Indian Journal of Chern is try Vol. 398, September 2000, pp. 674 - 679

Chemical structure of the core region of enteropathogenic Escherichia coli 0125, 0142 and 0158 lipopolysaccharide

Anup Kumar Datta* & Sumanta Basut

Department of Biological Chemistry, Indian Association for the Cultivation of Science, Jadavpur, Calcutta 700 032, India

Received 21 May 1998; accepted (revised) 31 December 1999

The core structures of E. coli 01 25/0142/0158 lipopolysaccharide has been invest igated using methylation analysis/mass spectrometry, chemical degradations and one-dimensional IH and l3C NMR spectroscopy. E. coli 0125 is found to be identical with R-I and 0142/0158 with R-3 type of enterobacterial core structure.

Over 173 serovars of Escherichia coli, based on serologically recognised LPS O-polysaccharide specific ities have been recorded '. The enteropatho­genic Escherichia coli group is subdivided into two classe~: Class I: members of which exhibit a localised adherence to Hep-2 cells and Class II : members of which exhibit either diffuse adherence or no adherence to Hep-2 cells2

. The surface of Gram­negetive bacteria, of which lipopolysaccharide is a major component, mediates its interaction with the hose. Lipopolysaccharides generally consists of three moieties: O-specific polysacc haride, a high molecular weight and major fraction determines the serological specificity, which is linked to central heteropoly­saccharide, the core, which is in turn, linked to a central acylated oligosaccharide of lipidic character, ca lled lipid-A. Recently, the core region of the LPS has gained interest due to its immunogenic and antigenic activi ties and its role in biological activities of lipid A 4. In contrast to O-antigen , the core structures exhibit a lowe,[ degree of diversity. The core region of the LPS is particularly important for maintaining the physiology of the bacteria. It consists of an inner and outer region, inner region contains heptose and KDO, however, outer region contains D­glucose, D-galactose, and in some cases N-acetyl-D­glucosamine. Dntill now, six different core structures (R-1, R-2, R-3, R-4, K-12 and B) have been identified in E. coliS. In this note, we report the results of chemical studies on the core region of lipopoly­saccharide of some enteropathogenic Escherichia coli.

tDeceased on 8th March, 1998

Experimental Section Bacterial culture. The enteropathogenic strain of

Escherichia coli 0125, 0142 and 0158 used in thi s study was obtained from Central Research In stitute. Kasauli , India. The bacteria were grown at 3rC in beef extract-peptone medium (150 mL, pH 7.2) containing beef extract (1 %), peptone (l %), sodium chloride (0.5%) and agar (1.5 %) in Roux bottles for 48 hr. The cells were harvested at an early stationary phase by gently shaking the cultures with 0.85% saline and isolated by centrifugation. The bacterial cells were then thoroughly washed wi th water and freeze dried.

Preparation of lipopolysaccharide and lipid free core oligosaccharide. The LPS from E. coli 0142 and 0158 strain was isolated using hot phenol-water method6

. Pure LPS was obtained by ultracentri ­fugation followed by gel permeation chromatography. The LPS (100 mg) was hydrolysed by 1 5% (vol/vol) acetic acid (20 mL) at lOODC for 2 hr on a boiling water bath and following the removal of the precipitated lipid A (20 mg) by centrifugation , the centrifugates were lyophilised. Gel permeation chromatography of the lyophilisate on a column of Sephadex G-50 gave a high molecular weight 0-specific polysaccharide, a core oligosaccharide, and a fraction containing phosphate and 2-keto-3-deoxY-D­manno-octulosonic acid (KDO).

Analytical methods. Neutral sugars were liberated by hydrolysis with 2 M trifluoroacetic acid at 120DC for 2 hr. The products were then converted to their alditol acetates and analysed by gas-liquid chromato­graphy (GLC) on a Hewlett Packard Model 5890 series II gas chromatograph equipped with a flame ionisation detector. Peaks were identified by co-

DA TT A el al. : CORE STRUCTURES OF E. COLI LIPOPOLYSACCHARIDE 675

chromatography with authentic standards. The alditol acetates were resolved on a glass column (1.8 m x 6 mm) containing 3% ECNSS-M on Gas Chrom Q (100-120 mesh) at l70°C and HP-I fused silica column (0.25 mm x 30 m) at 160°C for 2 min followed by an increase of 2°C/min to 240°C. The head pressure of the column was 105 Kpa. Amino sugars were detected with a glass column packed with 3% Poly-A 103 on Gas Chrom Q (100-120 mesh) at 200°C after hydrolysis with 4 M HCI at 100°C for 6 hours. Paper chromatography was performed on Whatman No.1 and No.3 MM papers . The solvent systems were (1) 8:2: 1 (voUvoUvol) ethyl acetate­pyridine-water, (II) 6:4:3 (voUvoUvol) I-butanol­pyridine-water (upper layer), and (III) 5:5: 1:3 (voUvollvol) ethyl acetate-pyridine-acetic acid-water. 2-Keto-3-deoxY-D-manno-octulosonic acid (KDO) was detected by thiobarbituric acid method? after hydrolysing the samples with 1 M HCI at 100°C for 30 minutes . Gel filtrations were performed on columns of Sephadex G-100 (70 x 1.5 cm) and Sephadex G-50 (70 x 1.5 cm) using a Gilson fraction collector (Model 201), and the eluate was monitored with a Waters Associates Differential Refractometer (Model 403) fitted with a recorder. Optical rotations were measured by using a Perkin-Elmer 241 MC spectropolarimeter.

Methylation analysis. Samples (2-4 mg) were methylated according to the method of Ciucanu and Kerek8 and the products were isolated by partition between dichloromethane and water. The products were further purified by passage through a Sep-pak C I8 cartidge9

. Methylated products were hydrolysed by 2 M trotluoroacetic acid at 120°C for 2 hr, reduced with sodium borohydride, acetylated with acetic anhydride in pyridine, and analysed by GLC or GLC­MS or both.

Periodate oxidation and Smith degradation. Core oligosaccharide (35 mg) in 0.1 M sodium metaperiodate solution (10 mL) was kept in the dark for 3 days at 24°C. The excess of periodate was destroyed by the addition of ethylene glycol (0.25 mL) and the resulting polyaldehyde was reduced with sodium borohydride (200 mg) at 24°C for 16 hr. Excess of borohydride was neutralised with 50% acetic acid and dialysed against distilled water for 2 days. The product was recovered by lyophilisation of the dialysate. The polyol was treated with 0.5 M CF)COOH (10 mL) at 24°C for 24 hr. The degradation product was fractionated on a column of Bio-Gel P-2 (200-400 mesh).

Partial periodate oxidation. The 0125 core oligosaccharide was dissolved in water at 5°C and 0.2 M NaI04 at 5°C added. The solution was kept in the dark at 5°C for 6 hr and product worked-up as above.

N-deacetylation-deamination. The COS (16 mg) was dissolved in water (l mL) and dimethyl sulphoxide (5 mL). The solution was stirred with sodium hydroxide (400 mg) and thiophenol (200 mL) under nitrogen for 20 hr at 100°C. The excess sodium hydroxide was neutralised with 2 M HCI and dialysed against distilled water. The dialysate was centrifuged to remove the insoluble solids and the supernatant Iyophilised. The product was purified by gel chromatography on Bio-Gel P-2 column using water as eluant. Aqueous solutions of sodium nitrite (5%, 2 mL) and acetic acid (33%, 2 mL) were added to a solution of N-deacetylated COS (10 mg) in water and the mixture was kept for 45 min at room temperature. The deaminated COS was isolated by passing through a column of Bio-Gel P-2 followed by Iyophilisation.

Dephosphorylation. The COS was dephosphory­lated with aqueous HF (48%, 4°C, 48 hr) .

Enzymatic hydrolysis. COS or degraded products was treated with a-D-glucosidase/a-D-galactisidase in 0.07 M Na3P04 buffer (PH 6.8) or by ~-D~

glucosidase/~-D-galactosidase in 0.05 M NaOAc buffer (PH 5.0) for 16 hr at 37°C in presence of toluene.

Results and Discussion E. coli 0125, 0142 and 0158 strains were grown

in large quantities on Beef extract-peptone medium. The LPS was isolated in a pure state by hot phenol­water method in a yield of 10% of the dry bacterial weight. The crude LPS after ultracentrifugation followed by cetavlon precipitation found to be free from protein and nucleic acid. The purified LPS on mild acid hydrolysis (2% AcOH) yielded an insoluble lipid-A, precipitated out, and Sephadex G-50 gel filtration of the concentrated water soluble products afforded O-specific polysaccharide (40%, 39% and 42%), core oligosaccharide (15%, 14% and 16%) and a fraction containing phosphate and KDO (5%) for 0125, 0142 and 0158 LPS respectively. Analytical data of 0142/0158 COS material revealed the presence of D-glucose, D-galactose, 2-icetamido-2-deoxY-D-glucose and L-glycero-D-manno-heptose in the molar ratio 3: 1: 1 :2.7 (Table I) while 0125 COS material revealed the presence of D-glucose, D­galactose, L-glycero-D-manno-heptose in the molar

676 rNDiAN J CHEM, SEC B, SEPTEMBER 2000

ratio 3:2:2.7 along with the usual components of the core material like phosphorous, ethanolamine and 2-keto-3-deoxY-D-manno-octulosonic acid (KDO) (Table II) . The optical rotation values for 0125 core oligosaccharide (COS) are {[a]D = +47° (c 0.5, water)}, 0142 core oligosaccharide (COS) {[a]D=+190° (c 0 .5, water)} and for 0158 COS {[a]D= +191 ° (c 0 .5, water)}. The absolute configura­tion of the sugar were determined by preparative paper chromatography on Whatman No.3 MM paper. Heptose was identified as L-glycero-D-manllo-heptose by gas-liquid chromatography.

Methylation analysis of 0142/0158 core indicated the presence of terminal glucose, terminal N­acetylglucosamine, 2- and 3-substituted glucoses, 2,3-disubstituted galactose and 3,7-disubstituted heptose

Table I - Analytical data of the core oligosaccharide 0142/0158

Core D-Glc D-Gal D-GlcNAc LD-Hep D-Man 01 igosaccharide

Original 3.0 1.0 1.0 2.7 Deamination 3.0 1.0 0.2 6 Smith degraded 1.0 0.9 0. 1 1.0 0.8 DeaminatediSmith 1.0 0.2 0.1 1.0 1.0 degraded

*Phosphorous, ethanolamine and KDO content were not detennined

Table II - Analytical data of the core oligosaccharide 0125

D-Glc D-Gal LD-Hep D-Man

Core oligosaccharide 3.0 1.0 2.7 Original Smith degraded partial 3.1 1.0 0.8 Smith degraded 3.4 1.0 1.0

*Phosphorous, ethanolamine and KDO content were not determined

(Table III). Methylated products derived from phos­phorylated heptoses were obt.ained in non-stoichio­metric amounts. After dephosphorylation of core, terminal heptose was formed. and the yield of 3-substituted heptose increased. These results proved that the branched and the 3-substituted heptoses are phosphorylated at C-7 and at C-4, respectively. The difference between core and deaminated core is a considerable decrease of the terminal N­acetylglucosamine, and 2,3-disubstituted. galactose and the presence of a corresponding amount of 2-substituted galactose in deaminated core. Methylation analysis of Smith degraded and dearrunatedlSrruth degraded revealed that Smith degraded core seems to be a tetrasaccharide composed of terminal galactose, 3-substituted glucose, heptose and man nose, and deaminatediSmith degraded core a trisaccharide composed of terminal glucose, 3-substituted heptose and mannose. The yield of the methylated product of the man nose component in Smith degraded core was low due to its phosphorylation. The results of periodate oxidation and methylation analysis lead to the formulation of structures for the degradation products and eventually for the oligosaccharides (Table IV). The branch point for terminal non­reducing heptose residue remains undetermined. For this reason, two alternative structures of core, and consequently of each of the degradation products have been suggested in their formulae. By analogy to Enterobacteriaceae it seems most probable that in E. coli core the branch point for the heptose residue vicinal to the hexose region of the core.

The IH NMR spectrum (Figure 1) of 01421 0158 core exhibits all of the anomeric proton resonances in the region 4.9-5 .9 ppm, which is characteristic of a­anomers. Other characteristic signals are 2.03 for N-

Table III - Methylation analysis of the core oligosaccharide 01421 0158 before and after chemical degradations

Methylated sugar Origi nal Deaminated Smith degraded Deaminated/ Dephosphorylated

2.3,4,6-Me4-GIc 1.0 2,3,4,6-Me4-Gal 2,4.6-Mer GIc 1.0 3,4,6-MerGlc 1.0 2,4.6-Mer Man 3,4,6-MerGal 4,6- Me2-Gal 1.0 3,4,6-Mer GlcNAc 0.9 2.3 ,4,6,7 -Mes-Hep 2,4,6.7 -Mc4-Hep 0.1 2.6.7-MerHep 04 2.3 4 ,6-Me4-Hep 0.2 2,4 ,6-Mer Hep 0.8

1.0

1.0 1.0

0.8 0.1 0.2

0.4

0.4

0.9 1.0

0.2

1.0

Smith degraded

1.0

1.0

1.0

1.0

1.0 1.0

0.6 0.5 0.7 0.5

0.4

DA ITA e/ at.: CORE STRUCTURES OF E. COLI LIPOPOLYSACCHARIDE 677

Table IV -Structure of the 0142/ 0158 core oligosaccharide and its degradation products [ Dashed lines ( ..... ) indicate alternative lecations. [dOdA] indicates the remainder of dOdA after·Smith degradation]

Oligosaccharide

Native core

Deaminated core

Smith degraded core

aoGlcNAc 1 .J-" 3

Structure

LaoHepp 1

LabHepp, 1

7 " ' ...... , ' .

.' ,/ 30Manpl~3LaDHeppl~[d OdA] ., \\,

\. 3LaoHeppl~30Manpl~[d OdA]

DeaminatedlSmith degraded core

,-' 30M anpl~3LaoHeppl~[d OdA]

aoGlcp -:< 3LaoHeppl~30Manpl~[d OdA]

5.80 5.40 5.00 4.60

ppm 4.20 3,80

I , 1 .

\tj~"II , A,

~ ~ II 1\ ·:oI;JL

3.40

Figure 1-500 MHz IH NMR spectrum of 0 158 core oligosaccharide

acetyl group. The 13C NMR spectrum (Figure 2) shows all of the anomeric carbons in the region 100-104.3 ppm, signals at 50.5, 175.7 for N-acetyl group. The results obtained by this procedures showed consistently the structure of 01421 0158 core oligosaccharide is an R-3 type of enterobacterial core structures.

The IH NMR spectrum (Figure not shown) of 0125 core showed signals of one proton at 4.77 ppm, JI.2 = 7 Hz and signals for other anomeric protons at 4.86-5.82 ppm. This results indicated that it contains

one f)-linked hexosyl residue, but other hexosyl and heptosyl residues are a-linked. 13C NMR spectrum (Figure not shown) showed all the anomeric carbons in the region 99-105 ppm, other characteristic signals are the same as observed for 0142/0158 core except that of the D-glucosamjne residue. On Smith degradation of COS, two D-galactopyranosyl residues, terminal D-glucopyranosyl and terminal heptosyl residue should be selectively eliminated. In the IH spectrum of this product, all signals for anomeric proton appeared at low field, 4.95-5.34 ppm which

678 INDIAN 1 CHEM, SEC B, SEPTEMBER 2000

)

I

I I I

-------.-----------.----------~----------~----------~--------~------1 1~1-.---

16 0 140 120 100 80 60 10

S (ppm) Figure 2 - 125 MH z DC NMR spectrum o f 01 58 core oli gosaccharide

fu rther supports that the innermost D-glucose res idue

is a -linked. Two galactopy ranosy l res idues contain c is-ori ented vicinal hyd roxy ls and these are readil y ox idi sed by periodate than the terminal D­g lucopyranosy l g roup. It was, there fore, poss ible to remove them se lective ly by a Smith degradation, in which a short reaction time was used for the periodate ox idation. lJl the 'H spectrum of the product , the

signal for the B-linked hexosyl res idue at 4 .66 ppm, h~ = 7 Hz was present, indicating that the te rmina l D-glucopyranosyl res idue in the core is actually B­linked. In agreement with these conclusio n D-glucose was re leased when the degraded product was treated with BD-glucosidase but not whe n it was treated with

aD-glucos idase . The ori ginal R-l core was inert to both these enzy mes, but it is often observed that a te rminal g lucosy l group, present as a branch, is di ffic ult to hydrol yse off enzy matica ll y. T o establi sh the sequence and ano meric confi guration , the enzymatic degradation o f core by aD-ga lactosidase was performed. It was fo und that the nati ve COS were res istant to a high degree to thi s treatment. Dephos­phorylated core, however, re leased 60% of the D-

Table V - Methylat ion analys is of the core ol igo accharidc 0125 before and after chcmi cal degradat ions

Mcthylated sugar Original Smith Part ial Smith degraded degraded

'2.3,4,6-Mc4-G lc 1.1 1.0 1.0 2,3,4.6- Mc4-Gal 1.0 2,4.6-Mcr Glc 1.2 1.0 1.2 3.4.6-Mer Gal 0.8 1.0 2.4.6-MerMan 0.2 3,4.6-Mer Gal 4.6- MCr Glc 1.0 0.3 2,3,4.6.7-Mes-Hcp 0 9 0.4 2.4,6.7-Mc4- Hcp 0.8 1.0 0.5 2,6,7-Mcr Hcp 2.3.4.6- Mc4-Hcp 2.4.6-Mcr Hcp 0.8 0.4

*2.3 .4.6-Mc4-Glc = 2.3.4,6-tctra-O-methyl-D-glucosc

galactose present after aD-glucos idase ac ti on. The enzymatic da ta together with methylati on ana lys is (Table V) and 'H NMR analysis proved that an

aDGal p-(l~2)-aDGal p sequence occ urs in the core.

All these result s obtained by these proced ures showed th ~ structure of 0125 core 01 igosacc haride is an R-l type e nterobacterial core structure.

7

LaDHepp

I -- , , , , ,

aDGalp l ~2aDGalp l~2aDGlcp l ----j3aDG lcp l ~3LaDHeppl ~3LaDHepp l ~ d OclA (01 25 core ol igosaccharide)

DA 1T A et al.: CORE STRUCTURES OF E.COLf LIPOPOLYSACCHARIDE 679

Acknowledgement The authors express their sincere thanks to

Professors N. Roy, B.P. Chatterjee, A.K. Guha of our department for their constant help and encouragement during the work and to Mrs Lily Sarkar for microbiological work. Anup Kumar Datta extends his gratitude to CSIR, New Delhi , India for providing him with a Junior Research Fellowship. The work was partially supported with financial assistance from grant No.SP/SOID39/94, Department of Science and Technology.

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