Carbohydrate Research 354 (2012) 102–105

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Carbohydrate Research

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Structural studies and biosynthetic aspects of the O-antigen polysaccharidefrom Escherichia coli O174

Carolina Fontana a, Magnus Lundborg a, Andrej Weintraub b, Göran Widmalm a,⇑a Department of Organic Chemistry, Arrhenius Laboratory, Stockholm University, S-106 91 Stockholm, Swedenb Karolinska Institute, Department of Laboratory Medicine, Division of Clinical Microbiology, Karolinska University Hospital, S-141 86 Stockholm, Sweden

a r t i c l e i n f o a b s t r a c t

Article history:Received 29 November 2011Received in revised form 16 February 2012Accepted 22 February 2012Available online 3 March 2012

Keywords:Escherichia coliLipopolysaccharideNMRBiological repeating unit

0008-6215/$ - see front matter � 2012 Elsevier Ltd. Adoi:10.1016/j.carres.2012.02.020

⇑ Corresponding author.E-mail address: gw@organ.su.se (G. Widmalm).

The structure of the repeating unit of the O-antigenic polysaccharide (PS) from Escherichia coli O174 hasbeen determined. Component analysis together with 1H and 13C NMR spectroscopy experiments wereemployed to elucidate the structure. Inter-residue correlations were determined by 1H,13C-heteronuclearmultiple-bond correlation and 1H,1H-NOESY experiments. The PS is composed of tetrasaccharide repeat-ing units with the following structure:

Cross-peaks of low intensity were present in the NMR spectra consistent with a b-D-GlcpNAc-(1?2)-b-D-GlcpA(1? structural element at the terminal part of the polysaccharide, which on average is composedof �15 repeating units. Consequently the biological repeating unit has a 3-substituted N-acetyl-D-galac-tosamine residue at its reducing end.

� 2012 Elsevier Ltd. All rights reserved.

Escherichia coli is a Gram-negative, facultative anaerobic rod,and part of the normal intestinal microflora. E. coli is one of themost common causes of food-borne gastroenteritis in humans.There are six different pathotypes of E. coli that may be involvedin diarrheal diseases.1 Verocytotoxin-producing E. coli (VTEC), alsoreferred to as Shiga toxin-producing E. coli (STEC) is considered to-day as one of the most important human pathogens in developedcountries. Humans infected with STEC show symptoms, such asabdominal pain and watery diarrhea, and a number of patients de-velop a life-threatening disease, such as hemorrhagic colitis (HC)and hemolytic-uremic syndrome (HUS).2 E. coli O174:H27 has pre-viously been designated as E. coli OX3.3 The isolate investigated inthe present study does not harbor any VTEC virulence genes; how-ever, most of the isolates belonging to the O174 serogroup havebeen characterized as VTEC.3 Scheutz et al.3 also described a strongcross-reactivity between E. coli O174 and E. coli K101 as well as toE. coli O21. The structures of the former capsular polysaccharide(CPS) and the latter O-antigen polysaccharide have been elucidated

ll rights reserved.

earlier.4,5 In this study we present the structure of the E. coli O174O-antigen which is similar to, but not identical to, the CPS fromE. coli K101, thereby clarifying the nature of the cross-reactivity be-tween the two strains.

E. coli O174 was grown in an LB (Luria-Bertani) medium. TheLPS was isolated from the bacterial membrane by hot phenol/waterextraction and delipidated under mild acid conditions to yield thepolysaccharide (PS), which was purified by gel-permeation chro-matography. Sugar analysis of the PS revealed galactose, 2-ami-no-2-deoxyglucose and 2-amino-2-deoxygalactose as the majorcomponents. Determination of the absolute configuration of thesugars utilized authentic standards and showed D-Gal, D-GlcNand D-GalN, as well as a D-GlcA.

In the 1H NMR spectrum of the E. coli O174 PS there are fourresonances that can be assigned to anomeric protons with 1Hchemical shifts of 4.81, 4.68, 4.53 and 4.51 ppm (Fig. 1). The corre-sponding sugar residues were denoted A–D, respectively. Reso-nances are also found, inter alia, at dH 2.04 (3H) and 2.02 (3H)indicating that the amino sugars are N-acetylated. The multiplicityedited 1H,13C-HSQC spectrum of the PS (Fig. 2) shows in the regionfor anomeric resonances four cross-peaks corresponding to

3.5 4.0

55

65

75

85

4.6 4.8

102

104

1H /ppm

13C

/ppm

A1 B1

C1

D1

A1' B1'

B4 C3 B2

D3

Figure 2. Selected regions of the multiplicity-edited 1H,13C-HSQC NMR spectrum ofthe O-antigen PS from E. coli O174. Resonances from anomeric and substitutionpositions are annotated. (Bottom) The anomeric region in which the cross-peaksfrom residues in the terminal part of the polysaccharide are labeled A0 and B0; (top)the region for the ring and hydroxymethyl atoms in which the cross-peaks from C-6are in red.

2.5 3.0 3.5 4.0 0.2 5.4

HDO

A B C D

A'

1H /ppm

Figure 1. The 1H NMR spectrum of the O-antigen polysaccharide from E. coli O174.

C. Fontana et al. / Carbohydrate Research 354 (2012) 102–105 103

hexopyranosyl residues. In the 13C NMR spectrum 28 resonanceswere observed (data not shown), confirming that the O-antigenof E. coli O174 is built of tetrasaccharide repeating units. The chem-ical shifts of the sugar residues were assigned using a combinationof 1D and 2D NMR techniques. 1H and 13C chemical shifts of thefour sugars are compiled in Table 1. All residues are b-linked sincethe 1JC1,H1 coupling constants are 161–165 Hz. From the analysis of1H,1H-TOCSY spectra and 1H and 13C chemical shifts, residues Aand B have the gluco-configuration whereas residues C and D havethe galacto-configuration. Residue B has five protons in its spin-system and is consequently b-D-GlcpA. The gluco-configuration inthis residue was also confirmed by measurement of the 3JH-4,H-5

coupling constant (�9 Hz). Residues A and C have C-2 chemicalshifts that correspond to b-D-GlcpNAc and b-D-GalpNAc residues,respectively. Residue D is thus b-D-Galp. A band-selective con-stant-time 1H,13C-HMBC experiment6 (Fig. 3a), with a selectivepulse centered at the carbonyl resonance frequencies, was usedto confirm the NMR assignments via 2JC,H and 3JC,H couplings fromcarbonyl groups. The resonances at dC 175.54 and 175.42 have cor-relations to dH 3.67 (H-2 in A) and dH 4.01 (H-2 in C), respectively,

confirming the N-acetyl groups at the C-2 positions in residues Aand C. The existence of the glucuronic acid was confirmed by cor-relations from dC 175.15 to dH 3.69 (H-5 in B) and 3.78 (H-4 in B).

The substitution positions for the sugar residues were identifiedfrom the 13C NMR glycosylation shifts.7 Residue B has significantglycosylation shifts DdC 6.60 and 7.57 for C-2 and C-4, respectively.These results show that residue B is disubstituted and correspondsto ?2,4)-b-D-GlcpA-(1?. A 1H,13C-H2BC spectrum (Fig. 3b) con-firmed the NMR assignments via 2JC,H couplings from the resonanceat dC 81.60 (C-2 in B) to dH 3.65 (H-3 in B) and 4.68 (H-1 in B), andfrom the resonance at dC 80.26 (C-4 in B) to dH 3.65 (H-3 in B) anddH 3.69 (H-5 in B). Residues C and D were assigned to be ?3)-b-D-GalpNAc-(1? and ?3)-b-D-Galp-(1?, respectively, due to thelarge C-3 glycosylation shifts of DdC 9.09 and 11.18, respectively.

The sequence of the sugar residues in the O-antigen repeatingunit was determined from 1H,1H-NOESY and 1H,13C-HMBC exper-iments. Three-bond heteronuclear correlations were observedfrom the anomeric protons to the respective carbon atoms atthe substitution positions that define the sequence of the sugarresidues in the repeating unit (Fig. 3c) and correlations from ano-meric carbons to protons at glycosyloxylated carbons (Table 1).Consequently, the structure of the repeating unit of the O-antigenPS from E. coli O174 is:

The inter-residue correlations (Table 1) observed in the 1H,1H-NOESY spectrum were consistent with the proposed structure. Inaddition, a long-range NOE correlation was observed from theacetamido methyl group at 2.04 ppm (residue A) to the resonanceat 3.65 ppm (H-3 in B), which is in good agreement with a 3Dstructure of the polysaccharide predicted by CarbBuilder,8 in whichthe distance between atoms was estimated to be about 4.5 Å.

In the multiplicity-edited 1H,13C-HSQC NMR spectrum (Fig. 2)two cross-peaks of low intensity, denoted A0 and B0, were observedat dH1/dC1 4.85/102.46 and 4.71/102.94 (3JH-1,H-2 8.4 and 7 Hz,respectively). The corresponding spin systems were partially deter-mined by further analysis of the 1H,1H-TOCSY experiments ofincreasing mixing times, showing for the A0 spin system H-2 toH-6 protons of 4.85, 3.68, 3.56, 3.38, 3.48, 3.72, and 3.94 ppm, aswell as 3.68 ppm for H-5 in B0 having JH-4,H-5 = 9.4 Hz and a two-bond heteronuclear correlation to C-6 at 176.4 ppm. These experi-ments revealed correlations that correspond to N-acetylglucosa-mine and glucuronic acid residues similar to A and B, but withslightly altered chemical shifts. In addition, the 1H,13C-HMBC spec-trum revealed a cross-peak between H-1 in residue A0 and C-2 inresidue B0 (dH-2/dC-2 3.58/81.60 in B0). Furthermore, the 1H chemicalshifts of H-30 and H40 were <3.6 ppm whereas H-3 and H-4 of?2,4)-b-D-GlcpA(1? in the PS they are >3.6 ppm (cf. Table 1).These results indicate that the terminal part in the PS is b-D-GlcpNAc-(1?2)-b-D-GlcpA(1? and, consequently, the biologicalrepeating unit in the O-antigen is defined, having a ?3)-b-D-Galp-NAc-(1? residue at its reducing end. Integration of the resonancesat 4.85 and 4.81 ppm in the 1H NMR spectrum revealed that the PSpreparation consisted of 15 repeating units on average.

The high structural similarity of the PS from E. coli O174 and theCPS from E. coli K101 may then explain the observed cross-reactiv-ity,4 as these two polysaccharides only differ in one of the substi-tution positions at the D-GlcpA residue. The cross-reactivity

4 332

β ββ

β

4 333

β ββ

β

4 332

β ββ

β

4

β

O21

O174

K101

Figure 4. Comparison using CFG notation of the structures of the O-antigen PS fromE. coli O21 (top), O174 (middle) and the CPS of E. coli K101 (bottom).

Table 11H and 13C NMR chemical shifts (ppm) at 58 �C of the resonances from the O-antigenic polysaccharide of E. coli O174 and inter-residue correlations from 1H,1H-NOESY and 1H,13C-HMBC spectra

Sugar residue 1H/13C Correlation to atom (from anomeric atom)

1 2 3 4 5 6 Me CO NOE HMBC

b-D-GlcpNAc-(1? A 4.81 [8.3] 3.67 3.56 3.37 3.47 3.71, 3.93 2.04 H2, B C2, B(0.09) (0.02) (0.00) (�0.09) (0.01) (�0.02)102.76 {164} 56.86 74.54 71.13 77.01 61.96 23.11 175.54 H2, B(6.91) (�1.00) (�0.27) (0.07) (0.19) (0.11) (0.01) (0.05)

?2,4)-b-D-GlcpA-(1? B 4.68 3.61 3.65 3.78 3.69 H3, Da C3, D(0.03) (0.31) (0.12) (0.24) (�0.03) H4, D103.19 {165} 81.60 75.30 80.26 77.06 175.15 H3, D(6.42) (6.60) (�1.23) (7.57) (0.13) (�1.32)

?3)-b-D-GalpNAc-(1? C 4.53 4.01 3.83 4.17 3.70 3.77, 3.80 2.02 H4, B C4, B(�0.15) (0.11) (0.06) (0.19) (�0.02) (�0.04)101.54 {161} 51.72 81.10 68.70 75.64 61.94 23.50 175.42 H4, B(5.25) (�3.08) (9.09) (�0.15) (�0.34) (0.05) (0.38) (�0.36)

?3)-b-D-Galp-(1? D 4.51 3.68 3.68 4.10 3.64 �3.72 H3, C C3, C(�0.02) (0.23) (0.09) (0.21) (�0.01) (0.08)104.71 {161} 70.38 84.96 68.52 75.53 61.85 H3, C(7.34) (�2.58) (11.18) (�1.17) (�0.40) (0.01)

3JH-1,H-2 values are given in Hertz in square brackets and 1JH-1,C-1 values in braces. Chemical shift differences as compared to the corresponding monosaccharides are given inparentheses.26

a Overlap with H5, B.

4.5 4.6 4.7 4.8

80

84

B2C3B4

D3

A1

B1

C1 D1

3.73.83.94.0 2.0 2.1

175.2

175.6 A(CO)

B6

C(CO)

B5 B4

C2 A2

C(NAc) A(NAc)

13C

/ pp

m

1H / ppm

a

c

B2C3B4

B1C4 C2

B3B5

B3

3.63.84.04.24.44.6

80

82

b

Figure 3. Selected regions of the (a) band-selective constant-time 1H,13C-HMBC, (b)1H,13C-H2BC and (c) 1H,13C-HMBC NMR spectra of the O-antigen PS from E. coliO174 with pertinent annotations.

104 C. Fontana et al. / Carbohydrate Research 354 (2012) 102–105

observed between E. coli O174 and E. coli O21 may also be ex-plained in terms of the stereochemical structural similarities ofthe O-antigen polysaccharides,5 that is, gluco- and galacto-configu-rations for the sugars in and around the branch-point residue(Fig. 4).

In E. coli two biosynthesis pathways have been identified, viz.,the one typical for heteropolysaccharides denoted Wzy-polymer-ase-dependent and that commonly occurring for homopolysaccha-rides denoted ABC-transporter dependent.9 The WecA transferaseslink undecaprenol-phosphate and UDP-GlcNAc thereby formingGlcNAc-PP-Und that subsequently is used as an acceptor in the bio-synthesis of the enterobacterial common antigen or an oligosac-charide. The sequential additions of sugar residues to thisacceptor lead to an oligosaccharide with typically four or five res-idues in the case of E. coli and gene cluster analysis of the putativeglycosyl transferases is becoming an integral part of structural

determination of O-antigen polysaccharides.10,11 The oligosaccha-ryl-PP-Und is then translocated from the cytoplasmic side of theinner membrane by a protein designated Wzx to the perplasmicside. These Und-PP-linked O-units are subsequently polymerized,which requires the Wzy protein, by transfer of the reducing endof the nascent polymer to the oligosaccharyl-PP-Und acceptor. Thismay then from a given oligosaccharide result in a number of differ-ent O-antigen polysaccharides since for example, the position ofpolymerization may be different; the O-antigen polysaccharidesfrom E. coli O5ab and O5ac are examples of this.12

For the E. coli O157 O-antigen which has a tetrasacchariderepeating unit with a D-GalpNAc residue at the reducing end of thebiological repeating unit it was shown that the a-D-GalpNAc-PP-undecaprenyl acceptor, used in the preassembly of the tetrasaccha-ride oligosaccharide on the cytoplasmic side of the inner membrane,is synthesized from a GlcNAc-PP-Und precursor by the action of anepimerase, the gene of which is denoted Z3206.13 The gene sequencewas shown to have 100% identity to the E. coli O55 gne and E. coli O86gne1 of E. coli O55 and E. coli O86, respectively, the O-antigen

C. Fontana et al. / Carbohydrate Research 354 (2012) 102–105 105

structures of which have been determined and have D-GalpNAc atthe reducing end of the biological repeating units. For the develop-ment of a PCR assay for detection of the E. coli O174 and O177 humanpathogens part of their genomes were sequenced and annotated.14

Genes for O-antigen synthesis in the region between JUMPStartand gnd were putatively annotated and for E. coli O174 three genescorresponding to glycosyl transferases were found and the O-anti-gen polysaccharide was predicted to be composed of tetrasaccha-ride repeating units, in excellent agreement with the resultspresented herein. We analyzed the available E. coli O174 genomeusing BLAST,15 but were not able to find any corresponding similar-ity or sequence identity to the Z3206 gene. However, the plausibleGalNAc-PP-Und furnishing D-GalpNAc residue at the reducing endof the biological repeating unit of E. coli O174 may well be synthe-sized from a GlcNAc-PP-Und by a GlcNAc-PP-Und epimerase afterthe formation of GlcNAc-PP-Und by WecA, in a similar way to thebiosynthesis in E. coli O55, O86, and O157. Moreover, it would beinteresting to elucidate the actions of glycosyl transferases16 inE. coli O174 and K101 and the relationships between O-antigen poly-saccharide17 and capsular polysaccharide18 structures.

1. Experimental

1.1. Bacterial strain, conditions of growth and preparation ofpolysaccharides

The test strain for E. coli O174:H27, strain 2531-54, was origi-nally isolated from a case of human diarrhea and was obtained fromthe International Escherichia and Klebsiella Centre (WHO), StatensSerum Institut, Copenhagen, Denmark. The strain is negative for vir-ulence genes normally present in E. coli causing diarrheal diseasesin humans. The bacterium was grown, the LPS isolated and the del-ipidated polysaccharide purified as previously described.19

1.2. Component analyses

The PS was hydrolyzed with 2 M trifluoroacetic acid at 120 �C for2 h. The sample was then reduced with NaBH4 and acetylated, afterwhich it was analyzed with GLC. The absolute configuration of thesugar components was determined by GLC analysis of their acety-lated (+)-butyl glycosides derivatives ((+)-2-butanol, AcCl, 80 �C,overnight) essentially as described using also racemic 2-butanol.20

1.3. GLC analyses

The alditol acetates and the acetylated butyl glycosides of glu-curonic acid and glucosamine were separated on a PerkinElmerElite-5 column with hydrogen as the carrier gas (25 psi) using atemperature program of 150 �C for 2 min, 3 �C min�1 up to220 �C and then 10 min at 220 �C. The injector and detector tem-peratures were set to 220 and 250 �C, respectively. The butyl glyco-side derivatives of galactose and galactosamine were separated ona PerkinElmer Elite-225 column with hydrogen as the carrier gas(25 psi) using a temperature program starting at 130 �C, 5 �C min�1

up to 150 �C, 7 �C min�1 up to 220 �C and then 20 min at 220 �C.The injector and detector temperatures were set to 140 and250 �C, respectively. The columns were fitted to a PerkinElmerClarus 400 Gas Chromatograph equipped with flame ionizationdetectors. Retention times of the derivatives were compared withthose of authentic reference compounds.

1.4. NMR spectroscopy

NMR spectra of the PS (1 mg) in D2O solution (0.55 mL) wererecorded at 58 �C on a Bruker Avance III 700 MHz spectrometer

equipped with a 5 mm TCI Z-Gradient CryoProbe. Chemical shiftsare reported in ppm using internal sodium 3-trimethylsilyl-(2,2,3,3-2H4)-propanoate (TSP, dH 0.00) or external 1,4-dioxane inD2O (dC 67.40) as references.

The assignments of the 1H and 13C resonances for the PS fromE. coli O174 were obtained by 2D NMR spectroscopy using a mul-tiplicity-edited 1H,13C-HSQC experiment,21 1H,1H-TOCSY experi-ments22 with mixing times of 20, 40, 70, and 100 ms, a 1H,13C-H2BC experiment23 with a constant time delay of 33 ms and aband-selective constant-time 1H,13C-HMBC experiment.6 The latterwas recorded over a spectral region of 5.0 ppm in the direct dimen-sion and 9.0 ppm in the indirect dimension, with 2048 � 256 datapoints and 256 scans per t1-increment. A 50 ms delay was used forthe evolution of long-range couplings and a selective 13C excitationpulse (Q3 Gaussian cascade) of 2.5 ms was applied at the center ofthe carbonyl region. The inter-residue correlations were assignedusing a 1H,13C-HMBC24 experiment with a 63 ms delay for evolu-tion of the long-range couplings and 1H,1H-NOESY experiments25

with mixing times of 50 and 100 ms. The chemical shifts werecompared to those of the corresponding monosaccharides.26

Acknowledgements

This work was supported by grants from the Swedish ResearchCouncil and The Knut and Alice Wallenberg Foundation. Theresearch leading to these results has received funding from theEuropean Commission’s Seventh Framework Programme FP7/2007-2013 under grant agreement no. 215536.

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