FEMS Immunology and Medical Microbiology 41 (2004) 133–139

www.fems-microbiology.org

Structure of the O-polysaccharide and serological cross-reactivityof the Providencia stuartii O33 lipopolysaccharide containing

4-(N -acetyl-DD-aspart-4-yl)amino-4,6-dideoxy-DD-glucose

Agnieszka Torzewska a, Nina A. Kocharova b, George V. Zatonsky b,Aleksandra Blaszczyk a, Olga V. Bystrova b, Alexander S. Shashkov b,

Yuriy A. Knirel b, Antoni Rozalski a,*

a Department of Immunobiology of Bacteria, Institute of Microbiology and Immunology, University of Lodz, Banacha 12/16, 90-237 Lodz, Polandb N. D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Leninsky Prospekt 47, 119991 Moscow, GSP-1, Russia

Received 4 December 2003; received in revised form 5 February 2004; accepted 23 February 2004

First published online 10 April 2004

Abstract

The O-polysaccharide of Providencia stuartii O33 was obtained by mild acid degradation of the lipopolysaccharide and the

following structure of the tetrasaccharide repeating unit was established:! 6)-a-DD-GlcpNAc-(1! 4)-a-DD-GalpA-(1! 3)-a-DD-GlcpNAc-(1! 3)-b-DD-Quip4N(Ac-DD-Asp)-(1!, where DD-Qui4N(Ac-DD-Asp) is 4-(N -acetyl-DD-aspart-4-yl)amino-4,6-dideoxy-DD-glu-

cose. Structural studies were performed using sugar and methylation analyses and NMR spectroscopy, including conventional 2D1H, 1H COSY, TOCSY, NOESY and 1H, 13C HSQC experiments as well as COSY and NOESY experiments in an H2O–D2O

mixture to reveal correlations for NH protons. The O-polysaccharide of P. stuartii O33 shares an a-DD-Glcp NAc-(1! 3)-b-DD-Quip4N(Ac-DD-Asp) epitope with that of Proteus mirabilis O38, which seems to be responsible for a marked serological cross-

reactivity of anti-P. stuartii O33 serum with the lipopolysaccharide of the latter bacterium. P. stuartii O33 is serologically related

also to P. stuartii O4, whose O-polysaccharide contains a lateral b-DD-Qui4N(Ac-LL-Asp) residue.

� 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Keywords: Lipopolysaccharide; O-antigen; Bacterial polysaccharide structure; Serological relationship; Providencia stuartii

1. Introduction

Gram-negative bacteria of the genus Providencia are

divided into five species, including Providencia alcali-

faciens, Providencia rustigianii, Providencia stuartii,

Providencia heimbachae, and Providencia rettgerii [1].

They are facultative pathogens that under favourable

conditions cause enteric diseases, wound and urinary-tract infections. Particularly, P. stuartii has been re-

cognised as a pathogen with an increasing involvement

in urinary-tract infections primarily in nursing home

patients with long-term urinary catheters in place [1].

* Corresponding author. Tel.: +48-42-6354464; fax: +48-42-6655818.

E-mail address: rozala@biol.uni.lodz.pl (A. Rozalski).

0928-8244/$22.00 � 2004 Federation of European Microbiological Societies

doi:10.1016/j.femsim.2004.02.007

These infections are frequently persistent, difficult to

treat and may even result in fatal bacteremia [2]. The

serological classification scheme of three Providencia

species, P. alcalifaciens, P. rustigianii, and P. stuartii,

used in serotyping of clinical isolates of these bacteria, is

based on the lipopolysaccharide (LPS, O-antigen, en-

dotoxin) and flagella (H-antigens) and includes 62 se-

rogroups [3,4]. Immunochemical studies of ProvidenciaO-antigens aim at creation of the molecular basis for the

serological classification and cross-reactivity of Provi-

dencia strains and related bacteria, including Proteus.

Recently, structures of the O-polysaccharides of the LPS

of Providencia serogroups O4, O5, O7, O14, O16, O18,

O19, O21, and O23 have been elucidated [5 (and refer-

ences therein),6,7,8 ,9]. Here, we report on the structure

of the O-polysaccharide of P. stuartii O33 and its

. Published by Elsevier B.V. All rights reserved.

134 A. Torzewska et al. / FEMS Immunology and Medical Microbiology 41 (2004) 133–139

serological relatedness to the O-antigens of P. stuartii

O4 and Proteus mirabilis O38.

2. Materials and methods

2.1. Bacterial strain and growth

Providencia stuartii O33:H1 strain 8078 came from

the Hungarian National Collection of Medical Bacteria

(National Institute of Hygiene, Budapest). Bacteria were

cultivated under aerobic conditions in nutrient broth

supplemented with 1% glucose. Bacterial mass washarvested at the end of the logarithmic growth

phase, centrifuged, washed with distilled water, and

lyophilised.

2.2. Isolation and degradations of the lipopolysaccharide

and the polysaccharide

The lipopolysaccharide was isolated from bacterialcells by phenol/water extraction [10] and purified by

treatment with cold aqueous 50% CCl3CO2H; the

aqueous layer was dialysed and freeze-dried.

A high-molecular-mass polysaccharide was prepared

by degradation of the lipopolysaccharide with aq. 2%

HOAc at 100 �C for 7 h followed by GPC of the water-

soluble portion on a column (60� 2.5 cm) of Sephadex

G-50 (S) in 0.05 M pyridinium acetate buffer, pH 4.5 (4mL pyridine and 10 mL HOAc in 1 L water) with

monitoring the elution using a Knauer differential re-

fractometer. The yield of the polysaccharide was 6.6% of

the lipopolysaccharide weight.

2.3. Monosaccharide and amino acid analyses

The polysaccharide was hydrolysed with 2 MCF3CO2H (120 �C, 2 h). GalA was identified using a

Biotronik LC-2000 sugar analyser as described previ-

ously [6]. Amino sugars were converted into the alditol

acetates [11] and analysed by gas liquid chromatography

(GLC) on a Hewlett–Packard 5880 instrument with a

DB-5 capillary column using a temperature gradient of

160 �C (3 min) to 290 �C at 10 �Cmin�1. Amino com-

ponents were analysed using a Biotronik LC-2000 ami-no acid analyser using standard sodium citrate buffers.

The absolute configurations of the monosaccharides and

aspartic acid were determined with GLC of the acety-

lated glycosides with (+)-2-octanol [12] and acetylated

(+)-2-octyl ester under the same chromatographic con-

ditions as above.

2.4. Methylation analysis

Methylation was performed as described previously

[13] and a part of the methylated polyssacharide was

reduced with LiBH4 in aq. 70% 2-propanol (25 �C, 2 h).

After hydrolysis with 2 M CF3CO2H (120 �C, 2 h), the

partially methylated monosaccharides were reduced

with NaBH4, acetylated and analysed with GLC–MS

on a Hewlett–Packard 5890 chromatograph equippedwith a DB-5 fused-silica capillary column and combined

with a NERMAG R10-10L mass spectrometer, using a

temperature gradient of 160 �C (1 min) to 250 �C at

3 �C �min�1.

2.5. NMR spectroscopy

Spectra were recorded using a Bruker DRX-500spectrometer at 32 �C in D2O or a 9:1 (v/v) H2O–D2O

mixture. Prior to the measurements in D2O, the samples

were lyophilised twice from D2O. Chemical shifts are

reported related to internal acetone (dH 2.225; dC 31.45).

Standard pulse sequences were used for gradient-se-

lected gsCOSY, gsTOCSY (MLEV-17), gsNOESY, 1H,13C gsHMBC and gsHSQC–TOCSY experiments. An1H, 13C gsHSQC experiment was performed with a pulsesequence that allows multiplicity editing [14]. A mixing

time was set to 200 ms in NOESY and 150 ms in

TOCSY and HSQC–TOCSY experiments; a 60-ms de-

lay was used in an HMBC experiment.

2.6. Serological techniques

Rabbit polyclonal anti-P. stuartii O33 and anti-P.stuartii O4 sera were obtained by immunization of New

Zealand white rabbits with heat killed bacteria as de-

scribed [15]. Passive hemolysis with alkali-treated LPS

and enzyme-immunosorbent assay (EIA) with LPS, as

well as inhibition of this test as antigen, DOC-PAGE

and Western blot and absorption of antisera were per-

formed as described previously [16].

3. Results and discussion

3.1. O-Polysaccharide structure elucidation

Sugar analysis using GLC of the alditol acetates de-

rived after full acid hydrolysis of the polysaccharides

showed the presence of GlcN. Analysis of the polysac-charide hydrolysate using sugar analyser revealed GalA

and, using an amino acid analyser, GlcN and aspartic

acid. GLC–MS of the acetylated alditols showed the

presence of GlcN and of a small amount of 4-amino-4,6-

dideoxyglucose (Qui4N). Further studies demonstrated

that the latter is a component of the repeating unit and

its poor release upon hydrolysis could be accounted for

by either decomposition under acidic conditions or/andby a stability of the amidic bond of N-linked aspartic

acid. GLC of the acetylated (+)-2-octyl glycosides and

(+)-2-octyl ester indicated that GalA, GlcN and aspartic

D1A1

B1

C1CO

C3B4

D3C6

D4A2

C2

Asp2

Asp3

CH CON3

D6

Fig. 1. 125-MHz 13C NMR spectrum of the O-polysaccharide of P. stuartii O33. Arabic numerals refer to carbons in sugar residues denoted as shown

in Table 1.

A. Torzewska et al. / FEMS Immunology and Medical Microbiology 41 (2004) 133–139 135

acid have the DD configuration. The DD configuration of

Qui4N was established by analysis of the 13C NMR

chemical shifts in the polysaccharide using known reg-ularities in glycosylation effects [17].

The 13C NMR spectrum of the polysaccharide

(Fig. 1) displayed signals for four anomeric carbons at d98.4–104.2; one non-substituted and one O-substituted

HOCH2-C groups (C-6 of GlcN) at d 61.3 and 69.6,

respectively; one CH3-C group (C-6 of Qui4N) at d 17.9;

three nitrogen-bearing sugar carbons (C-2 of GlcN and

C-4 of Qui4N) at d 53.3–58.1; other sugar ring carbonsat d 69.9–81.8; one aspartyl group (C-2 at d 53.0, C-3 at

d 39.6); three N -acetyl groups (CH3 at d 23.5–23.8); and

six CO groups at d 173.5–177.9 (C-6 of GalA, C-1 and

C-4 of Asp and three CH3CON). The 1H NMR spec-

trum contained signals for four anomeric protons at d4.46–5.31; one CH3-C group at d 1.15 (doublet, J5;6 5.7

Hz, H-6 of Qui4N); one aspartyl group at d 2.58, 2.67

(AB part of an ABX system; both H-2) and 4.54 (X partof an ABX system; H-3); and three N -acetyl groups at d1.99–2.08.

The O-polysaccharide has a tetrasaccharide repeating

unit containing two residues of DD-GlcN and one residue

Table 11H and 13C NMR data of the O-polysaccharide of P. stuartii O33 (d)

H-1 H-2 H

! 6)-a-DD-GlcpNAc-(1! A 4.90 3.92

! 4)-a-DD-GalpA-(1! B 5.31 3.87

! 3)-a-DD-GlcpNAc-(1! C 5.04 3.97

! 3)-b-DD-Quip4N-(1! D 4.46 3.46

Ac-DD-Asp 4.54 2.58

2.67

C-1 C-2 C

! 6)-a-DD-GlcpNAc-(1! A 100.8 55.0

! 4)-a-DD-GalpA-(1! B 102.2 69.9

! 3)-a-DD-GlcpNAc-(1! C 98.4 53.3

! 3)-b-DD-Quip4N-(1! D 104.2 73.7

Ac-DD-Asp 177.9 53.0

each of DD-GalA and Qui4N, as well as four acyl resi-

dues, including one aspartyl and three acetyl groups.

Methylation analysis of the polysaccharide resulted inidentification of partially methylated derivatives from 3-

substituted GlcN and 6-substituted GlcN. When the

methylated polysaccharide was carboxyl-reduced prior

to hydrolysis, in addition to the amino sugars, 2,3-di-O-methylgalactose was identified, which was evidently

derived from 4-substituted GalA.

The monosaccharide residues were designated as A–D

according to their sequence in the repeating unit (seebelow). The 1H and 13C NMR spectra of the polysac-

charide were assigned using gradient-selected 2D NMR

experiments, including gsCOSY, gsTOCSY, gsNOESY

and H-detected 1H, 13C gsHSQC (Table 1). The as-

signments were confirmed by a 1H, 13C gsHMBC ex-

periment, which enabled also the assignment of the CO

signals. In the TOCSY spectrum, anomeric protons for

GlcN C and Qui4N D showed correlations with H-2,3,4,5,6 and those for GlcN A and GalA B with H-

2,3,4,5 and H-2,3,4, respectively. The remaining protons

of GlcN A and GalA B were assigned by H-5,H-6a,6b

correlations in the COSY spectrum and a H-4,H-5

-3 H-4 H-5 H-6(6a) H-6b CH3CO

3.72 3.64 4.22 3.88 4.07 2.08

3.94 4.32 4.16

3.91 3.73 4.19 3.78 3.80 1.99

3.75 3.74 3.53 1.15

2.02

-3 C-4 C-5 C-6 CH3CO CH3CO

72.6 70.8 72.7 69.6 23.8 176.0

70.3 81.8 73.0 175.4

81.7 71.6 72.6 61.3 23.5 175.4

78.5 58.1 72.5 17.9

39.6 173.5 23.5 174.8

136 A. Torzewska et al. / FEMS Immunology and Medical Microbiology 41 (2004) 133–139

correlation in the gsNOESY spectrum, respectively. The

spin systems of the amino sugars were distinguished by

correlation of the protons at the nitrogen-bearing car-

bons (H-2 of GlcN and H-4 of Qui4N) with the corre-

sponding carbons.A relatively large 3J1;2 values of �8 Hz and the

characteristic position of the H-1 signal indicated the b-pyranose form of Qui4N, whereas smaller 3J1;2 values of3–4 Hz showed that the three other constituent mono-

saccharides are a-linked. The absence from the 13C

NMR spectrum of any signals for non-anomeric ring

carbons at a lower field than d 82 confirmed the pyra-

nose form of all monosaccharide residues [18].Downfield displacements of the signals for the linkage

carbons, including C-3 of GlcN C and Qui4N D, C-4 of

GalA B and C-6 of GlcN A, to d 81.7, 78.5, 81.8 and

69.6, respectively, i.e., by �4–10 ppm as compared with

their positions in the corresponding non-substituted

monosaccharides [18–20], showed that the polysaccha-

ride is linear and revealed the substitution pattern in the

repeating unit. The modes of the substitution of themonosaccharides was confirmed and their sequence in

the repeating unit established by a gsNOESY experi-

ment (Fig. 2), which revealed the following interresidue

correlations between the anomeric protons and protons

at the linkage carbons: GlcN A H-1,GalA B H-4; GalA

B H-1,GlcN C H-3; GlcN C H-1,Qui4N D H-3 and

Qui4N D H-1,GlcN A H-6a,6b.

A NOESY experiment with a polysaccharide samplein a 9:1 (v/v) H2O–D2O mixture revealed a correlation

between NH-4 of Qui4N and CH2 (H-3) of Asp at d8.14/2.58 and 8.14/2.67, which demonstrated a spatial

ppm

3.43.53.63.73.83.94.04.14.24.3 ppm

4.4

4.5

4.6

4.7

4.8

4.9

5.0

5.1

5.2

5.3

D1,3 D1,5D1,A6

A1,B4 A1,2A1,B5

C1,D3C1,2

B1,C3 B1,2

Fig. 2. Part of a 500-MHz gsNOESY spectrum of the O-polysaccha-

ride of P. stuartii O33 showing correlations for anomeric protons. The

corresponding parts of the 1H NMR spectrum are shown along the

axes. Arabic numerals refer to protons in sugar residues denoted as

shown in Table 1.

proximity of these protons and, therefore, the attach-

ment of Asp by COOH-4 to N-4 of Qui4N.

Based on the obtained data, we concluded that the O-

polysaccharide of P. stuartii O33 has the structure

shown in Fig. 3. A peculiar feature of the polysaccharideis the presence of DD-aspartic acid, which was found for

the first time in the O-polysaccharide of P. mirabilis O38

[21,22] and a glycoconjugate from Treponema medium

ATCC 700293 [23]. Recently, LL-aspartic acid has been

found in the O-polysaccharide of P. stuartii O4 [9,21]. In

all Providencia and Proteus polysaccharides, aspartic

acid is N-acetylated and attached by carboxyl-4 to the

same monosaccharide, 4-amino-4,6-dideoxy-DD-glucose,whereas in Treponema medium the non-acetylated amino

acid is linked by carboxyl-1 to 4-amino-4,6-dideoxy-DD-

galactose.

3.2. Serological studies

Serological studies with rabbit polyclonal anti-P.

stuartii O33 and anti-P. stuartii O4 sera were performed

to reveal the molecular basis of the immunospecificity

and the antigenic relationship of the bacteria. Both sera

were tested in passive hemolysis test (PHT), enzyme-

immunosorbent assay (EIA) and Western blot with theLPS of Providencia and Proteus strains, whose O-poly-

saccharides include DD-Qui4N(AcAsp) or DD-Qui4N. An-

ti-P. stuartii O33 serum strongly cross-reacted with the

LPS of P. mirabilis O38 in both passive hemolysis test

and enzyme-immunosorbent assay (Table 2). In enzyme-

immunosorbent assay binding of both homologous and

heterologous LPS was observed in a broad range of

antigen concentrations and the course of the bindingcurves was nearly identical (data not shown).

Studies with absorbed anti-P. stuartii O33 serum and

inhibition test confirmed the close relationships between

the LPS of P. stuartii O33 and P. mirabilis O38. The

alkali-treated LPS of P. mirabilis O38 removed most

antibodies from anti-P. stuartii O33 serum (after ab-

sorption the reciprocal titre of the reaction with the

homologous LPS dropped to 16,000 and 12,800 in EIAand PHT, respectively), whereas absorption with other

heterologous LPS, including the LPS of P. stuartii O4,

influenced the reactivity only insignificantly. The reac-

tivity in enzyme-immunosorbent assay of anti-P. stuartii

O33 serum with the homologous LPS was strongly in-

hibited not only by the homologous LPS and the iso-

lated O-polysaccharide but also by those of P. mirabilis

O38 (Table 3).In Western blot anti-P. stuartii O33 serum recognised

both slow and fast migrating bands of the homologous

LPS corresponding to high- and low-molecular-mass

LPS species with and without O-polysaccharide chain,

respectively (Fig. 4). It also cross-reacted strongly with

high-molecular-mass LPS species of P. mirabilis O38,

Table 2

Reactivity of anti-P. stuartii O33 and anti-P. stuartii O4 sera in passive hemolysis test and enzyme-immunosorbent assay

Antigen from Reciprocal titres with

anti-P. stuartii O33 serum anti-P. stuartii O4 serum

PHT EIA PHT EIA

P. stuartii O33 102,400 512,000 3200 8000

P. stuartii O4 51,200 4000 51,200 2,048,000

P. mirabilis O38 25,600 256,000 <100 <1000

P. stuartii O18 6400 1000 100 1000

Alkali-treated LPS and LPS were used as antigen in passive hemolysis test and enzyme-immunosorbent assay, respectively. Data of the

homologous antigen are shown in italics.

Table 3

Inhibition of the reactivity in enzyme-immunosorbent assay of anti-P.

stuartii O33 serum with the homologous LPS

Inhibitor Minimal inhibitory dose (ng)

P. stuartii O33 LPS 2.4

OPS 2.4

P. stuartii O4 LPS >5000

OPS >5000

P. mirabilis O38 LPS 2.4

OPS 19.5

P. stuartii O18 LPS >5000

LPS and OPS stand for lipopolysaccharide and polysaccharide,

respectively.

Providencia stuartii O33 (this work)

A B C D

→6)-α-D-GlcpNAc-(1→4)-α-D-GalpA-(1→3)-α-D-GlcpNAc-(1→3)-β-D-Quip4N(Ac-D-Asp)-(1→

Providencia stuartii O4 [9]

β-D-Quip4N(Ac-L-Asp) 1↓6

→6)-β-D-Galp-(1→3)-β-D-GlcpNAc-(1→3)-β-D-Galp-(1→6)-β-D-GlcpNAc-(1-

Proteus mirabilis O38 [22]

AcEtnP6

→6)-α-D-Glcp-(1→3)-α-D-GalpA-(1→4)-α-D-GlcpNAc-(1→3)-β-D-Quip4N(Ac-D-Asp)-(1→

Providencia stuartii O18 [7]

→6)-α-D-GlcpNAc-(1→4)-β-D-GlcpA-(1→3)-α-D-GalpNAc-(1→4)-β-D-Quip3NAc-(1

Fig. 3. Structures of the O-polysaccharides of the cross-reactive LPS. DD-Qui4N(Ac-DD-Asp) and DD-Qui4N(Ac-LL-Asp) stand for 4-(N -acetyl-DD-aspart-

4-yl)amino- and 4-(N -acetyl-LL-aspart-4-yl)amino-4,6-dideoxy-DD-glucose, respectively; Quip 3NAc stands for 3-acetamido-3,6-dideoxy-DD-glucose and

AcEtnP for 2-acetamidoethyl phosphate.

A. Torzewska et al. / FEMS Immunology and Medical Microbiology 41 (2004) 133–139 137

thus indicating that a common epitope(s) resides on the

O-polysaccharide chain. Comparison of the O-polysac-

charide structures of the cross-reactive LPS (Fig. 3)

showed that they share an a-DD-Glcp NAc-(1! 3)-b-DD-Quip4N(Ac-DD-Asp) disaccharide, which is evidently in-

volved in the common epitope.

We observed a significant cross-reactivity in passive

hemolysis test also between anti-P. stuartii O33 serumand the LPS of P. stuartii O4, whereas in enzyme-im-

munosorbent assay they reacted only insignificantly

(Table 2). The variance between the results obtained in

the two assays could be due to a different exposure of the

Fig. 4. Sodium deoxycholate polyacrylamide gel electrophoresis (A) and Western blot with anti-P. stuartii O33 serum (B) or anti-P. stuartii O4 serum

(C) of the LPS of P. stuartii O33 (lane 1), P. mirabilis O38 (lane 2), P. stuartii O4 (lane 3), P. alcalifaciens O5 (lane 4) and P. stuartii O18 (lane 5).

138 A. Torzewska et al. / FEMS Immunology and Medical Microbiology 41 (2004) 133–139

antigens on the carriers used (sheep red blood cells in

PHT and plastic surface in EIA). In Western blot with

anti-P. stuartii O33 serum a faint staining was observed

for high-molecular-mass LPS species of P. stuartiiO4 but

a stronger staining for low-molecular-mass species. This

finding indicated that most cross-reactive antibodies inanti-P. stuartii O33 serum are directed against an epi-

tope(s) shared by the core regions of the LPS, whose

structure remains to be determined. Anti-P. stuartii O4

serum cross-reacted with the LPS of P. stuartii O33 only

weakly in PHT in EIA (Table 2) but recognised both

types of LPS species of this bacterium in Western blot

(Fig. 4C). Therefore, the O-polysaccharides of P. stuartii

O33 and P. stuartii O4 possess cross-reactive epitopestoo, which, most probably, are associated with a

Quip4N residue N-acylated with Ac-DD-Asp and Ac-LL-

Asp, respectively. A lower level of the cross-reactivity as

compared with the pair P. stuartii O33/P. mirabilis O38

(see above) could be accounted for by the absence of any

common neighboring monosaccharide or/and different

absolute configurations of aspartic acid.

Finally, anti-P. stuartii O33 serum cross-reactedweakly with the LPS of P. stuartii O18 in passive he-

molysis test but almost not at all in enzyme-immuno-

sorbent assay (Table 2). In Western blot, it bound

weakly to high-molecular-mass LPS species of P. stuartii

O18, thus indicating the location of cross-reactive

epitope(s) resides on the O-polysaccharide chains.

Comparison of the O-polysaccharides structures of

P. stuartii O33 and P. stuartii O18 [7] (Fig. 3) showedsome similarity between their repeating units, which

may be responsible for the cross-reactivity. However,

the cross-reactivity of the P. stuartii O18 LPS as well as

that of P. stuartii O4 is considerably lower as compared

with the reactivity of the homologous LPS and, there-

fore, classification of the P. stuartii strains studied into

separate O-serogroups is expedient.

Acknowledgements

This work was supported by Grant 02-04-48118 of

the Russian Foundation for Basic Research and grant

505/386 of the University of Lodz. We thank Mgr M.

Wykrota for excellent technical assistance.

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