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Am J Physiol Lung Cell Mol Physiol 282: L751–L756, 2002;
10.1152/ajplung.00383.2001.
Identification of Pseudomonas aeruginosa flagellin
as an adhesin for Muc1 mucin
ERIK P. LILLEHOJ, BEOM T. KIM, AND K. CHUL KIM
Department of Pharmaceutical Sciences, University of Maryland
School of Pharmacy, Baltimore, Maryland 21201
Received 28 September 2001; accepted in final form 23 October 2001
bacteria; glycoprotein; cystic fibrosis; airway; infection
is a gram-negative opportunistic pathogen associated with pneumonia in immunocompromised patients and chronic lung disease,
such as cystic fibrosis. The molecular interactions of P.
aeruginosa with respiratory epithelial cells is complex
and likely involves multiple types of ligand-receptor
contacts (reviewed in Refs. 1, 17, 36, 38, 39). Several P.
aeruginosa adhesins have been described, including
pili (9, 16, 22, 42), flagella (12, 39), and lipopolysaccharide (LPS) (16). Some of these studies have emphasized
the importance of epithelial cell glycosphingolipids,
such as asialoGM1, for binding to bacterial pili (41). In
addition, specific binding of flagellar proteins to secreted respiratory mucins appears to be physiologically
relevant to P. aeruginosa colonization of the lung (3, 8).
P. aeruginosa adherence to secreted mucins prompted
us to investigate whether cell-associated mucins also
serve as receptors for bacterial adhesion. Five genes
PSEUDOMONAS AERUGINOSA
Address for reprint requests and other correspondence: K. C. Kim, Dept.
of Pharmaceutical Sciences, School of Pharmacy, University of Maryland,
20 N. Pine St., Baltimore, MD 21201.
http://www.ajplung.org
encoding membrane-tethered mucins have been identified (15, 20, 30, 46–48), among which MUC1 mucin is
abundantly expressed by respiratory epithelial cells,
such that its strategic location in the upper airways
permits interaction with inspired microorganisms. Indeed, we demonstrated previously that P. aeruginosa
specifically binds to hamster Muc1 mucin, judging
from in vitro saturable binding kinetics and competitive inhibition by the homologous ligand (24). In the
study reported here, we examined the molecular nature of the P. aeruginosa adhesin and identified flagellin, the major flagellar protein, as responsible for binding to Muc1 mucin.
METHODS
Materials. All materials were purchased from Sigma (St.
Louis, MO) unless stated otherwise. Rabbit antiserum to
PAK flagellin was kindly provided by Dr. Dan Wozniak
(Wake Forest University, Winston-Salem, NC). Rabbit antiserum to PAK pilin was kindly provided by Dr. Randy Irvin
(University of Alberta, Edmonton, AB, Canada). Mouse
monoclonal antibody (M4E8) to PAK LPS (serotype 6) was
kindly supplied by Dr. Joseph Lam (University of Guelph,
Guelph, ON, Canada).
P. aeruginosa. PAK is a wild-type, nonmucoid, piliated,
and motile strain. Its isogenic mutant PAK/NP is a derivative
of PAK in which the pilA gene encoding the pilin protein is
replaced by homologous recombination with a mutant gene
interrupted by a tetracycline resistance cassette (41). Strains
PAK/fliC (12) and PAK/fliD (3) are nonmotile derivatives in
which the fliC gene encoding flagellin or the fliD gene encoding the flagellar cap protein is replaced with homologous
genes interrupted by gentamicin resistance cassettes. PAK,
PAK/NP, and PAK/fliC were kindly provided by Dr. Alice
Prince (Columbia University, New York, NY). PAK/fliD was
a generous gift from Dr. Reuben Ramphal (University of
Florida, Gainesville, FL).
CHO-Muc1 and CHO-X cells. The hamster Muc1 expression plasmid pcDNA/Muc1 was constructed by insertion of a
full-length hamster Muc1 cDNA (34) into the pcDNA3 vector
(Invitrogen, San Diego, CA) (24). The pcDNA/Muc1 plasmid
and pcDNA3 vector alone were transfected separately into
Chinese hamster ovary (CHO) cells (CCL61, American Type
Culture Collection, Manassas, VA) using the cationic liposome Lipofectin (Life Technologies, Rockville, MD), and sta-
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1040-0605/02 $5.00 Copyright © 2002 the American Physiological Society
L751
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Lillehoj, Erik P., Beom T. Kim, and K. Chul Kim.
Identification of Pseudomonas aeruginosa flagellin as an adhesin for Muc1 mucin. Am J Physiol Lung Cell Mol Physiol
282: L751–L756, 2002; 10.1152/ajplung.00383.2001.—We reported previously that Muc1 mucin on the epithelial cell
surface is an adhesion site for Pseudomonas aeruginosa
(Lillehoj EP, Hyun SW, Kim BT, Zhang XG, Lee DI, Rowland
S, and Kim KC. Am J Physiol Lung Cell Mol Physiol 280:
L181–L187, 2001). The present study was designed to identify the adhesin(s) responsible for bacterial binding to Muc1
mucin using genetic and biochemical approaches. Chinese
hamster ovary (CHO) cells stably transfected with a Muc1
cDNA (CHO-Muc1) or empty plasmid (CHO-X) were compared for adhesion of P. aeruginosa strain PAK. Our results
showed that 1) wild-type PAK and isogenic mutant strains
lacking pili (PAK/NP) or flagella cap protein (PAK/fliD) demonstrated significantly increased binding to CHO-Muc1 cells,
whereas flagellin-deficient (PAK/fliC) bacteria were no more
adherent to CHO-Muc1 than CHO-X cells, and 2) P. aeruginosa adhesion was blocked by pretreatment of bacteria with
antibody to flagellin or pretreatment of CHO-Muc1 cells with
purified flagellin. We conclude that flagellin is an adhesin of
P. aeruginosa responsible for its binding to Muc1 mucin on
the epithelial cell surface.
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FLAGELLIN BINDS TO MUC1 MUCIN
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nescence reagent (Amersham Pharmacia Biotech, Arlington
Heights, IL).
Statistical analyses. The number of P. aeruginosa adhered
to CHO cells was calculated in the following order: 1) determination of the number of bacteria bound to cells in each well
by converting disintegrations per minute to actual number of
bacteria, 2) determination of the number of epithelial cells at
the end of the assay using a separate parallel group not
exposed to bacteria, and 3) assessment of the significance of
the difference between groups using Student’s t-test for unpaired samples or analysis of variance. Differences were
considered significant at P ⬍ 0.05.
RESULTS
P. aeruginosa lacking flagellin exhibit diminished
adherence to CHO-Muc1 cells. Prior studies have documented P. aeruginosa pili, flagella, and cap protein in
adherence to epithelial cell glycolipids and secreted
mucins (3, 12, 22, 39, 41, 42). To examine the possible
involvement of these bacterial components in binding
to Muc1 mucin, we compared adherence of wild-type
PAK with isogenic mutants devoid of pilin (PAK/NP),
flagellin (PAK/fliC), or cap protein (PAK/fliD) to
CHO-X and CHO-Muc1 cells. As shown in Fig. 1, binding of wild-type PAK, PAK/NP, and PAK/fliD to CHOMuc1 cells was nearly twice that to CHO-X cells. In
contrast, binding of flagellin-defective PAK/fliC to the
two cells was equivalent. Because PAK/fliC and PAK/
fliD are nonmotile, the lack of adherence of PAK/fliC to
CHO-Muc1 cells due to its lack of motility was ruled
out. Thus P. aeruginosa flagellin appeared to constitute the molecular adhesin binding to Muc1 mucin.
Fig. 1. Adherence of Pseudomonas aeruginosa to Chinese hamster
ovary (CHO) cells transfected with Muc1 cDNA (CHO-Muc1) requires flagellin. CHO cells transfected with empty plasmid (CHO-X)
or CHO-Muc1 cells were incubated with 35S-labeled P. aeruginosa
wild-type (PAK/WT) or isogenic mutants devoid of pilin (PAK/NP),
flagellin (PAK/fliC), or flagellar cap protein (PAK/fliD), and the
numbers of bacteria adhered were measured. Open bars, bacteria
binding to CHO-X cells; solid bars, bacteria binding to CHO-Muc1
cells. Each data point represents mean ⫾ SE of 3 (PAK/fliD) or 6
(PAK/WT, PAK/NP, and PAK/fliC) wells. Number of bacteria bound
to CHO-X cells (control) was set to 100% for the purpose of comparison. Absolute numbers of bacteria bound per cell were as follows:
2.69 ⫾ 0.06 CHO-X ⫹ PAK/WT, 5.14 ⫾ 0.18 CHO-Muc1 ⫹ PAK/WT,
0.64 ⫾ 0.05 CHO-X ⫹ PAK/NP, 1.11 ⫾ 0.08 CHO-Muc1 ⫹ PAK/NP,
0.23 ⫾ 0.01 CHO-X ⫹ PAK/fliC, 0.22 ⫾ 0.01 CHO-Muc1 ⫹ PAK/fliC,
0.18 ⫾ 0.02 CHO-X ⫹ PAK/fliD, and 0.37 ⫾ 0.06 CHO-Muc1 ⫹
PAK/fliD. *Significantly different (P ⬍ 0.05).
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ble clones were identified and characterized. A clone expressing hamster Muc1 mucin was referred to as CHO-Muc1 and
a clone derived from the vector alone as CHO-X. The latter
was used as a negative control in the P. aeruginosa binding
assay. Both cell lines were maintained in Ham’s F-12-Dulbecco’s modified Eagle’s medium containing 200 ␮g/ml G418,
5% fetal bovine serum, 100 U/ml penicillin, and 100 ␮g/ml
streptomycin (all from Life Technologies).
P. aeruginosa binding assay. Adhesion of P. aeruginosa to
transfected CHO cells was performed according to the
method described by Rostand and Esko (40) and modified by
Lillehoj et al. (24). Briefly, cells were cultured for 20–24 h in
24-well microplates in an inoculum of 2 ⫻ 105 cells per well.
The monolayer was washed twice with phosphate-buffered
saline (PBS), pH 7.2, fixed for 10 min with 2.5% (vol/vol)
glutaraldehyde in PBS at room temperature, and washed
three times with PBS. Bacteria were metabolically radiolabeled in sulfate-free M9 medium containing 10 ␮Ci/ml
Na235SO4 (1,175 Ci/mmol, 100 mCi/ml, carrier-free; American Radiolabeled Chemicals, St. Louis, MO) for 16 h at 37°C,
washed twice, resuspended in PBS containing 2 mg/ml glucose, and quantified photometrically (0.50 optical density at
600 nm ⫽ 5 ⫻ 108 cells). Fixed CHO cells were incubated
with 2 ⫻ 107 P. aeruginosa/well in 0.5 ml for 40 min at 37°C
and washed three times with PBS, adhered bacteria were
lysed with 1.0 ml of 2% sodium dodecyl sulfate (SDS), and
radioactivity was measured by liquid scintillation counting.
In some experiments, bacteria were pretreated for 30 min at
37°C with PAK flagellin antiserum or cells were pretreated
with purified flagellin as potential binding inhibitors.
Purification of flagellin. P. aeruginosa PAK flagellin was
purified according to the procedure of Allison et al. (2).
Briefly, overnight liquid cultures in mineral salts medium
[7.0 g K2HPO4, 3.0 g KH2PO4, 1.0 g (NH4)2SO4, 50 mg
MgSO4 䡠 7H2O, 2.5 mg FeCl3 䡠 H2O, 7.4 mg L-methionine, and
4.0 g sodium succinate per liter, pH 7.0] were centrifuged at
5,000 g for 15 min at 4°C, and the bacterial pellet was
resuspended in 10 mM potassium phosphate, pH 7.0, and
blended at a low speed in a commercial blender (Black and
Decker, Towson, MD) for 30 s at room temperature to shear
the flagella. The suspension was centrifuged at 16,000 g for
15 min at 4°C to remove bacteria, and the resulting supernatant was centrifuged at 100,000 g in a centrifuge (model
L8-70, Beckman, Duarte, CA) with a 42.1 rotor for 3 h at 4°C.
In some experiments, flagellin was further purified by sequential acid pH dissociation of flagella, centrifugation at
100,000 g for 1 h at 4°C, and neutral pH reassociation of the
supernatant as described elsewhere (7, 43). Protein concentrations were determined by the Coomassie blue technique of
Bradford (6) with crystalline bovine serum albumin as standard (Bio-Rad, Richmond, CA). A mock flagellin preparation
was prepared in an identical manner using an extract of
PAK/fliC bacteria.
SDS-PAGE and immunoblot analysis. SDS-PAGE was
performed according to the method of Laemmli (19). Separated proteins were stained with 0.25% Coomassie blue or
transblotted to polyvinylidene difluoride membrane as described previously (23), and the membrane was blocked with
5% nonfat dry milk in 10 mM Tris 䡠 HCl, pH 7.4, containing
1.5% Tween 20 and reacted with rabbit antisera to PAK
flagellin or pilin, each diluted 1:3,000 in blocking buffer, or
monoclonal antibody to PAK LPS diluted 1:20 in blocking
buffer. After the membrane was washed, bound antibody was
detected with horseradish peroxidase-conjugated goat antirabbit IgG or goat anti-mouse IgG (KPL, Gaithersburg, MD)
diluted 1:10,000 in blocking buffer and enhanced chemilumi-
FLAGELLIN BINDS TO MUC1 MUCIN
Fig. 2. P. aeruginosa binding to CHO-Muc1 cells is inhibited by
flagellin antiserum. 35S-labeled P. aeruginosa strain PAK (4 ⫻ 107/
ml) was pretreated for 30 min at 37°C with PBS (A and B) or 6.0 (C)
or 20 (D) ␮l/ml rabbit antiserum to PAK flagellin, and binding to
CHO-X (A) and CHO-Muc1 (B–D) cells was measured. Each data
point represents mean ⫾ SE of 4 wells.
AJP-Lung Cell Mol Physiol • VOL
Fig. 3. SDS-PAGE and immunoblot analysis of P. aeruginosa-purified flagellin. A: flagella were prepared by differential centrifugation.
Unpurified whole PAK cell extract [0.5 ␮g (lanes 1 and 3) and 25 ␮g
(lane 5)] or isolated flagella [2.5 ng (lanes 2 and 4) and 2.5 ␮g (lane
6)] were resolved on a 15% polyacrylamide gel and transblotted to
polyvinylidene difluoride membrane (lanes 1–4) or stained with
Coomassie blue (lanes 5–7). Transblotted proteins were reacted with
rabbit antisera to PAK flagellin (lanes 1 and 2) or pilin (lanes 3 and
4). The position of flagellin (53 kDa) is indicated at left. The 3 bands
in whole cell extract (lane 3) reactive with pilin antiserum represent
pilin monomer, dimer, and tetramer (14). Lane 7 contains prestained
protein size markers indicated at right in kDa. B: flagella prepared
by differential centrifugation were treated by sequential acid pH
dissociation, ultracentrifugation, and neutral pH reassociation as
described previously (7, 43). Unpurified whole PAK cell extract (1.0
␮g, lane 1), partially purified flagellin prepared by differential centrifugation as described above (0.1 ␮g, lane 2), or further purified
flagellin (0.1 ␮g, lane 3) was analyzed by immunoblotting with PAK
LPS monoclonal antibody. Positions of prestained protein size markers are indicated at right in kDa.
flagellin compared with PBS-treated cells (P ⬍ 0.05 at
0.02 ␮g/ml and P ⬍ 0.01 at 0.2 ␮g/ml). To control for
nonspecific inhibition, a mock flagellin preparation
was generated from flagellin-deficient PAK/fliC bacteria in the same manner used to prepare flagellin from
wild-type PAK. Pretreatment of CHO-Muc1 cells with
0.2 ␮g/ml mock flagellin did not affect P. aeruginosa
binding compared with PBS-treated cells (Fig. 4E, P ⬎
0.05). On the basis of the foregoing results, we conclude
that P. aeruginosa flagellin is an adhesin for Muc1
mucin.
DISCUSSION
We reported previously that expression of hamster
Muc1 mucin on the surface of transfected CHO cells
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Flagellin antiserum inhibits P. aeruginosa binding
to CHO-Muc1 cells. To confirm the role of flagellin as
an adhesin for Muc1 mucin, wild-type PAK were preincubated with flagellin antiserum for 30 min before
the binding assay. As shown in Fig. 2, flagellin antiserum blocked P. aeruginosa binding to CHO-Muc1
cells in a dose-dependent manner. Compared with PBS
treatment, preincubation with flagellin antiserum decreased PAK binding by 61.2% at 6.0 ␮l/ml (P ⬍ 0.001)
and completely abolished binding at 20 ␮l/ml (P ⬍
0.0001). Bacterial adherence after pretreatment with
flagellin antiserum also was significantly reduced compared with adhesion after preincubation with equivalent amounts of normal rabbit serum (P ⬍ 0.004 at 6.0
␮l/ml and P ⬍ 0.0002 at 20 ␮l/ml; data not shown).
Purified flagellin inhibits P. aeruginosa binding to
CHO-Muc1 cells. Flagellin was purified from wild-type
PAK by differential centrifugation as described previously (2) and analyzed by immunoblotting and Coomassie blue staining after separation by SDS-PAGE.
As shown in Fig. 3A, immunoblot analysis with rabbit
antiserum against PAK flagellin revealed a prominent
53-kDa immunoreactive band (lane 2) that comigrated
with a Coomassie blue-stained band detected in the
same flagellin preparation (lane 6). Importantly, no
protein bands were detected by immunoblotting with
antiserum against PAK pilin (lane 4), indicating the
absence of pilin (17.8 kDa), a protein often copurifying
with flagellin by the centrifugation technique (14).
However, immunoblot analysis using monoclonal antibody against PAK LPS revealed the presence of LPS
(Fig. 3B, lane 2) with a banding pattern essentially
identical to that previously reported (10). The majority
of LPS was removed by sequential acid pH dissociation, ultracentrifugation, and neutral pH reassociation
as described by Brett et al. (7) (Fig. 3B, lane 3). Although the recovery of flagellin from this additional
purification was low (⬃25%), essentially pure flagellin
was obtained. As shown in Fig. 4, adhesion of P. aeruginosa to CHO-Muc1 cells was significantly decreased
after pretreatment of cells for 30 min with purified
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FLAGELLIN BINDS TO MUC1 MUCIN
significantly augmented the binding of P. aeruginosa
strains PAK (nonmucoid) and CF-3 (mucoid) (24). Because the increase in bacterial adhesion was completely abolished by removal of the extracellular domain of Muc1 mucin, either by treatment of CHOMuc1 cells with human neutrophil elastase or deletion
mutagenesis, these results indicated that Muc1 mucin
is an adhesion site for P. aeruginosa and prompted us
to conduct the experiments described here to identify
the cognate bacterial adhesin. Our results with reduced bacterial adherence by flagellin-deficient bacteria and binding inhibition by flagellin antiserum and
purified flagellin collectively lead us to conclude that P.
aeruginosa flagellin is the adhesin interacting with
Muc1 mucin at the molecular level. We chose to employ
the PAK strain for the present studies, since it is
nonmucoid and, thus, more typical of P. aeruginosa
strains initially isolated from cystic fibrosis lungs (see
below). Although the amount of P. aeruginosa that we
observed binding to CHO-Muc1 cells (0.2–5.1 bacteria/
cell or 0.4–10 ⫻ 105 bacteria/well) may appear quantitatively low, it compares favorably with that previously
documented in the literature on several occasions. For
example, using 24-well plates with confluent monolayers, Feldman et al. (12) reported (2.0–30) ⫻ 105 colonyforming units per well of PAK wild-type, PAK/NP, or
PAK/fliC bound to 1HAEo⫺ cells. Although the structural features of Muc1 mucin accounting for its interaction with flagellin remain to be elucidated, the GalNAc␤(1–4)Gal carbohydrate sequence of asialoGM1,
previously suggested to mediate binding to P. aeruginosa pilin (44), is probably not involved, since Muc1
mucin glycosyl moieties lack this disaccharide structure (5). Moreover, because the PAK and PAO1 strains
exhibited adherence to CHO-Muc1 cells (data not
shown), both a-type (PAK) and b-type (PAO1) flagellin
are likely capable of binding to Muc1 mucin.
Numerous prior studies demonstrating the role of P.
aeruginosa flagella in the pathogenesis of various lung
diseases, both experimental (18, 21, 31) and clinical
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Fig. 4. P. aeruginosa binding to CHO-Muc1 cells is inhibited by
purified flagellin. CHO-X cells pretreated for 30 min at 37°C with
PBS (A) or CHO-Muc1 cells pretreated with PBS (B), 0.02 ␮g/ml
flagellin (C), 0.20 ␮g/ml flagellin (D), or 0.20 ␮g/ml mock flagellin
(from PAK/fliC extract, E) were incubated with 35S-labeled P. aeruginosa strain PAK, and binding of bacteria was measured. Each data
point represents mean ⫾ SE of 4 wells.
(25), support our observations. In a neonatal mouse
model of pneumonia, Tang et al. (45) showed that
flagella of P. aeruginosa strain PAO1 are among several bacterial virulence factors contributing to bacteremia, disease, and death. Although the P. aeruginosa
flagellum is a virulence factor associated with disease
pathogenesis, it also serves as a target for nonopsonic
bacterial clearance by host phagocytic cells (27). In a
later study, Feldman et al. (12) used PAK wild-type
and isogenic mutants to document reduced incidence of
pneumonia and absence of mortality associated with
murine lung infection by PAK/fliC compared with PAK
and PAK/NP infections. In a clinical setting, in vivo
selection against flagella production may be one mechanism by which P. aeruginosa persists in the cystic
fibrosis lung. Sequential bacterial isolations from infected patients revealed that initial isolates obtained
shortly after colonization were nonmucoid, flagellated,
and motile, whereas mucoid bacterial variants lacking
motility and resistant to phagocytosis emerged later
during the course of the disease (26). The mechanism
by which flagella synthesis is downregulated is not
known. However, it seems reasonable to suggest that
the initial interaction between flagella and the epithelial cell surface may be an early signal that activates
host innate defense responses, ultimately leading to
selection of nonmotile bacterial strains. In this regard,
Muc1 mucin on the epithelial cell surface may play an
important role in this process, considering the fact that
its cytoplasmic domain contains multiple phosphorylation sites implicated in signal transduction pathways
of inflammation (4, 28, 29, 34, 50).
One of the hallmark features of ligand-receptor interactions is the ability of unlabeled ligand to competitively inhibit radiolabeled ligand binding to its cognate receptor in a dose-dependant manner (49). We
therefore investigated the ability of P. aeruginosa
flagellin to block adhesion of 35S-labeled bacteria to
CHO-Muc1 cells. As shown in Fig. 4, purified flagellin
blocked P. aeruginosa binding to CHO-Muc1 cells.
Whereas binding decreased in a dose-dependent fashion up to 0.2 ␮g/ml, to our surprise, flagellin concentrations ⬎2.0 ␮g/ml enhanced P. aeruginosa binding to
control CHO-X cells, likely through as yet unidentified
interactions with nonmucin components present on the
cell surface. Interestingly, a similar enhancement of
attachment of Salmonella enteritidis to ovarian granulosa cells by purified flagellin was reported by Popielzrczyk et al. (37). Although flagellin was free of detectable bacterial protein contamination, minor levels of
LPS were apparent (Fig. 3B). However, this degree of
LPS contamination did not appear to be responsible for
inhibition of bacterial adherence, since an extract of
PAK/fliC bacteria (devoid of flagella) that was prepared
identically to wild-type flagellin and contained an
equal amount of LPS as estimated by immunoblotting
with LPS antibody was unable to block PAK binding to
CHO-Muc1 cells (Fig. 4).
Flagella are the most complex organelles known in
bacteria, assembled from three separate substructures
(filament, hook, and basal body), each containing a
FLAGELLIN BINDS TO MUC1 MUCIN
This work was supported by National Heart, Lung, and Blood
Institute Grant RO1 HL-47125 and a grant from the Cystic Fibrosis
Foundation.
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