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J. Biol. Chem., Vol. 277, Issue 37, 34568-34572, September 13, 2002
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From the
Received for publication, January 25, 2002
Curli are surface organelles of Escherichia
coli. These fibrous proteins, formed by polymerization of a
15-kDa subunit, are expressed by E. coli strains associated
with severe infections in humans. A remarkable property of curli is
their ability to interact with a wide range of human proteins,
interactions that contribute to the enhanced virulence of
curli-expressing E. coli. To define the protein-binding
region(s) of curli, we investigated the binding properties of
overlapping synthetic peptides covering the curli subunit. Two
peptides, one covering a 24-amino acid residue sequence in the
NH2-terminal half of the subunit (NNS24) and one
corresponding to the 26 COOH-terminal residues (VDQ26), were found
to bind a number of human proteins. Physiochemical analysis revealed
that NNS24 adopts a thermally stable Some strains of Escherichia coli and
Salmonella express fibrous surface proteins that aggregate
into matrix-like structures attached to the bacterial surface. In
E. coli these fibrous proteins are called curli (1) and in
Salmonella, thin aggregative fimbriae (2). They are closely
related structurally (1, 3), genetically (4, 5), and functionally (6,
7). Genes encoding a homologous structure were recently described also
in Shigella spp (8). Curli in E. coli are
composed of a major 15-kDa subunit protein, CsgA (4), and a minor
component, CsgB (9), acting as a nucleator in the formation of
insoluble curli aggregates at the bacterial surface (10). Temperature
and various stress conditions, such as low osmolarity and starvation,
regulate the expression of curli (11). A recent report describes the
complex network of regulatory factors that control the production of
both curli and thin aggregative fimbriae (12). Among clinical E. coli isolates, curli are expressed by most
enterohemorrhagic, enterotoxigenic, and sepsis strains, whereas
enteroinvasive and enteropathogenic strains do not express curli (4,
13). This difference in expression suggests a role for curli in
E. coli pathogenicity, a notion that is further supported by
findings that curli induce proinflammatory cytokines and that anti-CsgA antibodies are found in serum samples from patients with E. coli sepsis (14). Recent studies have also shown that curliated
E. coli when injected into mice activate the
NO/NOS2 arm of the innate immune system, resulting in blood
pressure fall (14). Finally, curliated E. coli, but not a
non-curliated mutant strain, has the ability to enter eukaryotic cells
(15).
A noteworthy property of curli is their broad protein-binding capacity.
A large number of human proteins have been shown to interact with
curli, including many plasma proteins and
MHC1 class I antigens (4, 7,
13, 16). Available data indicate that the CsgA subunit is responsible
for most of these interactions, which in some cases have been shown to
contribute to E. coli virulence. Thus, the
components of the contact system bind and assemble on curli fibers, and
as a consequence the proinflammatory peptide bradykinin is generated
and a hypocoagulative state is induced (17). Moreover, blocking of
contact system activation prevents curliated bacteria from inducing
severe lung lesions in rats (18). The connection between bacterial
virulence and the protein-binding properties of curli prompted us to
investigate whether the binding and functional properties of curli
could be mapped more precisely. In the present study we identify two
regions in the CsgA subunit that have broad protein binding activity
and the capacity to activate the contact system.
Peptide Synthesis--
A series of overlapping peptides (see
Fig. 2A), based on the sequence of the curli subunit CsgA,
were synthesized at Innovagen, Lund, Sweden. HPLC and mass spectrometry
were used to control the quality of the peptides.
Antibodies and Proteins--
Anti-fibrinogen antibodies were
from DAKO A/S, Glostrup, Denmark, and antibodies against high molecular
weight kininogen (H-kininogen) were raised in sheep as described (19).
Human fibrinogen, fibronectin, and serum albumin (HSA) were purchased
from Sigma. The preparations of human MHC class I antigen,
Slot Binding Experiments--
Synthetic peptides were dissolved
in phosphate-buffered saline (PBS; 0.15 M NaCl, 0.06 M phosphate, pH 7.2) and applied to PVDF membranes
(Immobilon, Millipore, Bedford, MA) using a Milliblot-D system
(Millipore). Membranes were blocked at room temperature for 1 h
with PBSAT (PBS + 0.02% sodium azide, 0.25% bovine serum albumin + 0.25% Tween 20). Various proteins were labeled with 125I
using the Bolton-Hunter reagent (20). Membranes were incubated with
radiolabeled protein for 3 h at room temperature, washed in PBSAT,
dried, and exposed to Kodak X-Omat AR film using regular intensifying
screens (Kodak, Rochester, NY).
Bacterial Binding Assay--
Binding of radiolabeled proteins to
intact bacteria was done as described (7). Basically, 2 × 108 bacteria were resuspended in PBS containing 0.1% Tween
20. The bacteria were incubated with 125I-labeled protein
(50-100 ng) in a total volume of 250 µl in polypropylene tubes at
20 °C for 1 h. After incubation, the bacteria were washed once
in 2 ml of PBS containing 0.1% Tween 20, and the radioactivity associated with the pellet was determined after centrifugation. Binding was expressed as the percentage of the added radioactivity.
Plasma Absorption Experiments--
The NNS24 and VDQ26 peptides
(10 mg) were coupled separately to 1 ml of CNBr-activated Sepharose 4B
(Amersham Biosciences) according to the manufacturer's instructions.
To suspensions of 200 µl of NNS24- or VDQ26-coupled Sepharose, 0.5 ml
of citrated fresh human plasma was added and incubated overnight at
4 °C. After incubation the Sepharose beads were washed in PBS, and
bound proteins were eluted with 100 µl of 0.1 M
glycine-HCl, pH 2.0. As a control, plasma was simultaneously absorbed
with glycine-Sepharose. After elution, the pH was adjusted to 7.4 by
adding 1 M Tris to the samples.
SDS-Polyacrylamide Gel Electrophoresis, Western Blotting, and
Immunoprinting--
Proteins were separated by 10% polyacrylamide gel
electrophoresis (PAGE) in the presence of 1% SDS (21). After
electrophoresis proteins were transferred onto nitrocellulose membranes
for 30 min at 100 mA (22). The membranes were blocked with PBS
containing 5% skim milk and 0.05% Tween 20, pH 7.4 (blocking buffer).
Immunoprinting of the transferred proteins was done as described (23).
A rabbit antibody against fibrinogen and a sheep antibody against
H-kininogen, diluted 1:1,000 and 1:5,000, respectively, in the blocking
buffer, were used. Bound antibodies were detected using
peroxidase-conjugated secondary antibodies against rabbit or sheep IgG
(Sigma) diluted 1:3,000 followed by a chemiluminescence detection
method as described by Nesbitt and Horton (24).
Diffusion-ordered Spectroscopy (DOSY)--
DOSY experiments on
NNS24 (50 µM in D2O) were carried out on a
Varian INOVA 500 (500 MHz) NMR spectrometer at 27 °C using bipolar
paired stimulation echo (BPPSTE) pulse sequence (25, 26). The duration
of pulsed field gradients was 1 ms; the amplitudes were increased in 11 steps from 18.59 gauss/cm to 74.35 gauss/cm, and 8192 transients (with
8 dummy scans) were acquired for each increment. Data analysis was
carried out using DOSY processing software (27).
Secondary Structure Analysis of the NNS24 Peptide--
Circular
dichroism (CD) spectra were recorded on a Jasco J-720
spectropolarimeter equipped with a thermostatted cell holder. The
spectra were recorded in the far-UV region (260-190 nm) in a cell with
a path length of 0.1 cm. The experiments were recorded in
H2O at 20 °C at concentrations of 50, 24, and 12 µM. Spectra were acquired at a scan speed of 20 nm/min
and a 2-s response time. Four scans were accumulated for each
experiment. The solvent dichroic absorbance was subtracted using Jasco
software. The thermal unfolding curve was run at a single wavelength
(216 nm) characteristic for Analysis of Bradykinin Release in Human Plasma following Addition
of Curli-derived Peptides--
Peptides NNS24, ALQ26, GFG24, and VDQ26
were added separately to human citrated plasma from healthy
individuals. The peptides were dissolved in 100%
Me2SO and diluted 10 times in water. 1, 10, or 100 µg of the peptides in 100 µl of Me2SO/water was added to 300 µl of plasma. The samples were incubated under rotation for 15 min at room temperature and analyzed for bradykinin content in an
enzyme-linked immunosorbent assay (Markit-M-Bradykinin, Dainippon
Pharmaceutical Co., Ltd., Osaka, Japan) according to the
manufacturer's instructions. The bradykinin concentration was
determined from the enzyme activity of peroxidase-labeled BK bound to
the anti-BK antibodies; detection limit is 4.9 pg/well. A standard
curve was prepared with five BK concentrations (4.9, 19.6, 78, and 313;
1250 pg/well). Samples were analyzed in duplicate and the results
expressed as ng/ml plasma.
A number of different human proteins have been shown to interact
with curli. In most of these experiments, radiolabeled proteins were
used in binding experiments with curli-expressing bacteria, whereas
isogenic curli-deficient mutants served as controls (4, 7, 13, 16). In
some cases, purified curli organelles and the CsgA subunit alone were
demonstrated to bind the tested proteins (4, 7, 16). In the present
study, initial experiments were performed to confirm and expand these
previous data. 125I-labeled human fibronectin, fibrinogen,
plasminogen, t-PA, H-kininogen, factor XII, MHC class I antigens,
To further investigate the binding properties of curli, we synthesized
a set of overlapping peptides covering the entire CsgA curli subunit.
The peptides (Fig. 2A) were
applied to filters, which were probed with the various radiolabeled
proteins tested for binding to intact bacteria (see Fig. 1). Fig.
2B shows the results obtained with 125I-labeled
fibrinogen. Distinct dose-dependent binding is seen to
peptides NNS24, KQF22, and VDQ26. The specificity was tested by
incubating the filter with an excess of unlabeled fibrinogen. This
completely inhibited the binding of radiolabeled fibrinogen to the
peptides (data not shown). Apart from HSA, which did not bind to any of
the peptides, and factor XII, which gave weak signals with peptides
KQF22 and VDQ26 but a strong signal with peptide NNS24, the other
radiolabeled proteins (fibronectin, plasminogen, t-PA, H-kininogen, MHC
class I antigens, and
Identification of Two Protein-binding and Functional Regions
of Curli, a Surface Organelle and Virulence Determinant of
Escherichia coli*
§,,,
, and
Section for Molecular Pathogenesis,
Department of Cell and Molecular Biology, Lund University,
SE-221 84 Lund, Sweden, ¶ Biovitrum, SE-112 87 Stockholm,
Sweden, and the Department of Medical Microbiology,
Dermatology, and Infection, Lund University Hospital, SE-221 85 Lund,
Sweden
ABSTRACT
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EXPERIMENTAL PROCEDURES
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-structure, and in solution the
peptide forms soluble multimers, predominantly octamers. Intact curli
are known to activate the proinflammatory and procoagulant contact
system, and when added to human plasma, the NNS24 and VDQ26 peptides
induced the release of the potent vasoactive peptide bradykinin. The
results map important curli functions to the regions corresponding to
the NNS24 and VDQ26 sequences.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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EXPERIMENTAL PROCEDURES
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2-microglobulin, factor XII, H-kininogen, plasminogen,
and tissue plasminogen activator (t-PA) used have been described
previously (7, 16, 17).
-sheet structures at a concentration of
50 µM. The temperature was increased from 5 to 90 °C
at a scan rate of 50 °C/h.
RESULTS AND DISCUSSION
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RESULTS AND DISCUSSION
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2-microglobulin, and HSA, were tested for binding to
curli-expressing E. coli and an isogenic curli-deficient
mutant. These results (Fig. 1) show that
all proteins except HSA interact with curli-expressing bacteria,
whereas none of the tested proteins had affinity for the mutant.
View larger version (23K):
[in a new window]
Fig. 1.
Binding of various radiolabeled human
proteins to curliated and non-curliated E. coli
bacteria. The binding of labeled protein to the bacteria was
expressed as percentage of the total amount of added radiolabeled
protein. The bars indicate mean values from at least two
experiments.
2-microglobulin) tested gave rise
to the same binding pattern as fibrinogen (data not shown). These
results suggest two protein-binding sites in the CsgA subunit, one in
the NH2-terminal region and one in the COOH-terminal
region.
View larger version (40K):
[in a new window]
Fig. 2.
Binding of fibrinogen to synthetic peptides
covering the CsgA curli subunit. Various amounts, 0.5-5 µg as
indicated, of synthetic peptides were applied to a PVDF membrane. The
membrane was blocked with bovine serum albumin and Tween 20 and probed
with 5 × 105 cpm of 125I-labeled
fibrinogen (20-50 ng protein). After 3 h of incubation the
membrane was washed extensively in PBSAT and exposed to
autoradiography. A, the sequences of the seven synthetic
overlapping peptides with their overlapping areas indicated in
bold. The peptides are designated after their three
NH2-terminal amino acid residues and the number of residues
they contain. B, a PVDF membrane, containing the synthetic
peptides depicted in A, probed with 125I-labeled
fibrinogen.
Physiochemical properties of NNS24 were investigated by circular
dichroism (CD) analysis and NMR. CD analysis of the peptide revealed
that NNS24 is predominantly in a -sheet conformation as indicated by
a single maximum and minimum at 197 and 216 nm, respectively
(Fig. 3A). The intensity of
the CD signal at 216 nm is correlated directly to the concentration of
the peptide indicating that the -sheet content is invariant within
this concentration range (12-50 µM). This conformation
is thermally relatively stable against thermal denaturation with a
melting point (Tm) of ~66 °C (Fig.
3B) compared with the average Tm of
proteins, which is 63 °C (28). These data demonstrate that the NNS24
peptide adopts a stable and folded conformation in solution. Amyloid
fibrils characteristically are composed of proteins rich in -sheets, and they bind Congo red. Also curli fibers are stained with this reagent (29). Interestingly, NNS24 and the two most COOH-terminal peptides, KQF22 and VDQ26, but not the other CsgA-derived peptides (see
Fig. 2A), were distinctly stained red when applied to PVDF membranes and incubated with Congo red (data not shown). These observations suggest also that peptides KQF22 and VDQ26 form -sheets and that the protein-binding regions of curli have this conformation in
common. CD analysis of these peptides could not be performed, as they
are soluble only in Me2SO. Although much less pronounced than peptides KQF22 and VDQ26, the NNS24 peptide has a tendency to
precipitate, suggesting that it could form soluble multimers. To study
the multimerization properties of NNS24, diffusion-ordered NMR
spectroscopy experiments were performed, resulting in a diffusion coefficient (D value) of 1.3 × 10
1 (± 0.034) m2/s in D2O at 2 °C, corresponding to
a molecular mass of ~20 kDa. The molecular mass of NNS24 determined
from its amino acid sequence is 2480.1 Da, demonstrating that the
peptide aggregates and forms multimers, predominantly octamers, in
solution. The multimerization of the protein-binding peptides of curli
indicates that these regions could also participate in the
polymerization of CsgA subunits and, together with the nucleator
protein CsgB (29), contribute to the formation of curli organelles.
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An important question raised by the broad protein-binding capacity of
curli is to what extent the multitude of possible protein interactions
actually takes place in the complex mixture of proteins surrounding
bacteria in vivo. Previous experiments (7, 17) in
which curli-expressing bacteria were incubated with human plasma showed
that a large number of different plasma proteins are indeed simultaneously bound to the bacterial surface through interactions with
curli. For instance, incubation with human plasma will result in the
binding and assembly of components of the contact system but also in
the binding of several other plasma proteins, such as plasminogen and
fibrinogen, to curliated bacteria (7, 17). To test whether the NNS24
and VDQ26 peptides, analogous to intact curli, could interact with many
different proteins when exposed to an excess of proteins in a complex
protein mixture, the peptides were coupled to Sepharose. Following
incubation with human plasma and extensive washing, proteins bound to
NNS24- or VDQ26-Sepharose were eluted by low pH and subjected to
SDS-PAGE and Western blot analysis. As shown in Fig.
4, A and B, STAIN,
lanes 2, several proteins were bound and eluted from NNS24-
and VDQ26-Sepharose. Compared with plasma (lanes 1) some
bands were accentuated, whereas other, such as the HSA bands around 66 kDa, were weaker or missing in the eluates (lanes 2). In
contrast, no proteins were absorbed from human plasma and eluted from
glycine-Sepharose, the negative control (lanes 3). These
results demonstrate a broad but selective binding of human plasma
proteins by the two curli-derived peptides.
|
, 
-chains of fibrinogen,
H-kininogen (HK), and H-kininogen degradation products are
also indicated. Low molecular weight kininogen (LK) is
derived by alternative splicing of the gene encoding H-kininogen.
Consequently, polyclonal antibodies to H-kininogen also identify low
molecular weight kininogen in plasma.
Sepsis and septic shock caused by Gram-negative bacteria is a serious and important clinical condition causing more than 100,000 deaths annually in the United States alone (30). Several observations have indicated that curli play a role in the pathogenesis of sepsis. First, E. coli strains isolated from patients with sepsis frequently express curli (13), and antibodies to CsgA are found in these patients (14). Second, when curliated bacteria are injected intravenously into mice or rats, they induce bleeding disorders, a fall in blood pressure, and lung lesions (17, 18, 31), symptoms that are common in cases of severe sepsis. The hypocoagulatory state of human plasma following exposure to curli is due to the binding of factor XII and fibrinogen to curli (17), and as shown in Fig. 4, A and B, BLOT I, fibrinogen is absorbed from plasma by NNS24- and VDQ26-Speharose. The contact system consists of the three serine proteinases, factor XI, factor XII, and plasma prekallikrein, and the non-enzymatic cofactor H-kininogen. When the system is assembled and activated, H-kininogen is cleaved by activated kallikrein to generate bradykinin, a highly potent proinflammatory peptide. On bacterial surfaces contact activation will result in the cleavage of H-kininogen into two major 45- and 65-kDa fragments (13), and in sepsis low levels of factor XII and intact H-kininogen correlate with a fatal outcome (32). It is therefore noteworthy that H-kininogen is absorbed from plasma by the Sepharose-coupled curli peptides and cleaved into fragments of 45 and 65 kDa (Fig. 4, A and B, BLOT II, lanes 2). These results suggest that the contact factors are bound and assembled on NNS24- and VDQ26-Sepharose and that the system is activated to generate bradykinin. Bradykinin is a primary mediator of inflammatory processes and induces pain, vasodilatation, and increased vascular permeability due to the local production of prostaglandins and nitric oxide (33). Consequently, a massive release of bradykinin by curliated bacteria could help to explain the hypovolemic hypotension seen in septic shock. To investigate whether the cleavage of H-kininogen shown in Fig. 4, A and B, BLOT II, actually results in the generation of bradykinin, peptides NNS24, GFG24, and VDQ26 were added separately to fresh human plasma. 1, 10, or 100 µg of the peptides in 100 µl of 10% Me2SO were added to 300 µl of plasma, and following incubation, the amount of bradykinin was determined by enzyme-linked immunosorbent assay. Neither the GFG24 peptide nor 10% Me2SO alone generated bradykinin above background level (<0.1 ng/ml plasma), whereas even 1 µg of NNS24 induced a significant increase (0.71 ± 0.03 ng/ml plasma). When 10 or 100 µg of NNS24 was added, this resulted in a massive generation of bradykinin (12.8 ± 3.4 and 57.0 ± 13.1 ng/ml plasma, respectively). The VDQ26 peptide did not induce bradykinin release at 1 or 10 µg, but at 100 µg the amount was clearly above background level (2.46 ± 0.15 ng/ml plasma). These results show that the fragmentation of H-kininogen induced by NNS24 and VDQ26 generates bradykinin.
In conclusion, the present work has identified and characterized two
protein-binding regions of curli, which are also responsible for the
activation of the pro-inflammatory contact system. The important role
played by curli in virulence and its unique protein-binding properties
should stimulate future investigations of the multipotent structural
entities included in the NNS24 and VDQ26 peptides.
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ACKNOWLEDGEMENT |
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Ingbritt Gustafson is acknowledged for excellent technical assistance.
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Addendum |
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After the initial submission of this manuscript, Chapman et al. (34) reported that purified CsgA subunits polymerize into amyloid curli fibers. It is likely that the NH2- and COOH-terminal CsgA regions defined in this study participate in this assembly process.
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FOOTNOTES |
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* This work was supported by grants from the Swedish Research Council (Projects 7480, 13413, and 14272), the Crafoord, Bergvall, Kock, Nilson, and Österlund Foundations, the Royal Physiografic Society in Lund, the Medical Faculty, Lund University, and Hansa Medical AB.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Dept. of Clinical Immunology, Göteborg University, Guldhedsgatan 10A, SE-413 46 Göteborg, Sweden. Tel.: 46-31-342-4895; Fax: 46-31-342-4621; E-mail: arne.olsen@immuno.gu.se.
Published, JBC Papers in Press, July 3, 2002, DOI 10.1074/jbc.M206353200
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ABBREVIATIONS |
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The abbreviations used are: MHC, major histocompatibility complex; BK, bradykinin; CsgA, curli subunit protein A; CsgB, curli subunit protein B; DOSY, diffusion-ordered spectroscopy; H-kininogen, high molecular weight kininogen; HPLC, high-pressure liquid chromatography; HSA, human serum albumin; PBS, phosphate-buffered saline; PVDF, polyvinylidene difluoride; t-PA, tissue plasminogen activator.
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