Identification of a Novel Core Type in Salmonella Lipopolysaccharide COMPLETE STRUCTURAL ANALYSIS OF THE CORE REGION OF THE LIPOPOLYSACCHARIDE FROM SALMONELLA

From the ‡Department of Mass Spectrometry, Bijvoet Center for Biomolecular Research, Utrecht University, NL-3508 TB Utrecht, The Netherlands, the §Department of Chemistry, Carlsberg Laboratory, DK-2500 Valby, Denmark, the ¶Max-Planck-Institute for Immunobiology, D-79108 Freiburg, Germany, and the iDivision of Medical and Biochemical Microbiology, Center for Medicine and Biosciences, Research Center Borstel, D-23845 Borstel, Germany

For the first time, the complete structure of a lipopolysaccharide (LPS) core region from Salmonella enterica has been identified that is different from the Ra core type generally thought to be present in all Salmonella LPS.The LPSs from two rough mutants and the smooth form of S. enterica sv.Arizonae IIIa O62, which all failed to react with an Ra core type-specific monoclonal antibody and were resistant to phage FO1, were analyzed after chemical modification using monosaccharide analysis, mass spectrometry, and NMR spectroscopy.In the novel core type, the terminal D-GlcNAc residue present in the Ra core type, is replaced by a D-Glc residue.The Ospecific polysaccharide is ␣134-linked to the second distal Glc residue of the core.Furthermore, phosphoryl substituents attached to O-4 of L-glycero-D-manno-heptose (Hep) I and II were identified as 2-aminoethyl diphosphate (on Hep I) and phosphate (Hep II).Lipopolysaccharides (LPSs), 1 major components of the outer membrane of Gram-negative bacteria, are composed of covalently linked structural domains that are differentiated by their structure, genetics, biosynthesis, and biological properties.They consist of the O-specific polysaccharide, the inner and outer core oligosaccharides, and the lipid A (1). Smooth form bacteria synthesize LPSs that are built up of all three structural regions, while LPSs from rough type bacteria comprise only the lipid A and the core region, due to genetic defects in the rfb locus, which encodes O-specific polysaccharide biosynthesis.Furthermore, mutations in the rfa locus encoding core biosynthesis may lead to truncated rough type LPSs (2,3).Such mutants were first identified in Salmonella enterica sv.Minnesota, and the analysis of their LPSs led to the elucidation of the structure of the Ra type core region (Fig. 1) (2).In contrast with the many diverse structures of O-specific polysaccharides identified in LPS from S. enterica (4), to date only one core type (the Ra core) has been identified in LPSs from S. enterica.
The current procedure for detecting Salmonella in food or other specimens involves the isolation and identification of the organisms using biochemical and serological methods that are time-consuming, labor-intensive, and also expensive.The application of immunological methods for detecting Salmonella either directly in food or in selective media could help to circumvent these problems.The success of an immunological approach depends on the availability of a specific antibody, and the outer core region was thought to be common to the LPSs of all S. enterica serovars and was thus suggested to represent a genus-specific antigen against which such an antibody could be raised.Two murine monoclonal antibodies (mAbs) T6 (5) and 105 (6) were obtained, the epitopes for which were identified in the distal part of the outer core region of S. enterica LPS.However, some serotypes, including S. enterica sv.Arizonae IIIa O62, failed to react with these mAbs (6 -9), suggesting the presence of a modified core oligosaccharide structure in some serotypes.mAb T6 is known to be specific for the T6 epitope, which comprises the ␣132-linked GlcNAc residue in the Ra type core.To investigate the possibility of developing mAb T6 as a polyvalent Salmonella serological reagent, and to gain information on the molecular diversity of the Salmonella LPS outer core structure, Tsang et al. (7,8) tested a broad variety of S. enterica strains for the presence of the T6 epitope.Ten serotypes of subspecies IIIa were identified that did not react with mAb T6.In early structural investigations of LPSs from S. enterica sv.IV and sv.Djakarta (9), the results of monosaccharide and methylation analyses led to the proposal of the existence of two novel LPS core structures in the LPS of S. enterica.Here, we describe the results of detailed structural analysis of the core region of LPSs from S. enterica sv.Arizonae IIIa O62, and of the linkage position of the O-specific polysaccharide.This report represents the first complete characterization of a novel LPS core type from S. enterica and allows the lack of reactivity with mAb T6 to be rationalized in terms of chemical structures.

EXPERIMENTAL PROCEDURES
Bacteria and Bacterial LPS-S.enterica sv.Arizonae IIIa O62 (smooth form), its rough mutants R4 and R5, and S. enterica sv.Minnesota R60 were obtained from the strain collection of the Max-Planck-Institut fu ¨r Immunbiologie, Freiburg, Germany.Bacteria were grown at 37 °C in a fermentor using media and growth conditions as described (10); washed successively with ethanol, acetone (twice), and ether; and then dried.From smooth bacteria and R4 and R5 mutants, the LPSs were isolated using the hot phenol/water method (11), and from the R4 mutant, LPSs were extracted in addition with phenol/chloroform/light petroleum (12) for purposes of comparison, as were LPSs from S. enterica sv.Minnesota R60 (Ra chemotype).The two differently extracted LPSs from mutant R4 are in the following designated as R4a (from phenol/water extraction) and R4b (from phenol/chloroform/light petroleum extraction).
Smooth form LPSs (202.1 mg) were hydrolyzed with 1% acetic acid (100 min, 100 °C), and the polysaccharide fraction (105.8 mg, 52.4% of the LPS) was isolated after centrifugation (100,000 ϫ g, 30 min) and lyophilization of the water phase and was separated using gel-permeation chromatography on Sephadex G50.The second fraction (15.0 mg, 7.4%) was further purified by semipreparative high performance anionexchange chromatography (HPAEC) at 4 ml min Ϫ1 as described (14,15) with the modification that the column was eluted using a gradient program of water to 120 mM aqueous sodium acetate (pH 6.0) over 25 min, followed by isocratic elution at 120 mM sodium acetate over 35 min.The purity of the fractions obtained was analyzed by analytical HPAEC (15), and the second fraction (fraction SR) was desalted on Sephadex G50, lyophilized (2.7 mg, 1.3% of the LPS), and used for further analyses.
Mild base treatment of 100 g of the smooth form LPS was carried out for MS analysis in 250 l of methanol and 250 l of ammonium hydroxide solution (25% NH 3 in water) overnight at ambient temperature followed by lyophilization.
The LPSs from S. enterica sv.Minnesota R60 (100 mg) were first dephosphorylated with 48% aqueous HF (5 ml, 48 h, 4 °C), then reduced and de-O-acylated (13).The sample (50 mg, 50% of the LPS) was de-N-acylated (10 mg ml Ϫ1 ) with 4 M KOH and separated using gelpermeation chromatography on Sephadex G10.The fractions obtained were lyophilized, and their purity and composition were investigated by 1 H NMR spectroscopy.The third fraction comprising the complete carbohydrate chain of the core-lipid A region was used for NMR analyses (yield: 2.3 mg (2.3% of the LPS)).
General and Analytical Methods-The conditions for gas chromatography (GC), gas chromatography-mass spectrometry (GC-MS), gel-permeation chromatography, and determination of the absolute configurations of monosaccharide residues were as reported (16), as well as the analytical methods for the determination of phosphates, GlcN, Kdo, and neutral monosaccharide residues (17).Acetylation and methylation together with the remaining steps of linkage analysis (hydrolysis, reduction, and acetylation) were carried out as described (18).
For methylation analysis of the Kdo region (19), the LPS from S. enterica sv.Arizonae mutant R5 was methylated according to Ref. 20, followed by evaporation of methyl iodide and then dialysis.This procedure was repeated, followed by carboxyl reduction (NaBH 4 ), dialysis, and a third methylation.The sample was hydrolyzed with 0.1 M trifluoroacetic acid (1 h, 100 °C), evaporated, carbonyl-reduced (NaB 2 H 4 ), and divided into two halves.One half was acetylated and analyzed by GC-MS.The other half was methylated with C 2 H 3 I, then hydrolyzed with 1 M trifluoroacetic acid (1 h, 120 °C), carbonyl-reduced (NaB 2 H 4 ), acetylated, and analyzed by GC-MS.For methylation analysis of the complete core region, S-form LPS was dephosphorylated, then reduced, and de-O-acylated.Three mg of this sample were methylated according to (20), after which methyl iodide was evaporated and the sample was dialyzed, followed by repetition of this procedure.The sample was divided into two halves, one of which was successively hydrolyzed in 90% aqueous formic acid (1 ml, 3 h, 100 °C) and 0.25 N sulfuric acid (H 2 SO 4 , 1 ml, 16 h, 100 °C), and the other in 4 M trifluoroacetic acid (1 ml, 4 h, 100 °C).After neutralization, the samples were N-acetylated (21), reduced (NaB 2 H 4 ), acetylated, and analyzed by GC-MS.For methylation analysis of the core region of mutant R5, its de-O-acylated LPS (4 mg) was methylated according to Ref. 20, followed by evaporation of methyl iodide and dialysis, and repetition of this procedure.The sample was hydrolyzed in 4 M trifluoroacetic acid (1 ml, 4 h, 100 °C), evaporated, reduced (NaB 2 H 4 ), acetylated, and analyzed by GC-MS.
Mass Spectrometry-Positive and negative ion mode fast-atom-bombardment mass spectrometry (FAB-MS) was carried out using the first two sectors of a Jeol JMS-SX/SX102A tandem mass spectrometer operating at 7 kV (mass range m/z 200 -3500), 8 kV (mass range m/z 200 -3000), or 10 kV (mass range m/z 200 -2400) accelerating voltage.The FAB gun was operated at an emission current of 10 mA with xenon as the bombarding gas.Spectra were scanned at a speed of 30 s for the full mass range specified by the accelerating voltage used, and were recorded and processed on a Hewlett-Packard HP 9000 data system.Tandem mass spectra (FAB-MS-MS) were obtained following collisioninduced dissociation (CID) in the third field free region using the same instrument under similar conditions with helium as collision gas at a pressure sufficient to reduce the parent ion to one half of its original intensity.In all experiments, the matrix used was thioglycerol (2-3 l) acidified by adding 1 l of concentrated trifluoroacetic acid in water (for underivatized samples) or 1 l of methanolic hydrochloric acid (for derivatized samples).Underivatized samples were dissolved in water, and derivatized samples were dissolved in methanol.The probe was loaded with 1 l of sample solution.
GC-MS was performed on a Fisons MD 800 mass spectrometer fitted with an Interscience 8000 gas chromatograph using an on-column injector with helium as the carrier gas.The monosaccharide derivatives were separated on a DB-5MS column (0.25 mm ϫ 30 m, J&W Scientific) using the following temperature program: 50 °C for 2 min, a gradient of 40 °C min Ϫ1 to 130 °C, 130 °C for 2 min, a gradient of 4 °C min Ϫ1 to 230 °C, and finally 230 °C for 2 min.Mass spectra were obtained under conditions of electron impact in the positive ion mode and were recorded by linear scanning of the mass range m/z 55-400 at an ionization potential of 70 eV.
Fraction SR (2.7 mg) was dissolved in 2.7 ml of water so that a final concentration of about 1 g l Ϫ1 was obtained.Negative mode electro-spray ionization (ESI) mass spectra were obtained on a VG Platform II single quadrupole mass spectrometer.Aliquots of 5 l of the sample were infused into a mobile phase of acetonitrile/water (50:50, v/v) and introduced into the electrospray source at a flow rate of 5 l min Ϫ1 .Spectra were scanned at a speed of 10 s for m/z 800 -2200, with a cone voltage of Ϫ80 V or Ϫ60 V and recorded and processed using the MassLynx software, version 2.0.Mass calibration was performed by multiple-ion monitoring of horse-heart myoglobin signals.
NMR Spectroscopy-Structural assignments were made from sample solutions in 2 H 2 O (about 3.8 mg ml Ϫ1 ) at p 2 H 7.1.Spectra were recorded in 5-mm tubes at 500.13 and 600.13 MHz for 1 H and 125.77 or 150.90 MHz for 13 C with a Varian Unity INOVA 500 or a Bruker AMX 600 spectrometer, and chemical shifts reported relative to internal acetone (2.23 ppm, 2 HOH at 4.67 ppm at 37 °C).Coupling constants (Ϯ0.5 Hz) were determined on a first-order basis.The 13 C resonances are reported relative to internal dioxane (67.4 ppm).A one-dimensional 1 H NMR spectrum was recorded on the Bruker AMX 600 spectrometer using 16,000 data points and a sweep width of 6250 Hz.
The phase-sensitive correlated spectroscopy (COSY) experiments were performed using double-quantum filtering (22,23) with the Bruker COSYPHDQ microprogram.In the F 2 dimension, 4000 data points were collected giving an acquisition time of 0.492 s.The data matrix was zero-filled in the F 1 dimension to give a matrix of 4000 ϫ 2000 points and was resolution-enhanced in both dimensions by a shifted sine-bell function before Fourier transformation.The total correlation spectroscopy (TOCSY) (24) and the nuclear Overhauser enhancement spectroscopy (NOESY) (25) experiments were carried out in the phase-sensitive mode according to the method of States et al. (26).A decoupling in the presence of scalar interactions (27) spin lock pulse sequence was used in the TOCSY experiment with a mixing time of 125 ms and a spin lock power of 7300 Hz.The NOESY experiments were performed with a mixing time of 200 ms.The intensities of NOESY cross-peaks were classified as either strong, medium, or weak using cross-peaks from intra-ring proton-proton contacts for calibration.
The 1 H, 13 C correlation experiment was performed in the inverse mode, as a heteronuclear multiple quantum coherence (HMQC) experiment (28).This uses the bilinear rotation decoupling (29) pulse sequence to suppress protons attached to 12 C with a pulse delay of 400 ms (30). 13C couplings were decoupled during acquisition using the globally optimized alternating phase rectangular pulse sequence (31) with a transmitter power corresponding to a 90°pulse of 106 ms.The experiment was performed in the phase-sensitive mode by the States timeproportional phase incrementation phase-cycling method (32) acquiring a total of 4000 points over a sweep width of 4545 Hz in F 2 and 1000 in F 1 over 13,500 Hz.Processing was performed using standard Bruker software after zero-filling to 2000 in F 1 .
Broad band 1 H-decoupled 31 P NMR spectra were recorded at 101.25 Hz on a Bruker DRX 250 at ambient temperature.Spectra were acquired using a sweep width of 10,100 Hz and 8000 data points.Pulses of 90°were applied for 9 ms with a pulse repetition time of 0.4 s and a pre-acquisition delay of 2 s.Chemical shifts were given in ppm relative to an external reference of 85% phosphoric acid.A proton-detected one-dimensional 1 H, 31 P HMQC spectrum was recorded on the same instrument at ambient temperature with an evolution delay of 60 ms using a sweep width of 2000 Hz and 4000 data points.The two-dimensional 1 H, 31 P HMQC experiment was carried out under the same conditions using a sweep width of 624 Hz.In the F 2 dimension, 1000 data points were collected giving an acquisition time of 0.82 s.The data matrix was zero-filled twice in the F 1 dimension to give a matrix of 1000 ϫ 256 points using linear prediction.
The COSY, TOCSY, NOESY, and HMQC spectra were assigned using the computer program PRONTO (33), which allows the simultaneous display of different two-dimensional spectra and the individual labeling of cross-peaks.The HSEA/Monte Carlo calculation was performed as reported (34).

RESULTS AND DISCUSSION
Compositional Analysis of LPS-The compositions and structures of the core region of S. enterica sv.Minnesota R60 (reviewed in Refs. 2 and 35) and of the O-specific polysaccharide of S. enterica sv.Arizonae O62 (36) have been reported.Monosaccharide and phosphate analyses of LPSs from S. enterica sv.Arizonae O62 rough mutants R4 and R5 (Table I) reveals the presence of Glc, Gal, L-glycero-D-manno-heptose (Hep), 3-deoxy-D-manno-oct-2-ulopyranosonic acid (Kdo), GlcN, organic phosphate, and small amounts of Rha (indicating a smooth LPS fraction) in LPSs from the rough mutants.GC analysis of the acetylated (R)-butyl glycosides showed that Glc, Gal, and GlcN residues possess the D-configuration.The Lconfiguration of Rha was determined by others (36).The fatty acids identified in the LPS preparations are 3-hydroxytetradecanoic acid, tetradecanoic acid, hexadecanoic acid, and dodecanoic acid.
The low relative molar ratios of GlcN (Table I) determined for the rough mutant LPS samples are noteworthy.The values suggest that one of the three expected GlcN residues is absent; since the lipid A moiety should possess two N-acylated GlcN residues, we assume that the GlcNAc residue in the outer core region (compare Fig. 1) is the one that is absent.In addition, the data indicate the presence of only one Glc residue.An additional quantification of Kdo after hydrolysis with 0.1 M sodium acetate buffer (pH 4.4, 100 °C, data not shown) (37) revealed one terminal Kdo residue in each LPS.
The success and fidelity of chemical manipulations were checked by monosaccharide analysis.These data (not shown) show no significant differences from those for the unmodified samples presented in Table I.
Mass Spectrometry of Rough Type LPS-Mass spectrometry was used to determine the details of the core structures present in the LPSs from mutants R4a, R4b, and R5.The dephosphorylated, reduced, and deacylated LPS preparations were desalted on Sephadex G50 and then examined using positive ion FAB-MS.Analysis of the early eluting G50 fraction obtained from the LPS from strain R5 (Fig. 2) yields a spectrum containing a series of intense [MϩH] ϩ pseudo-molecular ions in the region m/z 1300 -2050.The most intense ion at m/z 1845 corresponds to a species with a monosaccharide composition of Hex 3 Hep 3 Kdo 2 (HexNH 2 ) 2 , representing a species having a monosaccharide composition similar to that of the Salmonella Ra core with the lipid A but lacking one HexNH 2 and Hex residue.A related but very minor ion is present at m/z 2007, which would correspond to a species with the same composition as that giving rise to the ion at m/z 1845, but bearing an extra Hex residue.In addition, the ion at m/z 1683 corresponds to a species again related to the Ra core but this time lacking one   ϩ oxonium ion is present in the spectrum, while the ion for Hex 2 ϩ is notably absent.Thus, from our data, we conclude that the major component having a composition of Hex 3 Hep 3 Kdo 2 (HexNH 2 ) 2 , contains a branched hexose structure, as well as a branched heptose arrangement.
Methylation Analysis-Methylation analysis of the LPS from the R5 mutant (19) was performed to determine the linkages in the Kdo region.After hydrolysis of the methylated sample with 0.1 M trifluoroacetic acid, the derivative for a terminal (B in Fig. 4) Kdo was identified, although the derivative corresponding to a 4-substituted Kdo residue indicating a Kdo234Kdo branch was not observed.After additional strong acid hydrolysis with 1 M trifluoroacetic acid and further derivatization, a 4,5-disubstituted Kdo residue was detected.This product originates from the first Kdo residue (A) that links the core region to lipid A and is substituted on O-5 by the first Hep (C) residue and on O-4 by the second Kdo residue (B, product obtained after mild acid hydrolysis).On additional methylation analysis to determine the substitution pattern of hexoses and heptoses, derivatives corresponding to 3,7-disubstituted Hep, 3,6-disubstituted Hex, 3-substituted Hep, 6-substituted Hex, and terminal Hep and Hex residues were identified, indicating the presence of a Glc residue that is substituted on O-3 and O-6 by Gal.Derivatives corresponding to either 2-or 3-substituted Hex were not detected.These data confirm the presence of a branched Hex 3 outer core containing a 3,6-disubstituted Hex residue.In the outer core region of the minor component (see MS results above), the 3-linked Gal residue is absent (Rb 3 chemotype), which we assume is due to incomplete biosynthesis.The presence of such a minor Rb 3 LPS fraction (2) is consistent with the results of immunoblot analyses of various rough type LPSs from S. enterica (45), which indicated the presence of different terminal structures in each of these LPSs.
In summary, the core oligosaccharides from the LPSs of the rough mutants of S. enterica sv.Arizonae IIIa lack the distal GlcNAc and Glc residues found in the common Salmonella Ra core.This indicates that, in the rough mutants, biosynthesis or transfer of the distal two monosaccharide residues is absent.A minor component was identified that also lacks the 3-linked Gal residue (i.e.Rb 3 chemotype LPS), probably as a result of incomplete core biosynthesis.Thus, the major LPS fractions from the rough type strains investigated are of the Rb 2 chemotype (2).
Analysis of the Core Region from Smooth Form LPS-To determine whether the changes to the Ra core observed in the rough mutant LPS preparations are due to mutations leading to the Rb 2 chemotype or are indeed indicative of the occurrence of a novel core structure in Salmonella, the structure of the core region of smooth from LPS isolated from the parent strain (S. enterica sv.Arizonae IIIa O62) was analyzed.In addition, the position of the linkage between the core region and the Ospecific polysaccharide was investigated.Monosaccharide analysis of smooth form LPS revealed the presence of Rha, Ga-lANAc (constituents of the O-specific polysaccharide) Glc, Gal, Hep, GlcN, and Kdo residues.The structure of the repeating unit of the O-specific polysaccharide of this LPS has been reported (Fig. 4A) (36).After hydrolysis of the smooth form LPS with 1% acetic acid and centrifugation, the resulting mixture of poly-and oligosaccharides in the supernatant was separated by gel-permeation chromatography on Sephadex G50.Three fractions were obtained, the first of which eluted with the void volume contained the O-specific polysaccharide attached to the core region, and the third eluted in the region where mono-and disaccharides elute.Both fractions were not further investigated.The second fraction was expected to contain oligosaccharides of the SR-type, i.e. the complete core region substituted by one or few repeating units of the O-specific polysaccharide, and was therefore further separated using HPAEC.Five fractions were obtained which were desalted and investigated by 1 H NMR spectroscopy indicating that the second fraction contained only one compound that possessed the complete core and probably one repeating unit.This fraction was called "fraction SR" and used for structural analysis.
Mass Spectrometry of Fraction SR-FAB-MS and ESI-MS of fraction SR were carried out to determine the structure of the novel Salmonella core when linked to the O-specific polysaccharide.The positive ion mode FAB-mass spectrum (Fig. 5A) contains an intense series of poorly resolved pseudomolecular ion clusters in the m/z 3000 region.The ions in the cluster are separated by 22 mass unit increments and are centered at m/z 2957, 2979, 3001, 3021, 3043, and 3064.The increments of 22 mass units indicate that the LPS molecule bears several sodium adducts, consistent with the presence of several strongly acidic moieties.The most intense pseudomolecular ion cluster centered at m/z 3021 for a species bearing five sodium atoms and having a monosaccharide composition of HexANAc(DeoxyHex) 4 HexNAcHex 5 Hep 3 Kdo corresponds to one repeating unit of the O-specific polysaccharide and the full Ra core in which a HexNAc is replaced by a Hex residue (Fig. 4B) and includes three phosphates and an ethanolamine group.The other pseudomolecular ions represent the same species possessing from two to seven sodium atoms.Three additional ion clusters (labeled III, IV, and V) centered at m/z 3037, 3058, and 3080 derive from potassium-cationized pseudomolecular ions representing the same species.Minor pseudomolecular ions representing a species lacking one phosphate group (P, 80 mass units) are present centered at m/z 2898 ([M-P-2Hϩ3Na] ϩ ) and m/z 2920 (labeled II, [M-P-3Hϩ4Na] ϩ ).In addition, very minor ions that correspond to the species lacking the 2-aminoethyl phosphate group (PEA, 123 mass units) are observed at m/z 2856 ([M-PEA-2Hϩ3Na] ϩ ) and m/z 2877 (labeled I, [M-PEA-3Hϩ4Na] ϩ ).
Highly phosphorylated LPS molecules may be expected to Since both of the mass spectrometric experiments were carried out at resolutions of about 1000, this high mass LPS sample gives broad signals.To obtain well resolved signals and thus better mass assignments a higher resolution (resolving power of 3000) was used for a negative ion mode FAB-MS experiment.Prior to this, the sample was treated with ammonia in methanol to reduce the number of different ions formed by both reducing the amount of salt and excluding the possible presence of artifacts arising from acid treatment of the Kdo residue (38).Two weak but resolved signals were observed in the negative ion mode FAB-mass spectrum having their 12 C isotope peaks at m/z 2910.8 and 2932.8 (data not shown).These correspond precisely to the expected m/z values for the monoisotopic [M-H] Ϫ and [M-2HϩNa] Ϫ ions for the oligosaccharide HexANAc(deoxyHex) 4 HexNAcHex 5 Hep 3 Kdo including 2-aminoethanol and three phosphate groups.The mass spectrometric data are thus consistent with the presence of an LPS species bearing one GalANAcRha 4 GlcNAc O-specific polysaccharide repeating unit attached to a novel Salmonella core composed of five Hex residues in the outer core and lacking the terminal GlcNAc epitope.It is reasonable to postulate that the GlcNAc residue present in the Ra core is replaced in the core region of S. enterica sv.Arizonae O62 by a Glc residue.
NMR Spectroscopy of Fraction SR-Fraction SR was further subjected to NMR analyses to confirm the presence of a novel Salmonella core structure, to identify the linkage positions of the monosaccharide residues, to determine the manner of attachment of the O-specific polysaccharide to the core, and to define the nature and position of the phosphate substituents.The 1 H NMR (600 MHz) chemical shifts are given in Table II.The assignments are based on two-dimensional phase-sensitive COSY and TOCSY experiments.Anomeric configurations are assigned on the basis of the chemical shifts observed and J 1,2 values measured from the COSY experiment (Table III).Ring configurations are assigned on the basis of coupling constants as follows: J 2,3 ϳ 9 -10 Hz and J 3,4 ϳ 9 -10 Hz for ␣-gluco, J 2,3 ϳ 9 -10 Hz and J 3,4 ϳ 9 -10 Hz for ␤-gluco, J 2,3 ϳ 9 -10 Hz and J 3,4 ϳ 3-4 Hz for ␣-galacto, and J 2,3 ϳ 3-4 Hz and J 3,4 ϳ 8 -10 Hz for ␣-manno (in Hep, Rha).The NOESY data in Table IV confirm the assignments using intra-residue nuclear Overhauser effect (NOE) signals and allow the unambiguous determination of the monosaccharide sequence.Assignments of chemical shifts from 13 C NMR spectroscopy (Table V) are based on the HMQC experiment (500 MHz) together with the proton signal assignments.For comparison, the core oligosaccharide of S. enterica sv.Minnesota R60 (chemotype Ra) was also analyzed using the same methods as described above and the results are presented in parantheses in Tables II and V.The coupling constants and NOE data obtained for this sample are completely consistent with the published structure (2,35) and very similar to those obtained for fraction SR.
In the low field region of the 1 H NMR spectrum (4.6 -5.9 ppm) of fraction SR, signals for 14 anomeric protons, integrating each as a single proton, are present, together with some additional minor proton signals (Fig. 6).The major monosaccharide residues are identified as residues from the O-specific polysaccharides and the core oligosaccharide and are consistent with the results of monosaccharide analysis and mass spectrometry.Characteristic signals for H-3 of the major Kdo residue are present at 1.84 ppm (axial) and 2.17 ppm (equatorial).Except for the ␤-GlcNAc residue, all other residues are TABLE III J H,H and J H,P coupling constants (in Hz) observed for fraction SR from LPS of S. enterica sv.Arizonae IIIa O62 Values are given Ϯ0.5 Hz.Monosaccharide units A-Q (major fraction with ␣-Kdo as reducing terminus) and c-m (minor fraction with ␤-Kdo as reducing terminus) are as shown in Fig. 4.   IV) observed between protons L-1 and L-3 and L-5 (see Fig. 4 for labeling of residues), which are not observed for ␣-anomers.Therefore, this GlcNAc residue corresponds to the O-specific polysaccharide (36).All Rha, Hex, HexNH 2 , and Hep residues give NOE signals between their H-1 and the proton on the carbon atom to which they are glycosidically linked, from which the linkage positions were assigned.Interestingly, the NOE signals observed between protons L-1 and I-4 (see Fig. 7) identify the linkage of the ␤-GlcNAc L of the O-specific polysaccharide to O-4 of ␣-Glc I of the core oligosaccharide; thus, the O-specific polysaccharide is linked to the core via GlcNAc L rather than Rha M, as might be expected from the published structure of the repeating unit  Evidence for the presence of two components in fraction SR originating from ␣/␤ anomerization is also found in the NOESY spectrum.Most NOE signals observed are either between monosaccharide residues of the major portion (␣-Kdo-containing) or between those of the minor (␤-Kdo-containing).Interresidual NOE signals between major and minor residues are only found when minor counterparts of the major residues are absent (see Fig. 7).NOE signals for H-1 of ␣-Glc K are observed between H-4 of both ␣-Glc residues I and i, suggesting that ␣-Glc i has not arisen due to the partial absence of ␣-Glc K.This interpretation is consistent with the results obtained on MS analysis.The residues for which no minor version is observed probably have the same chemical shifts as the corresponding residue in the minor component.Core Substituents in Fraction SR-31 P NMR spectra were recorded to determine the composition and position of the phosphate groups substituted to the LPS.The proton-decoupled 31 P NMR spectrum of fraction SR (Fig. 8) contains equally intense resonances at Ϫ10.99 and Ϫ10.24 ppm, indicative of a diphosphodiester.In addition, a signal at 4.25 ppm is observed in the chemical shift range expected for phosphomonoesters.The twodimensional 1 H, 31 P-HMQC spectrum reveals that the phosphate signal at 4.25 ppm is coupled to a proton with a chemical shift of 4.34 ppm which is assigned to H-4 of Hep D. This result is consistent with the coupling pattern of the COSY cross-peaks of D-4 showing a coupling constant J H,P of 9 Hz.The signal at Ϫ10.99 ppm correlates with a broad signal in the two-dimensional spectrum around 4.6 ppm identified as H-4 of major Hep C and minor Hep c.Further evidence is found in the coupling pattern of the cross-peaks in the COSY spectrum of both, Hep C and c, that show an additional, large coupling JH, P 8 Hz.Furthermore, correlation of the second signal at Ϫ10.24 ppm to a proton with a chemical shift of 4.21 ppm is observed, consistent with a diphosphodiester.In the proton-detected one-dimensional HMQC spectrum, this proton appears together with the H-4 protons of Hep residues C and c.An additional, weak signal is observed in the spectrum at 3.3 ppm.In the COSY spectrum, this proton rise to only one cross-peak, that to the proton at 4.21 ppm.Compared with the 1 H chemical shifts of authentic 2-aminoethyl phosphate (4.08 ppm, CH 2 -P, 3.25 ppm, CH 2 -N3 ), these two signals can thus be assigned to the protons of a 2-aminoethyl group attached to diphosphate which in turn is attached to O-4 of Hep C and c.Evidence for the presence of a 2-aminoethyl group is also supported by the results of calculations of the proton and carbon chemical shifts for a 2-aminoethyl phosphodiester using the simulation programs ACD/HNMR and ACD/CNMR (47), respectively, which give results consistent with the experimental data.In general, charged substituents are present in non-stoichiometric proportions so that structural determination of phosphate substituents in the core region is difficult.In this study, however, purification of one component from a hydrolytic mixture using HPAEC has allowed the presence and position of phosphate substituents to be established.In conclusion, we have successfully identified the chemical structure of a novel core type of LPS from S. enterica.Its presence had been proposed based on serological and early chemical analyses (6 -9).The analysis of an SR-type oligosaccharide isolated from smooth form LPS from S. enterica sv.Arizonae O62 demonstrates unambiguously that the terminal ␣-D-GlcNAc residue which is present in the core regions of LPS from S. enterica sv.Minnesota or sv.Typhimurium is replaced by ␣-D-Glc.Furthermore, the site attachment of the O-specific polysaccharide to the core region was determined as the O-4 of the penultimate ␣-D-Glc residue (see Fig. 4), a site that is identical to that in LPS of S. enterica sv.Typhimurium (2).The chemical structure of the repeating unit of the O-specific polysaccharide from serotype O62 has already been published (36); however, we now show that this polysaccharide is linked to the core region via ␤-D-GlcNAc L and, thus, establish the monosaccharide sequence of the "biological repeating unit" in the LPS (see Fig. 4).Finally, the sites of phosphoryl substitution in the core region were identified as O-4 of Hep residues C (substituted by PPEA, also proposed for the core region of S. enterica sv.Minnesota and sv.Typhimurium (2)) and D (substituted by a phosphate residue, also proposed for the core region of S. enterica sv.Minnesota and sv.Typhimurium, albeit at an unknown position (2)).
Methylation analysis of S-form LPSs of S. enterica sv.Arizonae O62 reveals the presence of a 4-substituted Hex residue.Since the linkage of the O-specific polysaccharide to the core region (␤134 to Glc I) is the only linkage to an O-4 of a monosaccharide residue in this LPS, this derivative as well as that corresponding to a 2,4-disubstituted Hex residue must originate from Glc I, indicating that one fraction of the smooth form LPSs comprises an Ra analogue (complete core, Fig. 4) and another fraction an Rb 1 -type core region that lacks Glc K.A possible partial substitution of the core region by the terminal GlcNAc residue in various S. enterica serovars has been discussed (9), but no data have been published.Tsang et al. (9) proposed the presence of the Rb 1 -type core structure in LPS of S. enterica sv.Djakarta and suggested it as a third new Salmonella LPS core type.However, from our findings and from the discussion in Ref. 9, this core type may generally be present in a fraction of S. enterica smooth form LPSs, either as minor or even as a major component.This core type was observed mass spectrometrically ([MϩH] ϩ at m/z 2007) as a minor component in the LPS from one of the rough-type mutants from S. enterica sv.Arizonae IIIa.In S. enterica, the O-specific polysaccharides are first synthesized as undecaprenol-linked intermediates and then transferred as a whole to the core region (2,3).On the basis of the presence of the Rb 1 -type core region one may speculate that in the biosynthesis of S-form LPS of S. enterica sv.Arizonae O62, both the Ra analogue and the Rb 1 -type (which lacks Glc K) core regions may be used as acceptor molecules for this transfer.If this is so, then the terminal Glc (in S. enterica sv.Arizonae IIIa O62) and possibly also the GlcNAc (e.g. in S. enterica sv.Typhimurium) residues do not represent structural parameters important for this biosynthetic step.Alternatively, the terminal GlcNAc or Glc residues could be removed in a late step of smooth form LPS biosynthesis (9).These contrasting hypotheses await further investigation.
In addition to S. enterica sv.Arizonae IIIa O62, there exist other serovars of subspecies IIIa, which do not react with mAb T6 and are resistant to phage FO1 (7).Further investigations will show whether the LPSs from these serovars also possess the novel core structure as in S. enterica sv.Arizonae IIIa O62.
Abbreviations inStructure I are as follows: Hepp, L-glycero-D-manno-heptopyranose; Kdo, 3-deoxy-D-manno-oct-2-ulopyranosonic acid; PPEA, 2-aminoethyl diphosphate; R, O-specific polysaccharide.The presence of this novel core type in LPS of S. enterica should be taken into account in the development of a general antibody-based diagnostic system for Salmonella.

FIG. 1 .
FIG. 1. Structure of the Ra chemotype core oligosaccharide of Salmonella LPS (2, 35).A third Kdo residue ␣-234-linked to the second one is in most LPSs present in non-stoichiometric amounts.

HexNH 2
and two Hex residues.The remaining ions in the spectrum correspond to species originating from smaller related components that, in addition to lacking one HexNH 2 and one or two Hex residues, also lack one Kdo and/or a second HexNH 2 residue.Partial cleavage of monosaccharide residues during the chemical modifications is assumed to account for this heterogeneity.The acid-labile Kdo glycosidic linkage is partially cleaved on HF treatment, giving rise to the ions at m/z 1625 and 1463, representing species containing only one Kdo residue.The ions at m/z 1684 and 1464 also arise as artifacts of HF treatment 2 and represent species in which the reducing terminal HexNH 2 of the lipid A is cleaved prior to reduction.These species thus possess only one lipid A HexNH 2 residue and this became reduced.Similarly, the ions at m/z 1464 and 1302 correspond to species lacking both the Kdo and the lipid A HexNH 2 residue.The less intense ion observed at m/z 1359 probably arises by ␤-cleavage, and corresponds to a component with the monosaccharide composition Hep 3 Kdo 2 -(HexNH 2 ) 2 .Apart from small differences in relative ion intensities, the FAB-mass spectra of the two later eluting G50 fractions from R5, as well as those from the G50 fractions from R4a and R4b resemble that of the early eluting G50 fraction of R5.This indicates that the difference in extraction methods used to obtain R4a and R4b LPSs does not influence the types of LPS molecules isolated, but only their relative amounts.The de-O-acylated, dephosphorylated LPSs from strain R5 were further modified to reduce the heterogeneity of the preparation to obtain more detailed information on the arrangement of the hexoses in the outer core.Mild hydrolysis in 1% aqueous acetic acid was carried out to cleave the linkage between the inner core and lipid A, and to remove the remaining terminal Kdo residue.The positive ion FAB-mass spectrum (not shown) of the product reveals a reduction in heterogeneity following acid hydrolysis to yield a mixture of only two components.The major ion is observed at m/z 1301 for the protonated component Hex 3 Hep 3 Kdo.The minor ion at m/z 1161 reflects the sodium-cationized Hex 2 Hep 3 Kdo component.Both ions are accompanied by signals 18 mass units lower, probably corresponding to the presence of Kdo artifacts (38).To further improve sensitivity and to direct mass spectrometric fragmentation, allowing the sequence and branching patterns of the monosaccharide residues to be determined, the acid-hydrolyzed, de-O-acylated, dephosphorylated R5 LPS sample was per-Oacetylated.The positive ion mode FAB-mass spectrum (not shown) reveals a series of oxonium (or B-) ions(43,44).The two most intense of these are at m/z 1627 and 1987 and correspond to Hex 3 Hep 2 ϩ and Hex 3 Hep 3 ϩ, respectively, deriving from the Hex 3containing major component.The presence of these ions, together with the absence of an oxonium ion for Hex 3 Hep 1 ϩ , indicates that the third Hep residue in the core is terminal and linked to the Hep residue which is attached to the Glc residue (see Fig.1).The most abundant Hex 3 Hep 2 ϩ oxonium ion (m/z 1627) was used as the parent ion for a CID tandem mass spectrometric experiment (Fig.3) designed to allow determination of the arrangement of the Hex residues in the outer core.The most intense fragment ions at m/z 331 and 403 represent oxonium ions for terminal Hex and terminal Hep, respectively.The major ions formed from an acetylated derivative are known to originate from single glycosidic bond cleavages to form oxonium ions.Thus, if the Hex 3 outer core is linear, oxonium ions at m/z 331 (Hex 1 ϩ ), m/z 619 (Hex 2 ϩ ), and m/z 907 (Hex 3 ϩ ) may be expected to be formed, while a branched structure can only yield ions at m/z 331 (Hex 1 ϩ ) and m/z 907 (Hex 3 ϩ ) with no ion at m/z 619 for Hex 2 ϩ .The Hex 3

FIG. 4 .
FIG. 4. Structure of the repeating unit of the O-specific polysaccharide of serotype O62 (36) (A) and the novel core oligosaccharide identified in LPS from S. enterica sv.Arizonae IIIa O62 (B) .Monosaccharide residues are labeled A-Q as used in Tables II-V and throughout the text.Corresponding minor residues are labeled a-q.PPEA, 2-aminoethyl diphosphate; P, phosphate.

( 36 )
. The presence of the terminal ␣-Glc residue K 132-linked to ␣-Glc I is established by NOE signals and confirms the structure of a novel core oligosaccharide for Salmonella (see Fig.7).Interestingly, an NOE signal is observed between protons K-1 and F-4 that may indicate an unusual conformation of the outer core of the smooth form LPS in the region that might be responsible for recognition by specific antibodies.Consequently, an HSEA/Monte Carlo calculation for the tetrasaccharide Glc␣132Glc␣132Gal␣133Glc␣ was carried out to simulate the conformation of the outer core of the smooth form LPS. It nicely shows the short distance between H-1 of Glc K and H-4 of Glc F. In the minimum energy conformation, the distance is 2.25 Å and the average distance from the simulation is 2.70 Å.Both results fit very well with the NOE signal observed and thus give an indication for the conformation of the outer core of the smooth form LPS.Fraction SR contains one repeating unit of the O-specific polysaccharide (see MS data); its terminal residue, ␣-GalANAc Q gave only NOE signals to ␣-Rha P. The chemical shifts corresponding to protons of the O-specific polysaccharide are similar to those reported for serotype O62 by Vinogradov et al.(36) identifying the structure of the repeating unit present to be essentially that published, except that ␤-GlcNAc L is now identified as the residue linking the O-specific polysaccharide to the core.The presence of a major and a minor structure in fraction SR was confirmed by a 1 H,13 C-HMQC spectrum.The minor residues observed are ␣-Gal h, ␣-Glc i, ␣-Hep c, ␣-Hep d, ␣-Gal g, ␣-Rha m, ␣-Hep e, ␤-GlcNAc l, and Kdo a. Their intensities are about one third of those corresponding to the major residues.Comparison of the 1 H chemical shifts of the Kdo residues A and a with those for the two reference monosaccharides in Ref. 46 (allyl ␣-Kdo and allyl ␤-Kdo) shows that the mixture has probably arisen due to ␣/␤ anomerization of the reducing terminal (unreduced) Kdo residue.The chemical shifts for H-3eq (2.07 ppm) and H-4 (4.10 ppm) of allyl ␣-Kdo differ significantly from those for allyl ␤-Kdo (2.38 ppm and 3.71 ppm, respectively).Comparable differences are observed between the shifts for Kdo A and a, indicating that the presence of two Kdo residues is explained by ␣/␤ anomerization.From the chemical shifts, Kdo A corresponds to the ␣-anomer and Kdo a corresponds to the ␤-anomer.Comparison of the carbon shifts of A-4 and a-4 with those for the reference monosaccharides confirm this assignment.

FIG. 6 . 1 H
FIG. 6. 1 H NMR spectrum of fraction SR from S. enterica sv.Arizonae IIIa O62.The spectrum was recorded from a solution in 2 H 2 O (p 2 H 7.1) at 500.13 or 600.13 MHz at 37 °C.Assignments were made from COSY and TOCSY experiments.See Fig. 4 for labeling of the residues.

TABLE I
Monosaccharide and phosphate compositions of LPSs from strains R4 and R5 of S. enterica sv.Arizonae IIIa O62 Molar ratios are based on Gal ϭ 2.0 (absolute amounts in nmol/mg LPS).Kdo was determined after hydrolysis with 0.1 M sodium acetate buffer (pH 4.4, 1 h, 100 °C).

TABLE II 1
H NMR chemical shifts (in ppm) for fraction SR from LPS of S. enterica sv.Arizonae IIIa O62 and from S. enterica sv.Minnesota R60 (values given in parentheses) Spectra were recorded from a solution in 2 H 2 O (p 2 H 7.1) at 500.13 and 600.13 MHz at 37 °C.Chemical shifts are relative to internal acetone (2.23 ppm).Assignments were made from COSY and TOCSY experiments.Monosaccharide units A-Q (major fraction with ␣-Kdo as reducing terminus) and a-m (minor fraction with ␤-Kdo as reducing terminus) are as shown in Fig.4.

TABLE IV
NOE signals observed in the NOESY spectrum of fraction SR from LPS of S. enterica sv.Arizonae IIIa O62 Monosaccharide units C-Q (major fraction with ␣-Kdo as reducing terminus) and c-m (minor fraction with ␤-Kdo as reducing terminus) are as shown in Fig. 4. s, strong NOE; m, medium NOE; w, weak NOE.