involved in the process of bacterial adhesion to epithelial cells.
In conclusion, gonococci have developed several mechanisms that allow binding of human complement regulators. Factor H binds to sialic acid to mediate unstable serum resistance (12), whereas nonsialylated gonococci can use porin molecules to bind regulatory molecules C4BP and factor H (11). In addition, binding of C4BP can be mediated by pili. It seems likely that these different interactions allow the bacteria to escape complement attack.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Anna M. Blom, Department of Clinical Chemistry, Lund University, The Wallenberg Laboratory, Floor 6, University Hospital Malmö, S-205 02 Malmö, Sweden. E-mail address: anna.blom{at}klkemi.mas.lu.se
3 Abbreviations used in this paper: MCP, membrane cofactor protein; Arp4, streptococcal IgA/C4BP-binding M protein; C4BP, C4b-binding protein; CCP, complement control protein (domain); MBP, maltose-binding protein; GCB, GC base medium with Kellogg’s supplement.
Received for publication February 2, 2001. Accepted for publication March 23, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1H in proteolysis of C4b and C3b. J. Exp. Med. 150:267.-chain is crucial for C4b binding and factor I cofactor function. J. Biol. Chem. 274:19237.-chain is crucial for C4b binding and factor I-cofactor function. Biochem. J. 323:469.
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D. Perez-Caballero, I. Garcia-Laorden, G. Cortes, M. R. Wessels, S. R. de Cordoba, and S. Alberti Interaction between Complement Regulators and Streptococcus pyogenes: Binding of C4b-Binding Protein and Factor H/Factor H-Like Protein 1 to M18 Strains Involves Two Different Cell Surface Molecules J. Immunol., December 1, 2004; 173(11): 6899 - 6904. [Abstract] [Full Text] [PDF]
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T. Nordstrom, A. M. Blom, A. Forsgren, and K. Riesbeck The Emerging Pathogen Moraxella catarrhalis Interacts with Complement Inhibitor C4b Binding Protein through Ubiquitous Surface Proteins A1 and A2 J. Immunol., October 1, 2004; 173(7): 4598 - 4606. [Abstract] [Full Text] [PDF]
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 M. W. van der Woude and A. J. Baumler Phase and Antigenic Variation in Bacteria Clin. Microbiol. Rev., July 1, 2004; 17(3): 581 - 611. [Abstract] [Full Text] [PDF]
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A. M. Blom, B. O. Villoutreix, and B. Dahlback Mutations in {alpha}-Chain of C4BP That Selectively Affect Its Factor I Cofactor Function J. Biol. Chem., October 31, 2003; 278(44): 43437 - 43442. [Abstract] [Full Text] [PDF]
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N. V. Prasadarao, A. M. Blom, B. O. Villoutreix, and L. C. Linsangan A Novel Interaction of Outer Membrane Protein A with C4b Binding Protein Mediates Serum Resistance of Escherichia coli K1 J. Immunol., December 1, 2002; 169(11): 6352 - 6360. [Abstract] [Full Text] [PDF]
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T. Areschoug, M. Stalhammar-Carlemalm, I. Karlsson, and G. Lindahl Streptococcal beta Protein Has Separate Binding Sites for Human Factor H and IgA-Fc J. Biol. Chem., April 5, 2002; 277(15): 12642 - 12648. [Abstract] [Full Text] [PDF]
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E. Morfeldt, K. Berggard, J. Persson, T. Drakenberg, E. Johnsson, E. Lindahl, S. Linse, and G. Lindahl Isolated Hypervariable Regions Derived from Streptococcal M Proteins Specifically Bind Human C4b-Binding Protein: Implications for Antigenic Variation J. Immunol., October 1, 2001; 167(7): 3870 - 3877. [Abstract] [Full Text] [PDF]
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) competed
with trace amounts of 125I-labeled C4BP for
binding to immobilized pili. The 100% binding value was estimated in
the absence of fluid phase competitor. Shown is the mean of two
experiments performed in doublets; bars represent SD values.
B, Inhibition of interaction between C4BP and pili by C4 met
and Arp4. Increasing concentrations of C4 met (
