fbl is not involved in the invasion of eukaryotic epithelial and endothelial cells by staphylococcus...

R E S EA RCH L E T T E R

Fbl is not involved in the invasion of eukaryotic epithelial andendothelial cells by Staphylococcus lugdunensis

Florian Szabados, Lennart Marlinghaus, Miriam Korte, Sandra Neumann, Martin Kaase &Soeren G. Gatermann

Institute for Hygiene and Microbiology, Department of Medical Microbiology, Ruhr-University of Bochum, Bochum, Germany

Correspondence: Florian Szabados, Institute

for Hygiene and Microbiology, Department

for Medical Microbiology, University Bochum,

Universitatsstraße 150, Bochum,

Germany. Tel.: +49 0 234 32 26467;

fax: +49 0 234 32 14197; e-mail:

[email protected]

Received 26 October 2010; revised 8 August

2011; accepted 9 August 2011.

Final version published online 12 September

2011.

DOI: 10.1111/j.1574-6968.2011.02382.x

Editor: Jan-Ingmar Flock

Keywords

Staphylococcus lugdunensis; invasion; FACS;

5637; EA.hy 926.

Abstract

For several Staphylococci, such as Staphylococcus aureus, Staphylococcus sap-

rophyticus, and Staphylococcus epidermidis, invasion of eukaryotic cells has been

described and this mechanism has been considered an important part of the

infection process. The fibrinogen-binding protein (Fbl) of Staphylococcus lug-

dunensis, a homolog of the clumping factor A of S. aureus, has been described

as fibrinogen-binding adhesin and might promote invasion of cells. We there-

fore characterized several clinical strains of S. lugdunensis in terms of whole cell

fibrinogen and fibronectin binding and correlated these results with the inva-

sion of epithelial and endothelial cells by S. lugdunensis. We described for the

first time invasion of cells by S. lugdunensis. As invasion of cells by S. lugdun-

ensis was only partly inhibited by cytochalasin D in contrast to a complete

inhibition of invasion of cells by S. aureus, further invasion mechanisms are

likely to be present in S. lugdunensis. In addition, the Fbl of S. lugdunensis is

not involved in the invasion of cells as ruled out by an isogenic fbl mutant.

Introduction

Pathogen entry into eukaryotic cells plays an important

role in the understanding of infectious diseases at the

cellular level. This process has been termed bacterial inva-

sion (Finlay & Cossart, 1997). Invasion of non-phagocytic

host cells seems to be an effective mechanism for pre-

venting elimination and maintaining infection (Kubica

et al., 2008). A variety of gram-negative invasive bacteria,

such as Salmonella spp., have been described (Finlay &

Cossart, 1997). Some gram-positive organisms, such as

Listeria monocytogenes and Staphylococcus aureus, have

been also described as invasive. Moreover, for Staphylo-

cocci, invasion of eukaryotic cells has been observed not

only for S. aureus (Proctor et al., 1984), but also for

Staphylococcus saprophyticus (Szabados et al., 2008) and

Staphylococcus epidermidis (Khalil et al., 2007; Hirschhau-

sen et al., 2010). Invasion contributes to intracellular

persistence and seems to be an integral part of the infec-

tious process (Sinha & Fraunholz, 2009; Tuchscherr

et al., 2010). Fibronectin binding allows for S. aureus

invasion, via bridging to integrin a5b1 (Sinha et al.,

1999). Moreover, for S. aureus, the fibronectin-binding

proteins, FnBPA (and FnBPB), have been shown to be

prerequisite for invasion of endothelial cells (Que et al.,

2005; Kerdudou et al., 2006; Piroth et al., 2008; Sinha &

Fraunholz, 2009; Edwards et al., 2010). FnBP-homologs

have not been described for coagulase-negative staphylo-

cocci (other than S. aureus) so far. For S. epidermidis, an

Atl-dependent invasion mechanism via binding to heat

shock cognate protein 70 (Hsc70), has been described

(Hirschhausen et al., 2010). Invasion of epithelial cells

has also been described for S. saprophyticus, but the

underlying invasion mechanism has yet to be character-

ized (Szabados et al., 2008). Only two Staphylococcus

lugdunensis adhesins, the fibrinogen-binding protein (Fbl)

and the von Willebrand-factor-binding protein have

already been described (Mitchell et al., 2004; Nilsson

et al., 2004a, b; Geoghegan et al., 2010). The N2 and N3

regions of the Fbl have a sequence similarity of 62% to

that of the clumping factor A (ClfA) of S. aureus (Nilsson

et al., 2004a). The von Willebrand-factor-binding protein

is a homolog of protein A (Nilsson et al., 2004b).

Unexpectedly, data on fibronectin-binding adhesins and

FEMS Microbiol Lett 324 (2011) 48–55 ª 2011 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

MIC

ROBI

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invasins of S. lugdunensis are scarce. Binding to fibronec-

tin has previously been investigated in a small collection

of strains, but all of eleven isolates showed only weak

binding to fibronectin compared with that of strain

Cowan I of S. aureus (Paulsson et al., 1993). S. lugdunensis

has been described as being part of several niches of the

human skin flora (Bieber & Kahlmeter, 2010). The clini-

cal presentation of S. lugdunensis-caused infections is

similar to infections caused by S. aureus. Staphylococcus

lugdunensis can cause potentially fatal endocarditis, osteo-

myelitis, and skin and soft tissue infections (Vandenesch

et al., 1993; Pareja et al., 1998; Patel et al., 2000;

Hellbacher et al., 2006; Frank et al., 2008). Staphylococcus

lugdunensis is thought to be rarely isolated; nevertheless,

the low prevalence of S. lugdunensis in skin infection has

recently been questioned (Bocher et al., 2009). We there-

fore sought to analyze the invasion of epithelial and

endothelial cells (human urinary bladder carcinoma cell

line 5637 and the endothelial cell line EA.hy 926) using a

previously described fluorescence-activated cell-sorting

(FACS)-based invasion assay. We correlated these results

with the binding of clinical isolates of S. lugdunensis to

fibronectin.

Materials and methods

Bacteria and cell lines

The bacteria used were S. aureus Cowan I, Staphylococcus

carnosus TM 300 and eight clinical isolates of S. lugdun-

ensis: Stlu 12, Stlu 30, Stlu 33, Stlu 35, Stlu 36, Stlu 39,

Stlu 50, and Stlu 108 and the isogenic knockout mutant

Stlu 108Dfbl::ermB (Table 1). All S. lugdunensis isolates

used in this study were confirmed by two reference meth-

ods: S. lugdunensis specific tanA and fbl PCRs and by

MALDI-TOF MS, as described previously (Noguchi et al.,

2009; Szabados et al., 2010; Szabados et al., 2011).

Human urinary bladder carcinoma cell line 5637 (DSMZ,

Braunschweig, Germany) and endothelial cell line EA.hy

926 (DMSZ) were used throughout this study. Bacteria

were grown until the mid-logarithmic phase, as described

previously (Szabados et al., 2008).

Homologeous recombination of the fbl gene of

S. lugdunensis

The mutagenesis construct for the homologous recombi-

nation cloned into pBT2, and named pMB2503, has pre-

viously been described (Marlinghaus et al., 2011). The

homologous recombination of the fbl gene in strain Stlu

108 was also performed as previously described (Marling-

haus et al., 2011). The D fbl knockout mutant was con-

firmed by sequencing (data not shown).

Fluorescence activated cell sorting (FACS)-

based invasion assay

Human urinary bladder carcinoma cell line 5637 was cul-

tured in RPMI 1640 with phenol red (PAA, Pasching,

Austria). The endothelial cell line EA.hy 926 was cultured

in HAT medium (Invitrogen) with addition of HAT

medium supplement (hypoxanthine, aminopterin, and

thymidine). Both media were also supplemented with

10% heat-inactivated fetal calf serum (PAA) and 1 g L�1

pyruvate (Invitrogen) and 1.5 g L�1 glucose (Invitrogen).

Eighteen hours before inoculation with bacteria,

3 9 105 cells per well were seeded in 24-well plates (Gre-

iner Bio-one, Frickenhausen, Germany) in modified

RPMI with phenol red, as described previously (Szabados

et al., 2008). Experiments performed with viable bacteria

yielded the equivalent of 5 9 108 S. aureus Cowan I from

a suspension with an optical density (OD600 nm) of 1.0.

Staphylococci were adjusted to an estimated concentra-

tion of 2 9 108 CFU mL�1 cell culture medium and kept

at +4 °C until use. Three FACS experiments were per-

formed as previously described, on different days in

duplicates, and up to 5000 invasion events were counted,

unless described elsewhere. Staphylococcus aureus Cowan I

and S. carnosus TM 300 were measured in the same

experiment as a positive control and a negative control,

respectively. The arbitrary value of FITC-stained bacteria,

used as a surrogate for invasion of cells, was normalized

to the positive control S. aureus Cowan I to display the

relative invasiveness of the tested strains to the strongly

invasive S. aureus Cowan I.

Table 1. Bacterial stains and plasmids used in this study

Species Strains Properties Source

S. aureus Newman

S. aureus Cowan I

S. carnosus TM 300

S. lugdunensis Stlu 12 Wild type

Stlu 30 Wild type

Stlu 33 Wild type

Stlu 35 Wild type

Stlu 36 Wild type

Stlu 39 Wild type

Stlu 50 Wild type

Stlu 108 Wild type

MB105 Stlu 108

D fbl::ermB

This study

Plasmids

pBT2 Temp. shuttle

vector

Bruckner

(1997)

pMB2503 pBT2 + D fbl::ermB

(mutagenesis construct)

Marlinghaus

et al. (2011)


Fbl is not an invasin 49

Bacterial adherence to solid-phase fibrinogen

and fibronectin

Purified fibrinogen (plasminogen, von Willebrand-factor

and fibronectin depleted; Enzyme Research Laboratories,

South Bend, IL) was coated to a 96-well microtiter as

previously described (Szabados et al., 2011). For the

fibronectin binding, a precoated microtiter plate was used

(BD Biocoat™ Cellware Human Fibronectin; BD, Bed-

ford, MA). The binding experiments were performed as

previously described (Szabados et al., 2011). An OD550 nm

value of 0–0.06 was interpreted as negative, 0.07–0.15 as

intermediately positive (+), 0.15–0.3 as positive (++), and> 0.3 as strongly positive (+++). Staphylococcus aureus

Cowan I was used as positive control for fibrinogen and

fibronectin binding. A sample without bacteria and the

S. carnosus TM 300 were used as negative controls.

Extra/intracellular staining of bacteria by FITC-

biotin-avidin

Bacteria (1 9 108) were washed with PBS and suspended

in an estimated 1 lg mL�1 FITC and incubated for

30 min. Bacteria were washed three times with ice-cold

PBS. Sulfo-NHS-LC-biotin (Pierce Biotechnology, Rockford,

IL) was solved at a final concentration of 0.3 mg mL�1 in

PBS as previously described (Agerer et al., 2004). Samples

were washed three times with PBS, mounted with

embedding medium ProLong® Gold (Invitrogen) in glass

slides and sealed with nail polish. The glass slides were

examined using confocal microscope Leica DM IRE2

(Leica, Solms, Germany).

Transmission electron microscopy (TEM)

A suspension of human urinary bladder carcinoma

cells 5637 from the FACS assay was used. The lysis stepwas omitted and cells were centrifuged gently (1000 g)for 60 s and transferred into 500 lL D-PBS (PAA) andfixed with 500 lL glutaraldehyde 2.5% as previouslydescribed.

Results

Binding of clinical strains of S. lugdunensis to

fibrinogen and fibronectin

Only three of eight strains (Stlu 12, Stlu 50, and Stlu

108) showed binding to solid-phase fibrinogen (Fig. 1a)-

as seen in previous results (Szabados et al., 2011). Four

of eight strains (Stlu 30, Stlu 33, Stlu 36 and Stlu 108)

showed binding to solid-phase fibronectin (Fig. 1b). One

strain (Stlu 108) showed binding to immobilized fibrino-

gen and also to immobilized fibronectin. Recently, Fbl

has been shown to be the major fibrinogen-binding pro-

tein (Mitchell et al., 2004; Marlinghaus et al., 2011). To

impair adhesion due to fibrinogen binding, this isolate

was selected for a knockout of the fbl gene by homolo-

gous recombination and the knockout mutant was named

MB105 (Table 1). Fibrinogen binding was completely

abolished in the MB105 mutant in contrast to their fibro-

nectin-binding attributes (Fig. 1a and b).

Invasion of the human bladder carcinoma cell

line 5647

Clinical isolates of S. lugdunensis invaded the human

bladder carcinoma cell line 5647 relative to the invasion

of S. aureus Cowan I, which was defined as 100%. The

non-invasive S. carnosus TM 300 has been shown to have

a relative invasiveness of 11.6%. Some clinical isolates of

S. lugdunensis were internalized up to 6.7-fold compared

with S. carnosus, which is equivalent to a relative inva-

siveness of 78% of that of S. aureus Cowan I (Fig. 2a).

Invasion of the endothelial cell line EA.hy 926

Clinical isolates of S. lugdunensis invaded the endothial

cell line EA.hy 926. The invasion of S. aureus Cowan I

into the cell line EA.hy 926 was defined as 100%.

The non-invasive S. carnosus TM 300 has been shown

to have a relative invasiveness of 7.5% to that of S. aureus

Cowan I. Some clinical isolates of S. lugdunensis were

internalized up to 7.4-fold compared with S. carnosus,

which is equivalent to a relative invasiveness of 55% of

that of S. aureus Cowan I (Fig. 2b).

Intracellular location of S. lugdunensis

The invasion of epithelial and endothelial cells as deter-

mined by the FACS-invasion assay was confirmed by

characterizing the intracellular location of the bacteria. A

previously described intra/extracellular staining method

(Agerer et al., 2004) and TEM were thus used (Hamill

et al., 1986). FITC-stained and biotin-labeled bacteria

were submitted to the invasion experiment to stain

extracellular bacteria. After invasion of cells, extracellular

bacteria were stained with streptavidin-conjugated Alexa

647. Cells and bacteria (intra- and extracellular) were

investigated by confocal microscopy as previously

described (Agerer et al., 2004). Up to 10 FITC-stained

bacteria were found in selected planes of 5637 cells

(Fig. 3). To confirm the intracellular location of the

bacteria by a third method, human urinary bladder carci-

noma cell line 5637 treated with S. lugdunensis were sub-

mitted to electron microscopy. In TEM, S. lugdunensis

ª 2011 Federation of European Microbiological Societies FEMS Microbiol Lett 324 (2011) 48–55Published by Blackwell Publishing Ltd. All rights reserved

50 F. Szabados et al.

was detected inside human urinary bladder carcinoma

cells, surrounded by a phagosome-like membrane, similar

to pictures described for invasive S. aureus (Sinha et al.,

1999) and S. saprophyticus (Szabados et al., 2008) strains.

Up to 20 bacteria per cell were found in selected eukary-

otic cells (Fig. 4).

Fibrinogen binding is not involved in the

invasion by S. lugdunensis

Fibrinogen-binding adhesins have been described for a

variety of bacteria (Palma et al., 2001). One might expect

that adhesion to eukaryotic cells via binding to fibrinogen

could supposedly promote invasion. Nevertheless, an

effect of fibrinogen on the invasion of cells has not been

described for S. aureus. The invasion of the clinical

strains of S. lugdunensis seems to be dependent on their

ability to bind fibronectin rather than binding fibrinogen –

similar to S. aureus. This was also supported by the fact

that the wild type strain Stlu 108 and its fbl knockout

MB105 were similar with regard to their invasion of 5637

cells (Fig. 5a). Expectedly, binding of the MB105 mutant

to solid-phase fibronectin was also unaltered compared

with the Stlu 108 wild type. To confirm the importance

of fibronectin for the invasion of cells, an invasion experi-

ment without FCS was performed. Without the addition

of FCS to the medium, the invasion of cells was impaired

in S. aureus and also in S. lugdunensis – similar to results

previously described (Sinha et al., 1999). After the addi-

tion of 20 ng fibronectin, invasion of cells was restored in

S. aureus and also in S. lugdunensis. Notably, the addition

of cytochalasin D (10 and 25 lM) completely inhibited

the invasion of cells by S. aureus similar to previous

results (Sinha et al., 1999). Interestingly, the same con-

centrations of cytpchalasin D only partly inhibited the

invasion of cells by S. lugdunensis (Fig. 5b).

Fig. 1. Adherence of the clinical strains of

Staphylococcus lugdunensis to solid-phase

fibrinogen and fibronectin. The binding ability

of the bacteria to solid-phase fibrinogen (a)

and fibronectin (b) was determined. An

OD550nm value of 0–0.06 was interpreted as

negative, 0.07–0.15 as intermediately positive

(+), 0.15–0.3 as positive (++), and a value of

> 0.3 as strongly positive (+++).

Staphylococcus aureus Cowan I was used as a

positive control, Staphylococcus carnosus TM

300 and bacteria without cells were used as

negative controls. The binding was performed

in two independent experiments measured in

at least four wells at the same time. Error

bar ± SD.



Discussion

Recently, the ability of S. aureus to infect and survive in

professional phagocytes and non-phagocytic cells has been

described (Kubica et al., 2008). The intracellular persis-

tence of S. aureus plays an important role in its patho-

genesis (Sinha & Fraunholz, 2009; Tuchscherr et al.,

2010). Recently, invasion was also shown for S. epidermi-

dis (Khalil et al., 2007; Hirschhausen et al., 2010) and

S. saprophyticus (Szabados et al., 2008; Szabados et al.,

2009); therefore, invasion of eukaryotic cells may also be

an important pathogenicity factor in other coagulase-neg-

ative staphylococci (CoNS). The invasion of S. aureus has

been considered to involve an interaction between the

FnBPA and the a5b1-integrin eukaryotic cell (Sinha et al.,

1999) and has been measured in so called invasion assays

(Sinha et al., 1999; Pils et al., 2006; Szabados et al., 2008;

Szabados et al., 2009; Sinha & Fraunholz, 2009; Trouillet

et al., 2011). The invasion of eukaryotic cells by S. aureus

has been described by viable bacteria and also by formal-

dehyde-inactivated bacteria (Sinha & Fraunholz, 2009).

The invasion of 5637 cell by S. saprophyticus was

restricted to viable bacteria only (Szabados et al., 2008),

indicating differences in the invasion mechanism between

S. aureus and the coagulase-negative S. saprophyticus.

Moreover, for S. epidermidis, a novel Atl-dependent inva-

sion mechanism via binding to Hsc70 has recently been

described (Hirschhausen et al., 2010), suggesting that

additional or different mechanisms, by which invasion of

eukaryotic cells can occur, in staphylococci other than

S. aureus were present. For S. aureus, fibrinogen-binding

ClfA has been described as virulence factor (Palma et al.,

2001). In addition, the cooperation of fibrinogen and

fibronectin-binding proteins is essential during experi-

mental endocarditis (Que et al., 2005). Therefore, Fbl, as

a ClfA-homolog and other putative proteins, such as the

yet-to-be-described fibronectin-binding surface proteins

could promote S. lugdunensis invasion. In general, only

low fibronectin binding has been described (Paulsson

et al., 1993) and putative homologs to FnBP’s of S. aur-

eus have not yet been described for S. lugdunensis. The

binding of clinical strains of S. lugdunensis to solid-phase

fibrinogen varied within the strains independently of the

occurrence of the fbl gene (Szabados et al., 2011). The

Fig. 2. Invasion of epithelial and endothelial

cells. The mean arbitrary fluorescence value

was normalized to the mean value of the

positive control Staphylococcus aureus Cowan

I and given as relative invasiveness. Invasion of

human bladder carcinoma cell line 5637 cells

up to a relative invasiveness of 78% of that of

S. aureus Cowan I (a) was observed. Invasion

of endothelial cell line EA-hy.926 cells up to a

relative invasiveness of 55% of that of

S. aureus Cowan I (b) was observed. The

Staphylococcus lugdunensis strain numbers

were shown below the bars. Error bars ± SD;

filled symbols: Strains that were greater than a

threefold in their relative invasiveness to the

negative control were defined as invasive.



fibronectin binding also varied within the strains

(Fig. 1b), but the allocation of the fibronectin binding

seems to be expectedly independent of the fibrinogen

binding. The fibrinogen- and fibronectin-binding proteins

could be either differentially expressed or the expression

could be masked by the production of extracellular

matrix, such as a biofilm (Frank & Patel, 2007). Notably,

the relative invasiveness of S. aureus isolates into 293 cells

was dependent on the clinical strain. Some S. aureus

strains, such as S. aureus 8325-4, S. aureus Wood 46 and

S. aureus Newman, have been shown to have a relative

invasiveness of below 20% compared with S. aureus Co-

wan I and have been therefore defined as non-invasive

(Sinha et al., 1999). Interestingly, the S. aureus Newman

was also weak in binding to solid phase fibronectin, sup-

porting the hypotheses that S. aureus Newman is non-

invasive due to a weak fibronectin binding. Notably, the

strain S. aureus 8325-4 has recently been described as

invasive, compared with its isogenic fnbA and fnbB

knockout mutants (Trouillet et al., 2011), indicating that

invasion of cells is not only strain-dependent but also a

relative attribute. Limited data on very few strains of

S. aureus indicate that the degree of fibronectin binding

influences the invasion of eukaryotic cells (Sinha et al.,

1999). Nevertheless, fibronectin binding in S. lugdunensis

and correlated invasion attribute have not been investi-

gated in a larger collection of clinical isolates of S. aureus.

Moreover, the binding S. lugdunensis to solid-phase

(a) (b)

(c) (d)Fig. 3. Intracellular S. lugdunensis was

documented by a previously described staining

of intra- and extracellular bacteria. Bacteria

were unspecifically stained with FITC (a green

fluorescence), NHS-LC biotin was covalently

bound to the bacteria. After the invasion step,

biotinylated-components were stained by

streptavidin-conjugated fluorophor Alexa647

(b, red fluorescence), showing extracellular

bacteria. Human bladder carcinoma cell line

5636 cells were seen in transmitted light (c).

In (d), green and red fluorescence was

merged. Intracellular bacteria were

distinguished by a solely green fluorescence

compared with extracellular bacteria by a

green and red fluorescence.

Fig. 4. Intracellular localization of Staphylococcus lugdunensis

documented by TEM. Bacteria were surrounded by a membrane in

phagosome-like structures, similar to pictures of invasive Staphylococcus

aureus and Staphylococcus saprophyticus. Up to 20 bacteria per cell

were found in selected eukaryotic cells. The scale bar indicates 1.7 lm.



fibrinogen in our study was independent from the inva-

sion of cells. The fibronectin binding was also indepen-

dent of the fibrinogen-binding attribute, as shown by an

isogenic fbl knockout mutant. In addition, Fbl is not

involved in the invasion of cells, as shown by an isogenic

fbl mutant (Fig. 5). The invasion of cells was impaired in

S. aureus and S. lugdunensis if an experiment was per-

formed without FCS. The addition of 20 ng fibronectin

restored the impaired invasion of cells by S. aureus and

also by S. lugdunensis, similar to results that have previ-

ously been published for S. aureus (Sinha et al., 1999).

Interestingly, the addition of cytochalasin D completely

inhibited the invasion of cells by S. aureus Cowan I, but

only partly by S. lugdunensis strain Stlu 108 (Fig. 5). This

indicates that invasion of cells by S. lugdunensis was med-

iated by at least one other additional pathway.

In conclusion, we have shown, for the first time, that

clinical isolates of S. lugdunensis invaded the endothelial

cell line EA.hy 926 and the urinary bladder carcinoma cell

line 5637. The invasion of cells is similar, in some cases,

to that of S. aureus. Clinical strains which showed a bind-

ing to solid-phase fibronectin were invasive into the 5637

and EA.hy 926 cells. The isolate Stlu 108 with a strong

fibronectin binding, similar to that of S. aureus Cowan I,

was also invasive to a similar degree. The fibrinogen-

binding protein Fbl is not involved in the invasion of

cells by S. lugdunensis Stlu 108, as shown by an isogenic

fbl mutant. Our results indicate the presence of an inva-

sion mechanism, supposedly similar to that described for

S. aureus and one which contains a putative further cyto-

chalasin D-independent invasion mechanism.

Acknowledgements

We thank Anke Albrecht (Bochum) for excellent technical

assistance, Inge Schmitz (Institute of Pathology, Univer-

sity of Bochum) for electron microscopy, and Gurpreet

Khaira (Vancouver, Canada) for critically reading the

manuscript. The authors certify that there is no actual or

potential conflict in relation to this article.

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fbl is not involved in the invasion of eukaryotic epithelial and endothelial cells by staphylococcus...

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