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* Division of Infectious Diseases, Childrens Hospital, and Keck School of Medicine, University of Southern California, Los Angeles, CA 90027;
Department of Clinical Chemistry, Lund University, University Hospital Malmo, Malmo, Sweden; and
Institut National de la Santé et de la Recherche Médicale, Unité 428 University Paris V, Paris, France
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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 OmpA binds the
-chain of C4bp, which is composed of eight homologous complement
control protein (CCP) modules. Binding studies using mutants of
recombinant C4bp that lack one CCP at a time suggest that CCP3 is the
major site of interaction with OmpA. Furthermore, we demonstrate that
the N terminus of OmpA interacts with C4bp. Binding of C4bp to OmpA is
not significantly inhibited in the presence of either C4b or heparin
and is not salt sensitive, implying that it is hydrophobic in nature,
suggesting a novel interaction between OmpA and C4bp. A compelling
observation in this study is that synthetic peptides corresponding to
CCP3 sequences block the binding of C4bp to OmpA and also significantly
enhance serum bactericidal activity. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The complement system is the first line of defense against pathogenic micro-organisms. Activation of this system results in opsonization of the micro-organism for phagocytosis and formation of the membrane attack complex. Excessive activation of complement is inhibited by various regulatory proteins, both cell bound and fluid phase, which protect the host from attack by its own complement. The complement regulatory proteins include soluble plasma proteins, C4b binding protein (C4bp) and factor H (FH), and several membrane proteins.
Neisserial pathogens have been studied extensively for their serum
resistance mechanism (3). The sialylation of
lipooligosacchrides has been implicated as a mechanism of unstable
serum resistance in Neisseria gonorrhoeae, in which sialic
acid binds to FH, a critical regulator of the alternative pathway of
complement. In contrast to unstable serum resistance, several
gonococcal strains remain serum resistant under reduced sialylation.
This is termed stable resistance and has been shown to depend on the
binding of FH to Por1A (4). The binding of FH is confined
to loop 5 of Por1A, and the addition of loop 5 peptide in a serum
bactericidal assay enhances the killing of an otherwise completely
serum-resistant gonococcal strain (5). Furthermore, Por1A
as well as Por1B bind to C4bp, and blocking of this interaction renders
the bacteria serum sensitive (6). Finally, type IV pili of
Neisseria also bind C4bp; thus, gonococci have several
mechanisms that act cooperatively to mediate serum resistance
(7). C4bp is present in human plasma at concentrations of
200–250 µg/ml and is present in plasma in several forms, the major
one composed of seven identical 70-kDa subunits (-chains) and one
45-kDa subunit (-chain) (8). Most C4bp in blood exists
in complexes with protein S (C4bp-PS), a component of the vitamin
K-dependent protein C anti-coagulant system, and interacts with the
-chain of C4bp. Many isolates of Streptococcus pyogenes
have also been reported to bind C4bp (9). In this species
the NH2-terminal, highly variable region of
several members of the M family proteins is responsible for binding to
C4bp. In addition, C4bp binding has been demonstrated in all clinical
isolates of Bordetella pertussis expressing filamentous
hemagglutinin (10, 11).
Since the classical pathway is crucial in initiating complement
deposition on pathogenic bacteria, regulation of this pathway is an
efficient means for bacteria to evade initial host defense by serum
complement (12, 13). E. coli K1 has been shown
to activate the classical complement pathway via 018 LPS
(14); however, E. coli still survive in the
blood by avoiding complement attack. In the present study we
demonstrate that OmpA, which is a 35-kDa highly conserved protein among
Gram-negative bacteria, of E. coli K1 contributes to serum
resistance. Its N-terminal domain, encompassing aa residues 1–177,
crosses the membrane eight times, forming anti-parallel
-strands. The four extracellular loops are mobile and are not well
defined in the crystal structure (15). Our study
demonstrates that C4bp binds to the N-terminal loops of OmpA, which
leads to a decrease in serum killing, and that the interaction is
confined to CCP3 of the -chain of C4bp. Another salient feature of
this study is that the synthetic peptides that represent sequences from
the CCP3 domain of C4bp inhibit OmpA-C4bp interaction and increase
serum bactericidal activity.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All strains used in this study were derived from a cerebrospinal
fluid isolate of E. coli K1 strain RS218 (serotype
018:K1:H7) as described previously (16), and their
characteristics are depicted in Table I
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Bacteria were grown in brain heart infusion broth (Difco, Detroit, MI)
with appropriate antibiotics at the following concentrations; rifampin,
100 µg/ml (E44 and E58); tetracycline, 12.5 µg/ml (E91); and
ampicillin, 100 µg/ml (E105 and E111). Normal adult human serum (AHS)
was obtained from 10 healthy individuals. Sera were pooled, aliquoted,
and stored at -70oC until further use. For some
experiments, AHS was incubated at 56°C for 30 min to yield
heat-inactivated (72°C) serum.
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The purification of human C4bp-PS, FH, and the generation of recombinant human C4bp (rC4bp) and eight rC4bp molecules lacking individual CCPs were described previously (17, 18). Vitronectin from adult human serum was purified as described previously (19). Peptide sequences that represent portions of CCP3 and CCP8 domains of C4bp were synthesized on an automatic peptide synthesizer. The sequences of the peptides are as follows: CCP3-1, 132 DIRNGRHS 139; CCP3-2, 141 EENFYAYGF 149; CCP3-3, 156 DPRSFSLLGH 164; CCP3-4, 172 ENETIGVWRP 181; CCP8-1, 437 RKPELVNGR 445; CCP8-2, 449 DKDQYVEPEN 458; CCP8-3, 464 DSGYGVVGPQ 473; and CCP3-4, 480 NRTWYPEVPK 489. Polyclonal Abs to C4b, C4bp, C1q, and FH were obtained from Calbiochem (San Diego, CA). The mAbs 104 and 67 against C4bp were raised by Dr. B. Dahlbäck (Lund University, Malmo, Sweden). Polyclonal anti-OmpA Abs that recognize the N-terminal portion of OmpA (OmpA-N-Ab) were generated as previously described (16), and polyclonal anti-OmpA Ab (OmpA-C-Ab) that reacts to the C-terminal portion of OmpA was obtained from Dr. M. Inouye (Robert Wood Johnson Medical School, Piscataway, NJ).
Flow cytometry
We used flow cytometry for quantitation of binding of C4bp to E. coli. OmpA+ and OmpA- E. coli strains were grown overnight in appropriate antibiotics. The cells were centrifuged and washed twice with HBSS at room temperature. The bacteria were resuspended in HBSS, and the OD600 was adjusted to 1 x 109 CFU/ml. The bacterial suspension (140 µl) was incubated with AHS (60 µl) for 1 h at 37°C. The suspension was centrifuged at 8000 x g for 3 min, and the pellet was resuspended in HBSS. The suspension was further incubated with either anti-C4bp or anti-FH Abs (0.2 µg/ml) at room temperature for 30 min, followed by washing four times with HBSS. The bacterial pellets were further incubated with FITC-conjugated secondary Abs for 30 min at room temperature, washed, and resuspended in PBS. Flow cytometric analysis was conducted on a BD Biosciences (Mountain View, CA) instrument using CellQuest software.
Biotinylation of C4bp proteins and incubation with bacteria
The proteins to be biotinylated (C4bp-PS and rC4bp, 2 mg each)
were added to 0.1 M sodium bicarbonate buffer (pH 8.0) containing 0.5
mg/ml NHS-LC-Biotin (Pierce, Rockford, IL) at a final protein
concentration of 2 mg/ml. The mixture was incubated on ice for 1
h, followed by extensive dialysis against PBS, and was concentrated
using Centricon tubes (Millipore, Bedford, MA; 10-kDa cut-off). The
biotinylated proteins (2–5 µg) were incubated with the bacterial
pellet from a 5-ml overnight culture in a volume of 0.5 ml at 37°C on
a rotator for 1 h. The bacteria were then centrifuged, and the
pellets were washed three times with PBS containing 0.1% Triton X-100.
After a final wash, the bound proteins were released with Laemmli
buffer in the presence of -ME and analyzed by SDS-PAGE. The
separated proteins were transferred to nitrocellulose, and
immunoblotting was conducted using streptavidin coupled to peroxidase.
The protein bands were visualized by ECL reagent (Amersham Biosciences,
Piscataway, NJ).
Deglycosylation of C4bp
Recombinant C4bp (200 µg) was treated with 5 U of peptide N-glycosidase F in 50 mM sodium phosphate buffer (pH 7.8) at 37°C overnight (20). The protein was purified by gel filtration over Sephacryl S-300 HR. The peak containing C4bp was concentrated and tested for deglycosylation by blotting with wheat germ agglutinin, followed by anti-wheat germ agglutinin Ab immunoblotting.
Bactericidal activity
The susceptibility of various E. coli strains to complement-mediated killing was determined using nonimmune AHS at a final concentration of 40%. The bacteria (105) were suspended in HBSS containing CaCl2, followed by addition of AHS, and were incubated for 1 h with rotation. Routinely, heat-inactivated AHS (56°C for 30 min) was included as a control. In some experiments AHS was incubated with 100 nM EGTA for 15 min before performing the bactericidal assays. Various Abs and C4bp peptides were incubated with the bacteria in separate experiments at room temperature for 30 min before the addition of AHS to examine their effect on the bactericidal activity. Synthetic OmpA peptides were incubated with AHS at room temperature for 30 min before adding them to the bacteria. The effects of these reagents on bacterial survival were examined by the colony count method.
Heparin-Sepharose column chromatography of serum proteins
The AHS containing protease inhibitors was passed through a heparin-Sepharose column for a period of 2 h at 4°C. The column was then thoroughly washed with PBS, and bound proteins were eluted with PBS containing varying concentrations of NaCl. The proteins from each fraction were dialyzed against PBS and concentrated using Centricon tubes.
Labeling of peptides with fluorescein amine
The synthetic peptides were labeled using a fluorescein amine (FS) labeling kit (Panvera, Madison, WI). The labeling reagent is a succinimidyl ester of fluorescein, which selectively forms an amide bond with N-terminal amino group of peptides or proteins at neutral pH; this reagent reacts more rapidly and forms a more stable derivative than FITC. Briefly, each peptide (500 µg) was resuspended in coupling buffer and chilled to 4°C for 10 min. Ice-cold FS in DMSO (20 µM/20 µl) was added to the peptide solution and incubated at 37°C for 1 h. Following incubation, excess FS was quenched by addition of 1 M Tris-HCl (pH 8.0), and the mixture was incubated for an additional 30 min at room temperature. A portion of fluoresceinated peptide was purified by TLC on silica gel plates using n-butanol/acetic acid/water (4/1/1) as an irrigation solvent. The plates were dried, and fluoresceinated peptides were visualized under UV light. Each peptide band, which runs very close to the origin of application, was excised from the plate and then eluted from the silica gel with 50 mM Tris-HCl, pH 8.0. Each peptide (10 µg) was separately incubated with either E. coli strain E44 or E91 (1 ml culture pellet) at 4°C for 1 h, followed by extensive washing with Tris-HCl, and was subjected to flow cytometry as described above.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Weiser et al. (2) previously showed that expression
of OmpA on E. coli enhances serum survival. The study
used E44 and a TnPhoA mutant of E44, E58, in which
TnPhoA was inserted at the 3' end of the ompA
gene. Strain E58 has been classified as an OmpA deletion mutant by
Western blot analysis using OmpA-C-Ab. However, our studies employing
OmpA-N-Ab showed that truncated OmpA is still expressed on the surface
of E58 (16). Due to these observations we re-examined
bactericidal activity of serum on strain E44 along with a true
OmpA- strain E91, which showed no expression of
OmpA by Western analysis (Table I
). As depicted in Fig. 1, 60% of E44 survived in 40% AHS,
whereas only 25–30% of OmpA- E.
coli E91 survived. Interestingly, E58 showed a similar survival
rate as E91, indicating that expression of truncated OmpA is not
sufficient to confer serum resistance, perhaps due to the disturbed
orientation of OmpA. The HB101 strain, used as a control, barely
survived. In addition, incubation of E44 with heat-inactivated serum
(to denature the complement) resulted in the loss of bactericidal
activity. To further confirm that OmpA expression is important for the
serum resistance capacity of E. coli, E91 was complemented
with the ompA gene in a pUC19 plasmid (E105) and subjected
to AHS bactericidal activity. As a control, E91 containing vector alone
(E111) was used. Introduction of OmpA in E91 restored the serum
resistance capacity of E91 to the level of E44. In contrast, E91 with
only vector was not able to survive in a similar fashion in AHS. In
addition, AHS did not show any bactericidal activity toward E91 in the
presence of 100 nM EGTA, suggesting that the classical complement
pathway might be responsible for the serum killing activity. To further
confirm the role of the classical pathway, AHS-treated E44 was
subjected to flow cytometry using anti-C1q Ab, and the results
showed that a significant amount of C1q, a marker for the classical
complement pathway, had been deposited (Fig. 1B). E91 had
been coated with similar amounts of C1q (data not shown). Taken
together, these data indicate that although the classical pathway is
activated, intact OmpA expression may contribute to the increased serum
resistance of OmpA+ E. coli compared
with OmpA- E. coli.
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To understand the mechanism of serum survival of E.
coli, we next examined whether OmpA+
E. coli binds to any AHS proteins compared with
OmpA- E. coli strains.
OmpA+ E. coli, when incubated with
biotinylated serum proteins, bound to three proteins with apparent
molecular sizes of 65–70, 45–50 kDa, and, to a lower extent, 50–55
kDa (Fig. 2). In contrast,
OmpA- E. coli bound only the 50- to
55-kDa protein, suggesting that OmpA may be specifically responsible
for interacting with the two former serum proteins. However, HB101, a
laboratory E. coli strain that also contains OmpA on its
surface, showed only weak binding to both serum proteins.
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70 kDa) and anti-thrombin (AT;
50 kDa), which are known to bind heparin. Thus, AHS was subjected to
heparin-Sepharose chromatography, and the bound proteins were eluted
with increasing concentrations of NaCl. Each fraction contained several
proteins, as observed by SDS-PAGE, followed by Coomassie staining (gel
not shown), which were then biotinylated and incubated with
OmpA+ and OmpA- E.
coli. No proteins bound to OmpA+ E.
coli from the 0.1-M NaCl fraction, whereas two proteins (70 and
45–50 kDa), similar to the serum proteins, bound to AHS interacted
from the 0.2- to 0.4-M NaCl fractions (Fig. 2B). The binding was
significantly greater in the 0.2-M fraction than in the other
fractions. Since these two proteins bound heparin, we examined whether
OmpA interacts at the heparin binding region of these proteins.
Interestingly, incubation of E44 with AHS in the presence of excess
amounts of heparin did not block the binding of serum proteins to
OmpA+ E. coli (Fig. 2B,
lane 1). These results suggest that the two heparin-binding
proteins interact with OmpA+ E. coli
via an interaction site localized away from the heparin binding
site. C4bp binds directly to OmpA+ E. coli, and protein S does not influence the binding
Since heparin-Sepharose-bound proteins were observed to interact
with OmpA+ E. coli, we examined
whether vitronectin and AT bound to the bacteria. Purified vitronectin
(5 µg) and proteins from the 0.2-M NaCl fraction of the
heparin-Sepharose column were incubated with E44 as described, followed
by immunoblotting with anti-vitronectin Ab. The results show that
vitronectin did not bind E44. and the protein (70-kDa) binding to
E44 from the 0.2-M fraction is not vitronectin (Fig. 3A). Similarly, analysis of
biotinylated AT binding to OmpA+ E.
coli showed no association with the bacteria, suggesting that
heparin binding proteins from serum other than vitronectin and AT are
interacting with E44. C4bp, a key soluble phase classical pathway
regulatory protein, is also known to bind heparin. Therefore, we tested
whether plasma-purified C4bp-PS is able to interact with
OmpA+ and OmpA- E.
coli strains. As shown in Fig. 3B, biotinylated C4bp-PS
binds E44 in significantly greater quantities than it does E91. HB101
bound with far less efficiency to C4bp-PS despite the presence of OmpA.
It seems that the orientation of OmpA on the HB101 strain is different
from that of OmpA present on the E44 strain. In agreement with this
concept, the ompA gene from the HB101 strain can complement
the OmpA- E. coli strain, suggesting
that OmpA might acquire a special three-dimensional structure only on
E44 due to the presence of other molecules. This particular orientation
may enable E. coli K1 to interact with serum proteins and
confers serum resistance to complement.
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B). We next
attempted to determine whether the proteins that bound
OmpA+ E. coli strains from AHS could
be C4bp by preincubating the bacteria with excess amounts of
nonbiotinylated C4bp-PS (25 µg), followed by incubation with
biotinylated AHS. The bound proteins were released and analyzed as
described above. C4bp-PS pretreatment completely abolished the binding
of AHS proteins to OmpA+ E. coli,
suggesting that the protein bound to OmpA+
E. coli is indeed C4bp (Fig. 3C). To further
confirm these results, FACS analysis using anti-C4bp Ab was
performed. As shown in Fig. 4, E44 bound
significantly more C4bp compared with E91. We have also analyzed FH
binding to these strains, and the results are in concurrence with the
results of Western blots showing that E91 bound FH slightly better
than E44.
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-chains without -chain and protein S) was used
in the binding assays. We found that rC4bp binds in a fashion similar
to C4bp-PS (Fig. 3B). The rC4bp also blocked the binding of
AHS proteins to E44 when bacteria were pretreated with excess amounts
of protein (Fig. 3C). These results suggest that protein S
does not influence the binding of rC4bp to OmpA+
E. coli and that the binding region for OmpA on C4bp is
localized within the -chain.
To further characterize the interaction of rC4bp with OmpA, we tested
whether increasing salt concentrations had any influence on the
binding. In our experiments OmpA+ E.
coli suspended in PBS containing 0.1% BSA, 0.1% Tween 20, and
various concentrations of NaCl ranging from 50 to 500 mM were incubated
with biotinylated rC4bp. After 2 h of incubation at 37°C and
washing with PBS, the bound rC4bp was analyzed as described above. As
shown in Fig. 5, binding of rC4bp to
OmpA+ E. coli was not reduced even at
high NaCl concentrations. Thus, the rC4bp-OmpA interaction seems to
primarily rely on hydrophobic contacts, with only minor contributions
from electrostatic forces.
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We determined the region of human C4bp that bound OmpA using rC4bp
and eight mutant rC4bp proteins, each lacking one CCP at a time. The
characterization of these recombinant proteins was described previously
(17). Equal amounts of biotinylated CCP mutant proteins
were incubated with OmpA+ E. coli, and
the bound proteins were analyzed by SDS-PAGE. As shown in Fig. 6A, deletions of CCP3 and CCP8
of rC4bp affected the binding considerably. Particularly rC4bp
CCP3
molecules showed a dramatic reduction of affinity to OmpA. Other
deletions did not significantly affect the binding, suggesting that
CCP3 and CCP8 modules could be important for the interaction with
OmpA+ E. coli.
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A)
(21). To assess the potential role of glycosylation, rC4bp
was deglycosylated using peptide N-glycosidase F and
was incubated with E44. Although the binding was slightly affected by
deglycosylation (Fig. 6B), it was not completely abrogated,
suggesting that N-glycosylation plays a minimal role in the
interaction. In addition, we examined the effect of mAb 104, which
recognizes CCP1, on the binding of rC4bp to OmpA. This Ab has been
shown to inhibit the interaction of C4b to C4bp, and this interaction
was, in turn, mapped to CCP1 to -3 (22). Preincubation of
this Ab with rC4bp partially blocked the binding of rC4bp to
OmpA+ E. coli. Densitometric analysis
of the respective bands indicated a 20% decrease in the binding of
rC4bp to OmpA. The Fab' of mAb 104 showed no effect on the binding
(data not shown), suggesting that the blocking effect of mAb 104 on the
binding, despite its recognition site localized on CCP1, could clearly
be due to steric hindrance. Furthermore, mAb 67, which recognizes CCP4
of C4bp, showed no effect on the binding.
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5-fold excess of
C4b (assuming that each molecule of C4bp-PS binds seven C4b molecules)
showed no effect on the binding of C4bp to OmpA+
E. coli strain (Fig. 6C). These results suggest
that either the OmpA binding region on C4bp does not significantly
overlap with the binding site for C4b or that the affinity of
interaction between OmpA and C4bp is much higher than that for C4b-C4bp
binding, which is known to be in the micromolar range.
Because we found that the N-terminal portion of OmpA interacts with the
CCP3 and/or CCP8 regions of rC4bp, it was of interest to define
potential recognition sites on the C4bp molecule. To this end we
defined solvent-exposed regions on a three-dimensional model structure
for C4bp (Fig. 7A) and used synthetic peptides from both
CCP3 and CCP8 to study their effects on the binding of rC4bp to E44. A
total of eight peptides (four from each CCP) were synthesized, and each
peptide (50 µM) was preincubated with E44 on ice for 30 min, followed
by incubation with biotinylated C4bp. As shown in Fig. 8, peptides CCP3-1, CCP3-3, and CCP3-4
completely abolished the binding of rC4bp, whereas CCP3-2 did not show
any effect on the interaction. Interestingly, except for peptide
CCP8-2, which showed marginal inhibition, all other peptides derived
from CCP8 were ineffective in blocking the binding of rC4bp to E44. To
examine whether the inhibition was due to direct binding of the
peptides to OmpA+ E. coli, the
synthetic peptides were labeled with FS and examined for their binding
capacity by flow cytometry. As shown in Fig. 9, peptides CCP3-1, -3, and -4 bound E44
significantly more compared with E91 (one picture is shown as a
representative for all; Fig. 9, A and B). CCP3-2
peptide and all CCP8 peptides showed very weak or no binding (Fig. 9C), suggesting that the inhibitory effect of CCP3 peptides
that impede the rC4bp-OmpA+ E. coli
interaction is due to direct binding to OmpA.
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To identify the C4bp binding site on OmpA, we first examined the
effect of OmpA-N-Ab on the binding. The anti-Ab significantly
blocked the binding of C4bp-PS to E44, whereas neither
OmpA-C- Ab nor control Ab showed such inhibition
(Fig. 10), suggesting that C4bp-PS
binds to the N-terminal portion of OmpA. To further confirm that
hypothesis, we used two synthetic peptides, N
(Asn27-Gly-Pro-Thr-His-Glu32)
and G
(Gly65-Ser-Val-Glu-Asn69),
from N-terminal loops 1 and 2 of OmpA in the C4bp-PS binding assays.
Our previous studies by computer simulation (23) (Fig. 7B) showed that the N-acetylglucosamine 1,4
N-acetylglucosamine epitopes of HBMEC
glycoproteins interact with these sequences. In addition, these
peptides significantly inhibited the E. coli invasion of
human brain microvascular endothelial cells (16). As
expected, these peptides (200 µM) also blocked the binding of C4bp-PS
to E44, whereas, peptide H
(His19-Asp-Thr-Gly22), a
control peptide sequence present on loop 4, did not have any effect on
the interaction (Fig. 10). These results suggest that C4bp-PS binds
OmpA near or at the N-terminus.
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Our studies to date have suggested that OmpA of E. coli
is responsible for the binding of C4bp, a phenomenon that might
contribute to serum resistance of E. coli. To further
investigate this point, we examined whether inhibition of C4bp
interaction with OmpA+ E. coli
increases the bactericidal activity of serum. First, we verified the
effects of various Abs that block the binding of rC4bp on the
bactericidal activity. OmpA-N-Ab and OmpA-C-Ab were incubated with
OmpA+ E. coli on ice before the
bactericidal assays. OmpA-N-Ab contributed to a significant increase in
the serum killing activity of AHS (80%) compared with either
control Ab or OmpA-C-Ab (Fig. 11A). The killing of
OmpA+ E. coli was significant even at
20% AHS concentration when the bacteria were pretreated with OmpA-N-Ab
(data not shown). At a 20% serum concentration we did not observe
significant bactericidal activity under normal conditions. Since the
binding of OmpA-N-Ab could induce complement fixation via Fc portions
of Ab, we also used Fab' of the Ab to examine whether the enhanced
serum killing is due to blocking of C4bp binding to E. coli.
The serum bactericidal activity of AHS also increased 60% in the
presence of Fab', suggesting that both blocking of C4bp to E.
coli and complement activation by OmpA-N-Ab might be contributing
to the enhanced bactericidal activity of AHS. It is interesting that
mAb 104, which showed a partial inhibitory effect on the binding of
C4bp to OmpA+ E. coli displayed a
10–20% increase in bactericidal activity. mAb 67 showed no effect on
the bacterial killing. Incubation with various Abs alone did not alter
OmpA+ E. coli survival. Surprisingly,
these Abs also had some effect on OmpA- E.
coli. It is possible that these Abs bind
OmpA- E. coli nonspecifically and
activate the classical pathway of complement.
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B). In contrast, the presence of other C4bp
peptides had no effect on bactericidal activity. The C4bp peptides
CCP3-1, CCP3-3, and CCP3-4 showed no enhancement of
anti-bactericidal activity in the absence of AHS. Similarly, OmpA
peptides G and N showed 40% enhancement of serum killing activity
compared with peptide H. The C4bp peptides increased the sensitivity of
OmpA+ E. coli for serum killing at a
concentration of 100 µM, whereas OmpA peptides achieved a similar
effect at 200 µM. All reagents (the blocking Abs or the peptides) had
a profound serum killing capacity when the bacteria were incubated for
longer times with AHS (2 h; data not shown). These results indicate
that blocking the C4bp-OmpA interaction makes
OmpA+ E. coli more susceptible to
serum killing, which supports the idea that E. coli K1
evades serum bactericidal activity mostly by binding to C4bp, an
important fluid phase regulator of the complement system. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-chains. C4bp -chains are also known
to bind C4b (22), heparin (24),
Bordetella pertussis (10),
Streptococcus pyogenes (25), and
serum amyloid P component (26, 27). We demonstrate here
the specificity of the C4bp-OmpA interaction using several different
experimental approaches. First, OmpA-N-Ab and the peptides
corresponding to partial sequences of loops 1 and 2 of OmpA blocked the
interaction. Second, the CCP3- and CCP8-deleted mutants of rC4bp could
not bind OmpA, whereas other deletion mutants did not affect the
binding. Synthetic peptides that represented portions of CCP3 blocked
rC4bp binding to OmpA more efficiently than those of CCP8. This
blocking effect was due to direct binding of the peptides to OmpA,
suggesting that CCP3 contained a key binding site for OmpA.
Interestingly, mAb 104, which is directed against CCP1 of the -chain
of C4bp, did not cause significant inhibition of the rC4bp-OmpA
interaction. Yet, this Ab has been previously shown to fully block
binding between CCP1 of the -chain of C4bp and C4b
(22), N. gonorrhoeae (7), and
streptococcal M proteins (28), suggesting a novel site of
interaction between OmpA and C4bp. The moderate survival rates of
OmpA- E. coli, E91, could be due to
the presence of capsular polysaccharide K1, which is composed of sialic
acid residues. Several reports have shown that the sialic acid
expressed by many pathogenic bacteria binds to FH to avoid complement
attack. Alternatively, binding of small quantities of C4bp from AHS to
E91, as detected in the present study, might be another mechanism for
survival of this strain despite the absence of OmpA. It could be that
other molecules, such as S-fimbriae, also bind C4bp to a
lesser extent. Such an interaction of C4bp with type IV pili has been
demonstrated in N. gonorrhoeae in addition to Por1B/1A,
highlighting the role of more than one molecule binding to C4bp
(7). Our studies showed that heparin treatment of AHS did not affect the C4bp-OmpA interaction, which suggests that the binding sites for heparin (positive amino acids on the interface between CCP1 and CCP2 as well as on CCP2) and OmpA on C4bp might be different. In addition, excess amounts of C4b could not impede the rC4bp-OmpA interaction. Several studies have indicated that a cluster of positively charged amino acids at the interface between CCP1 and CCP2 in C4bp is necessary for binding to C4b (22, 24). In contrast, the binding of rC4bp to OmpA was completely abolished by the deletion of CCP3, whereas no effect was observed by the deletion of CCP2. This is in agreement with the results of heparin and C4b inhibition studies, highlighting the discovery of a new binding site for OmpA on C4bp. This binding activity differs from the interactions of other bacterial proteins, which bind to the CCP1-CCP2 of C4bp (27). Minimal or no effect of salt on the C4bp-OmpA interaction also stands in sharp contrast to the C4b-C4bp binding that was completely abolished at 300 mM NaCl. Previous studies on the binding of N. gonorrhoeae porin 1B and pili also showed that 0.2 M NaCl significantly blocked their interaction with C4bp, highlighting the role of ionic interactions. In contrast, the high affinity binding of C4bp to CCP1 of streptococcal M proteins and to N. gonorrhoeae por1A was proposed to be hydrophobic in nature because it was insensitive to high ionic strength (5, 28). It is likely that the binding of C4bp to OmpA involves hydrophobic contacts and hydrogen bonds, while the C4b-C4bp interaction could be essentially dependent on salt bridges and long range attractive electrostatic forces.
Our data to date suggest that CCP3 is probably the key binding site for OmpA. The role of CCP8 is unclear, since we found that the mutant lacking CCP8 showed decreased binding ability for OmpA, but no peptides from CCP8 were able to inhibit the interaction. Thus, the role of CCP8 requires further investigations, which we will undertake in the future. It is interesting to note that both CCPs contain N-glycosylation sites. However, the slightly reduced binding of deglycosylated C4bp to OmpA indicates that the interaction is essentially mediated by amino acids. Collectively, our data strongly suggest that CCP3 is crucial to the interaction, because three synthetic peptides from CCP3 that cluster closely significantly blocked the interaction of C4bp with OmpA. In addition, the direct binding of CCP3-1, -3, and -4 peptides to OmpA+ E. coli compared with CCP3-2 and CCP8 peptides supports the idea that the CCP3 module forms the key binding site for OmpA.
A compelling observation of this study is the ability of CCP3-1, CCP3-3, and CCP3-4 peptides to enhance serum killing activity, which is in good agreement with the finding that these peptides efficiently blocked the binding of rC4bp to E. coli. Interestingly, none of the CCP8 peptides showed any effect on AHS bactericidal activity, suggesting that OmpA may not or may only minimally interact with this module. OmpA-N-Ab is the most potent Ab in enhancing the AHS bactericidal activity. In our earlier studies we developed Abs to OmpA, which recognize only the C-terminal portion of OmpA, by immunizing animals with UV-killed whole bacteria. Similarly, anti-OmpA Abs raised by several other investigators also preferentially recognize the C-terminal portion, indicating that the C-terminal region of OmpA might be more immunogenic than the N-terminus. In addition, the inability of OmpA-C-Ab and control Abs to increase serum sensitivity could be due to inefficient binding of these Abs to bacteria to initialize the classical pathway. Thus, the N-terminal portion of OmpA, at least for E. coli K1 could be a suitable target for the construction of an effective vaccine that nullifies the binding of C4bp to permit complement attack.
In conclusion, our studies have demonstrated that OmpA of E. coli contributes to serum resistance by avoiding complement attack via binding to C4bp. The N-terminal portions of OmpA and CCP3 of C4bp are involved in the interaction. Neither high ionic strength nor C4b/heparin could block the binding of C4bp to OmpA, highlighting a novel site of interaction at the surface of C4bp molecules. OmpA-N-Ab and synthetic peptides mimicking CCP3 regions enhance the serum sensitivity of E. coli, suggesting that these regions could be potential targets for novel therapeutic approaches.
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2 Address correspondence and reprint requests to Dr. Nemani V. Prasadarao, Division of Infectious Diseases, MS #51, Childrens Hospital Los Angeles, 4650 Sunset Boulevard, Los Angeles, CA 90027. E-mail address: pnemani{at}chla.usc.edu
3 Abbreviations used in this paper: OmpA, outer membrane protein A; AHS, adult human serum; AT, anti-thrombin; C4bp, C4b binding protein; C4bp-PS, C4bp complex with protein S; CCP, complement control protein (domain); FH, factor H; FS, fluorescein amine.
Received for publication April 3, 2002. Accepted for publication October 4, 2002.
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