The structures of the lipooligosaccharide and capsule polysaccharide of Campylobacter jejuni genome sequenced strain NCTC 11168 Frank St. Michael, Christine M. Szymanski, Jianjun Li, Kenneth H. Chan, Nam Huan Khieu, Suzon Larocque, Warren W. Wakarchuk, Jean-Robert Brisson and Mario A. Monteiro Institute for Biological Sciences, National Research Council of Canada, Ottawa, Canada Campylobacter jejuni infections are one of the leading causes of human gastroenteritis and are suspected of being a pre- cursor to Guillain–Barre ´ and Miller–Fisher syndromes. Recently, the complete genome sequence of C. jejuni NCTC 11168 was described. In this study, the molecular structure of the lipooligosaccharide and capsular polysaccharide of C. jejuni NCTC 11168 was investigated. The lipooligosac- charide was shown to exhibit carbohydrate structures anal- ogous to the GM1a and GM2 carbohydrate epitopes of human gangliosides (shown below): The high M r capsule polysaccharide was composed of b- D -Ribp, b- D -GalfNAc, a- D -GlcpA6(NGro), a uronic acid amidated with 2-amino-2-deoxyglycerol at C-6, and 6-O-methyl- D -glycero-a- L -gluco-heptopyranose as a side- branch (shown below): The structural information presented here will aid in the identification and characterization of specific enzymes that are involved in the biosynthesis of these structures and may lead to the discovery of potential therapeutic targets. In addition, the correlation of carbohydrate structure with gene complement will aid in the elucidation of the role of these surface carbohydrates in C. jejuni pathogenesis. Keywords: lipooligosaccharide; capsule; electron spray ionization mass spectrometry; high-resolution magic angle spinning NMR; heptose. In humans, Campylobacter jejuni infection often gives rise to enteritis and, in some regions, this Gram-negative bacterium surpasses Salmonella, Shigella and Escherichia as the primary cause of gastrointestinal disease [1,2]. Moreover, C. jejuni infections have been linked to the more severe clinical outcomes caused by Guillain–Barre ´ [3,4] and Miller–Fisher syndromes [5]. The subsequent paralysis observed in Guillain-Barre ´ and Miller–Fisher syndromes is thought to be an autoimmune reaction due to molecular mimicry of gangliosides by C. jejuni lipooligosaccharides (LOS) [6,7]. In the pioneering studies carried out by Aspinall and coworkers on the cell-surface carbohydrates from Campylobacter species, it was observed that insoluble gels from phenol-water extractions of bacterial cells yielded mainly low M r LOS, with core oligosaccharide linked to lipid A, and the aqueous phases from such extractions furnished high M r glycans with extended polymers with no attachment to lipid A as seen in the teichoic acid-like P/PEtn GM1a GM2 fl 6 b-Gal-(1fi3)- b- D -GalNAc-(1fi4)-b- D -Gal-(1 fi3)-b- D -Gal-(1fi3)- L -a- D -Hep-(1fi3)- L -a- D -Hep-(1fi5)Kdo 32 2 4 ›› › › 21 1 1 a-Neu5Ac a- D -Gal b- D -Glc b- D -Glc 6-O-Me- D -a- L -glcHepp 1 fl 3 fi2)-b- D -Ribf-(1-5)-b- D -Galf NAc-(1-4)-a- D -GlcpA6(NGro)-(1fi 0 B B B B @ 0 B B B B @ 1 C C C C A Correspondence to J R. Brisson, Institute for Biological Sciences, National Research Council of Canada, Ottawa, Canada, K1A 0R6. Fax: + 1 613 952 9092, Tel.: + 1 613 990 3244, E-mail: jean-robert.brisson@nrc.ca; M. Monteiro, Wyeth Vaccines Research, 211 Bailey Road, West Henrietta, NY, 14586, USA. Fax: + 1 585 273 751, Tel.: + 1 585 273 7667, E-mail: Monteim@wyeth.com Abbreviations: CE, capillary electrophoresis; ESI-MS, electron spray ionization mass spectrometry; FAB, fast-atom bombardment; HMBC, heteronuclear multiple bond coherence; HMQC, heteronu- clear multiple quantum coherence; HR-MAS, high-resolution magic angle spinning; HSQC, heteronuclear single quantum coherence; KmR, kanamycin resistance; LOS, lipooligosaccharide; LPS, lipo- polysaccharide; OS, oligosaccharide. Dedication: The authors would like to dedicate this manuscript to Professor Gerald Aspinall. (Received 3 July 2002, revised 16 August 2002, accepted 21 August 2002) Eur. J. Biochem. 269, 5119–5136 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03201.x polymer from C. coli serotype HS:30, the repeating disac- charide from C. jejuni serotype HS:3, and the poly(tetra- glycosylphosphates) from C. lari [6]. Analysis of the genome sequence of C. jejuni NCTC 11168 revealed that the strain possessed a type II/III capsule locus found in other organisms such as E. coli K1 and Neisseria meningitidis group B [8,9]. This led to the realization that what was once believed to be high molecular weight lipopolysaccharides (LPS) was actually capsule and that capsule was the main serodeterminant of the Penner typing scheme [10,11]. Campylobacter LOS and capsule are important in adherence and invasion in vitro [10,12], colonization and disease in vivo [10], molecular mimicry of gangliosides [6,7], possible autoimmunity leading to Guillain–Barre ´ and Miller–Fisher syndromes [5,13], maintenance of cell surface charge [10], antigenic and phase variation [10,12,14–17], and serum resistance [10,15]. Also, C. jejuni NCTC 11168 was recently sequenced [8] and serves as a reference strain for the understanding of the genetics of this significant food-borne pathogen. We therefore elucidated the structures of these major cell surface carbohydrates to make functional analysis of the genes in the respective genetic loci possible. EXPERIMENTAL PROCEDURES Bacterial strains and plasmids C. jejuni NCTC 11168 (HS:2) was originally isolated from a case of human enteritis [18] and later sequenced by Parkhill et al.[8].E. coli DH10B (Invitrogen) was used as the host for the cloning experiments. Plasmid pPCR-Script Amp (Stratagene) was used as the cloning vector. Media and growth conditions C. jejuni NCTC 11168 was routinely grown on Mueller Hinton agar (Difco) under microaerophilic conditions at 37 °C. E. coli clones were grown on Luria S-gal agar (Sigma) at 37 °C. When appropriate, antibiotics were added to the following final concentrations: kanamycin, 30 lgÆmL )1 and ampicillin, 150 lgÆmL )1 . Generation of LOS and capsule Campylobacter biomass was harvested from overnight liquid cultures by centrifugation. Carbohydrates were isolated by the hot water/phenol extraction of bacterial cells [19] as a gel-like pellet upon ultracentrifugation of the aqueous phase. The LOS pellet was lyophilized, then purified on a column of Bio-Gel P-2 (1 cm · 100 cm) with water as eluent. Some of the LOS preparation was then treated with 1% acetic acid at 100 °C for 1 h with subsequent removal of the insoluble lipid A by centrifuga- tion (5000 g) to yield the core oligosaccharide (OS). The supernatant from ultracentrifugation was purified on Bio-Gel P-2 with water as eluent and lyophilized to obtain the capsule polysaccharide (PS-1). Sugar composition and methylation linkage analysis Sugar composition analysis was performed by the alditol acetate method [20]. The hydrolysis was performed in 4 M trifluoroacetic acid for 4 h at 100 °C followed by reduction with NaBD 4 in H 2 O overnight, then acetylated with acetic anhydride at 100 °C for 2 h using residual sodium acetate as catalyst. The alditol acetate derivatives were characterized by GLC-MS using a Hewlett-Packard chromatograph equipped with a 30- M DB-17 capillary column (180 °C to 260 °C at 3.5 °CÆmin )1 ). MS was performed in the electron impact mode and recorded on a Varian Saturn II mass spectrometer. Absolute configuration was assigned by characterization of the but-2-yl glycosides in GLC-MS [21]. Methylation analysis was carried out by the NaOH/ dimethylsulfoxide/CH 3 I procedure [22] using GLC-MS in the electron impact mode to characterize the sugars as above. A portion (1/4) of the methylated sample was used for fast-atom bombardment-mass spectrometry (FAB-MS) in positive ion mode. The Jeol JMS-AX505H mass spectrometer was used with a matrix of glycerol/thioglycerol 1 : 3 and 3 kV as the tip voltage. Smith degradation The polysaccharide sample ( 5 mg) was oxidized with 40 m M sodium metaperiodate in 0.1 M sodium acetate at 4 °C for 72 h [23]. The product was isolated on a Bio-Gel P-2 as per above then reduced with NaBD 4 and acidified with cation exchange resin (J. T. Baker). The product was then hydrolyzed with 1 M trifluoroacetic acid at 45 °C for 1 h, reduced with NaBD 4 and re-acidified. Then, finally the sample was fractionated on a Bio-Gel P-2 column and fractions analyzed. CE-ESI-MS and CE-ESI-MS/MS A crystal Model 310 capillary electrophoresis (CE) instrument (AYI Unicam) was coupled to an API 3000 mass spectrometer (Perkin-Elmer/Sciex) via a microIon- spray interface. A sheath solution (isopropanol/methanol 2 : 1) was delivered at a flow rate of 1 lLÆmin )1 to a low dead volume tee (250 lm internal diameter, Chromato- graphic Specialties). All aqueous solutions were filtered through a 0.45-lm filter (Millipore) before use. An electrospray stainless steel needle (27 gauge) was butted against the low dead volume tee and enabled the delivery of the sheath solution to the end of the capillary column. The separations were obtained on about 90-cm length bare fused-silica capillary using 10 m M ammonium acetate/ ammonium hydroxide in deionized water, pH 9.0, con- taining 5% methanol. A voltage of 20 kV was typically applied at the injection. The outlet of the capillary was tapered to c.15lm internal diameter using a laser puller (Sutter Instruments). Mass spectra were acquired with dwell times of 3.0 ms per step of 1 m/z )1 unit in full-mass scan mode. The MS/MS data were acquired with dwell times of 1.0 ms per step of 1 m/z )1 unit. Fragment ions formed by collision activation of selected precursor ions with nitrogen in the RF-only quadrupole collision cell, were mass-analyzed by scanning the third quadrupole. Construction and characterization of insertional mutants For construction of the Cj1428c mutant, genes Cj1427c to Cj1428c were PCR amplified from C. jejuni NCTC 11168 5120 F. St. Michael et al. (Eur. J. Biochem. 269) Ó FEBS 2002 with the following primer pair: Cj1427cF918 (5¢-AA CTTTCATCATTTTAAACGCTCTT-3¢)andfclR51 (5¢-TACAGCATTGGTAGAAAACTTACAA-3¢). For construction of the kpsM mutant, gene Cj1448c was PCR amplified with: kpsMF771 (5¢- TACCGCCGTTAAAGCT TGTCTATTA-3¢) and kpsMR73B (5¢- TATATATGGGT AGTTGGGGAGCCTA-3¢). For construction of the Cj1439c mutant, gene Cj1439c was PCR amplified with: glfF1081 (5¢-TTTTACAAAATAATAATGCCGATCT-3¢) and glfR6 (5¢-TGATTATTTAATTGTTGGTTCTGG A-3¢). The PCR products were ligated to pPCR-Script Amp according to the manufacturer’s instructions. A blunt- ended kanamycin resistance (KmR) cassette from pILL600 [24] was inserted into the filled-in BglII restriction site of Cj1428c to create pCSc28, into the NheI restriction site of kpsM to create pCSc48, and into the BsaBI restriction site of Cj1439c to create pCSc39. The orientation of the KmR cassette was determined by sequencing with the ckanB primer (5¢-CCTGGGTTTCAAGCATTAG-3¢) using terminator chemistry and AmpliTaq DNA polym- erase FS cycle sequencing kits (Perkin Elmer-Applied Biosystems) and analyzed on an Applied Biosystems 373 DNA sequencer. The mutated plasmid DNA was used for electroporation into C. jejuni NCTC 11168 [25] and KmR transformants were characterized by PCR to confirm that the incoming plasmid DNA had integrated by a double cross-over event. Reverse transcriptase-polymerase chain reaction It has previously been shown that gene insertion of the Campylobacter KmR cassette in a nonpolar orientation has no effect on transcription of downstream genes [26]. Sequencing results confirmed that the KmR insertion in both kpsM and Cj1439c was in the nonpolar orientation. However, the KmR cassette inserted in a polar orientation into Cj1428c so, we wanted to determine whether Cj1428c disruption had a polar affect on downstream genes. RNA was isolated from C. jejuni wild type and the Cj1428c mutant using the RNeasy mini kit (Qiagen). RT-PCR reactions, along with controls, were performed using the OneStep RT-PCR kit (Qiagen). First-strand synthesis was carried out as described by the manufacturer at 50 °C for 30 min. PCR conditions were 95 °C for 15 min followed by 30 cycles at 94 °C for 30 s, 59 °C for 4 min, and 72 °C for 30 s followed by a final annealing at 72 °C for 10 min using primers: Cj1427cF918 (5¢-AACTTTCATCATTTTAAAC GCTCTT-3¢) and Cj1427cR10 (5¢-AAAGTTTTAATTAC AGGTGGTGCAG-3¢). Deoxycholate-PAGE analysis and silver staining of polysaccharides Proteinase K-treated whole cells of C. jejuni were prepared according to the method of Salloway et al. [27] based on the original method of Hitchcock and Brown [28]. The samples were separated on 16.5% deoxycholate-PAGE [29]. After electrophoresis, the gels were silver stained according to the method of Tsai and Frasch [30]. However, gels were fixed for 2–5 h rather than overnight to prevent elution of the high molecular weight polysaccharides [31]. In addition, we have recently improved the silver staining procedure by visualizing the carbohydrates with commercially available developer (Bio-Rad [32]). Nuclear magnetic resonance NMR experiments were acquired on Varian Inova 600, 500 and 400 MHz spectrometers using a 5-mm triple resonance probe with the 1 H coil nearest to the sample and with a Z gradient coil. All measurements were made at 25 °C on 2–5 mg of sample dissolved in 0.6 mL of D 2 O, pH 6–7. Experiments in 90% H 2 O were carried out at pH 4–5. The methyl resonance of acetone was used as an internal reference at 2.225 p.p.m. for 1 H spectra and 31.07 p.p.m. for 13 C spectra. Standard homo and heteronuclear correlated 2D techniques were used for general assignments: COSY, TOCSY, NOESY, HMQC or HSQC, HMQC-TOCSY and HMBC. Selective 1D experiments were performed for the determination of accurate coupling constants and NOEs andtoperformthe1Danalogofa3D-TOCSY- NOESY experiment [33]. High resolution magic angle spinning (HR-MAS) experiments were performed using a gradient 4 mm indirect detection nano-NMR probe (Varian) with a broadband decoupling coil. Proton spectra of killed cell pellets were acquired as described previously [34]. Molecular modeling The conformational analysis for trisaccharide BCD of PS-1 was performed using the Metropolis Monte-Carlo method as previously described [35]. The PFOS potential was used [36]. Residue B was modeled as a glucuronic acid. Minim- ized coordinates for the monosaccharides were obtained using MM3(92) available from the Quantum Chemistry Program Exchange. The minimum energy conformation for each disaccharide was used as the starting conformation for the trisaccharide. The (O5–C5–C6–O6) torsion angle was restricted to the )60° conformer for the DL form of residue D andto+60° for the LD form. For the trisaccharide, 5 · 10 3 macro moves were used with a step length of 10° for the glycosidic linkage and pendant groups and a temperature of 10 3 K resulting in an acceptance ratio of 0.5. Distances were extracted from the saved coordinates at each macro move. The molecular model for the trisaccharides were generated using the minimum energy conformer. Molecular drawings were performed using Schakal97 from E. Keeler, University of Freiburg, Germany. Vacuum MD simulation were performed with the DISCOVER -3 program from Accelrys Inc. (MSI) using the AMBER FORCEFIELD version 1.0–1.6, with Homans’ param- eters applicable to saccharides [37], on a SGI Indigo 2 Solid Impact R10000 195 MHz. As before, the (O5–C5–C6–O6) torsion angle for residue D was restrained. The initial structure was subjected to a 300-step energy minimization (BFGS method), followed by 50 ps dynamics simulation at 298 K. Initial velocities were generated from a Maxwell– Boltzmann distribution, and the temperature was controlled by the direct velocity scaling method. The Verlet algorithm of integrating the Newton’s equations of motion was applied with 1 fs timestep for the simulation where a distance-dependent dielectric of the form er (r ¼ the distance between atoms) was used. Distances were extracted Ó FEBS 2002 LOS and capsule structures of C. jejuni NCTC 11168 (Eur. J. Biochem. 269) 5121 from a trajectory file of 5000 frames stored after each MD run. RESULTS Structural determination of the LOS The alditol acetate derivatives [20] of D -glucose ( D -Glc), D -galactose ( D -Gal), N-acetyl- D -galactosamine ( D -GalNAc) and L -glycero- D -manno-heptose ( LD -Hep), in the respective ratios of 3 : 2.3 : 1 : 1.8, were detected in both the lipid A-free core OS and intact LOS, by GLC-MS. The absolute configurations of the sugars mentioned above were deduced by the identification of but-2-yl chiral glycosides in GLC- MS [21]. Sugar linkage analysis (Table 1), by the methyla- tion procedure [22], on the LOS revealed the following sugar linkage types: two units of terminal D -Glc, one unit of each terminal D -Gal, 2,3-substituted D -Gal and 3,4-disubstituted D -Gal, traces of 4-substituted D -Gal, one unit of terminal D -GalNAc, trace amounts of 3-substituted D -GalNAc, one unit of 2,3-disubstituted LD -Hep, and trace amounts of 3,4-disubstituted LD -Hep. A parallel linkage analysis on the liberated core OS, after removal of lipid A with 1% acetic acid, afforded the same sugar linkage types, but in addition it showed a significant decrease of 3,4-disubstituted D -Gal and a greater amount of 4-substituted D -Gal (Table 1). To gain a quick insight into the overall composition of the LOS, a series of ESI-MS experiments were performed on the core OS. The ESI-MS spectrum (Fig. 1a) of the core OS showed a heterogeneous mixture (Table 2) with the pres- ence at the reducing-end of the anhydro form of 3-deoxy- manno-octolusonic acid (Kdo) as a distinct marker. The primary molecular ion at m/z 1759 [)18 (H 2 O)] correspon- dedtoacompositionofHex 5 ,Hep 2 , GalNAc, Kdo and 2-amino ethyl phosphate (PEtn), and ion m/z 1716 ()18) belonged to a composition of Hex 5 ,Hep 2 , GalNAc, Kdo and phosphate (P). Trace amounts of the phosphate-free core OS (Hex 5 ,Hep 2 , GalNAc, Kdo) at m/z 1636 ()18) was also observed, along with small amounts of m/z of 1877 and 1921, which corresponded to the addition of an extra hexose to both phosphorylated core OSs. For both molecular ions, a significant ion at m/z 2007 ()18) (Hex 5 , Hep 2 , GalNAc, Kdo, Neu5Ac, P) and m/z 2050 ()18) (Hex 5 ,Hep 2 , GalNAc, Kdo, Neu5Ac, PEtn) pointed towards the presence of sialic acid (Neu5Ac) in the core OS. Indeed, ESI-MS on core OS preparations that were obtained by a harsher treatment with 5% acetic acid, to intentionally remove any acid labile Neu5Ac, yielded the same primary ions as discussed above, but no ions containing sialic acid. Taking into account the previous observed variation between 3,4-disubstituted Gal and 3-substituted Gal in the core OS, before and after mild acid treatment, and the detection of the acid sensitive sialic acid in ESI-MS, suggested that Neu5Ac may be attached at O-3 of the 3,4-disubstituted Gal. The CE-MS/MS spectra for the components having a total mass of m/z 2050 or precursor ions at m/z 1026 (doubly protonated) are presented in Fig. 1b. The fragment ions observed at m/z 1848 and 1760 clearly indicated that one HexNAc residue and one Neu5Ac residue were present as terminal units. As shown in the Fig. 1b, the fragment ion of m/z 1848, arising from the loss of HexNAc, subsequently loses one hexose (m/z 1686), one Neu5Ac (m/z 1394.5), three hexoses (m/z 1232, 1070, 908) and finally one heptose residue (m/z 716). The fragment ion at m/z 366 suggested that the HexNAc was attached to a Hex unit, and the fragment ion at m/z 454 was indicative a Hex-Neu5Ac disaccharide. Moreover, fragment ion m/z 554 suggested the existence of PEtn-Hep-Kdo moiety. It was also observed that a minor glycoform, with an extra Hex (composition of Hex 6 HexNAc 1 PEtn 1 Hep 2 Kdo 1 ) was present in the LOS of C. jejuni NCTC 11168. The tandem mass spectrum of ion m/z 1005, which corresponded to the morpholine adduct of the LOS, is presented in Fig. 1c. The doubly charged ion yielded a fragment ion at m/z 1922 when the morpholine ion was cleaved off. The dissociation of the core oligosaccharide gave rise to consecutive losses of Hex, HexNAc, and five hexoses. In contrast with Fig. 1(b), in which the ion m/z 292 corresponded to a protonated Neu5Ac, no sialic acid fragment ion was found in this glycoform. For the core structural features of oligosaccharide from C. jejuni NCTC 11168, we have shown evidence for the proposed structure as indicated in the inset of Fig. 1b. Solid information regarding the sequences of sugar units was obtained by FAB-MS of the methylated core OS derivative. Figure 2 and Table 3 shows a series of A-type primary glycosyl oxonium ions, and secondary ions, of defined compositions at m/z 260fi228 (GlcNAc) + , m/z 376fi344 (Neu5Ac) + , m/z 464 (Gal, GalNAc), m/z 826 (GalNAc, Neu5Ac, Gal) + , m/z 872 (GalNAc,Gal 3 ) + , m/z 1029 (Hex 2 , GalNAc, Neu5Ac) + , m/z 1120 (GalNAc, Hex 3 , Hep) + , m/z 1233 (GalNAc, Hex 3 ,Neu5Ac) + , m/z 1324 (GalNAc, Hex 4 ,Hep) + , m/z 1407 (Hex 4 ,Hep 2 ,P) + , m/z 1477 (Hex 4 ,Hep,Hep,PEtn) + , m/z 1685 (GalNAc, Hex 4 , Neu5Ac, Hep 2 ) + , m/z 1857 (GalNAc, Hex 5 ,Hep 2 ,P) + and m/z 1928 (GalNAc, Hex 5 ,Hep 2 , PEtn] + . A double cleavage ion containing a phosphate moiety was also see at m/z 547 (Hep,Glc,P) + . Combining the FAB-MS sequence data (Fig. 2, Table 3) with the information obtained from the linkage analysis (Table 1) and from the selective ESI-MS experiments (Fig. 1, Table 2). The following provisional structural arrangement for the core OS region can be proposed (Hex ¼ Glc or Gal): Table 1. Methylation linkage analysis of C. jejuni NCTC 11168 intact LOS, core OS and Smith degradation products. Linkage type LOS OS Smith degradation product Glc-(1fi 22 Gal-(1fi 113 fi3)-Gal-(1fi 1 fi4)-Gal-(1fi Traces < 1 Traces fi2,3)-Gal-(1fi 11 fi3,4)-Gal-(1fi 1 < 1 Traces GalNAc-(1fi 1 1 Traces fi3)-GalNAc-(1fi Traces Traces fi2,3)-Hep-(1fi 11 – fi3,4)-Hep (1fi Traces Traces fi3)-Man-(1fi 3 fi5)-3d-Hexitol a 0.5 fi5)-3d-Hexitol b 0.5 a,b Two isomeric forms of 3-deoxy-1,1,2,6-tetra- 2 H-5-O-acetyl- 1,2,4,6-tetra-O-methyl-hexitol (from 5-substituted Kdo). 5122 F. St. Michael et al. (Eur. J. Biochem. 269) Ó FEBS 2002 A Smith degradation [23] was strategically performed on the core OS to disentangled the linkages at the branch points. Prior to periodate oxidation, the core OS was reduced with NaBD 4 for the incorporation of deuterium at the Kdo terminus so that this unit could be detected in the final product. Periodate oxidation of the reduced core OS was followed by reduction with NaBD 4 , mild acid hydro- lysis, and a final reduction with NaBD 4 . Sugar linkage analysis of the final product (Table 1) showed the presence of terminal Gal (from 3,4-substituted Gal), 3-substituted Gal (from 2,3-substituted Gal), 3-substituted Man-O-6- 2 H (from 2,3- and 3,4-substituted LD -Hep units) and two isomeric units of 3-deoxy-1,1,2,6-tetra- 2 H-5-O-acetyl- 1,2,4,6-tetra-O-methyl-hexitol (from 5-substituted Kdo), in the approximate ratios of 3 : 1: 3 : 0.5 : 0.5. There were also traces of 4- and 3,4-substituted Gal and terminal GalNAc; these two derivatives originated from an extended molecule that contained a hexose at the nonreducing terminus of the core {Hex-(1fi4)-GalNAc-(1fi4)[Neu5Ac-(1fi3)]- Gal… inner core}.Theisomerichexitolderivativeswere recognized as originating from a modified 5-linked Kdo termini as seen in all C. jejuni strains. The backbone Gal and LD -Hep units are thus joined by 1fi3 linkages and the inner most LD -Hep is linked to O-5 of Kdo. The FAB-MS Fig. 1. Electron spray ionization-mass spectr- ometry. C. jejuni NCTC 11168 core OS showing a heterogeneous mixture (a). CE-MS/ MS (+ ion mode, produces ions of m/z 1026) analysis of C. jejuni NCTC 11168 core OS (b). CE-MS/MS (+ ion mode, produces ions of m/z 1005) analysis of LOS from C. jejuni NCTC 11168 core OS (c). P/PEtn fl [ D -Hex-(1fi3)]±- D -GalNAc-(1fi3 or 4)- D -Gal-(1fi2 or 3)- D -Gal-(1fi2 or 3)- LD -Hep-(1fi3 or 4)- LD -Hep-(1fiKdo 3or4 2or3 2or3 3or4 ›› › › 21 1 1 Neu5Ac D -Hex D -Hex D -Hex Ó FEBS 2002 LOS and capsule structures of C. jejuni NCTC 11168 (Eur. J. Biochem. 269) 5123 spectrum (Table 3) of the methylated final product from the Smith degradation yielded some limited, but corroborating, sequence data (* ¼ deuterium) stemming from the terminus showing m/z 219 (Gal) + , m/z 424 [Gal-(1fi3)-Man*] + ,and m/z 289 (Neu5Ac*) + , m/z 493 [Neu5Ac*(1fi3)-Gal]*, and m/z 902 [Neu5Ac*(2fi3)-Gal-(1fi3)-Gal-(1fi3)-Man*] + . Two final products were thus recovered from the Smith degradation (shown below), one was terminated by a GM2 structure, and the major product was a linear Gal-Man- Man-3dhex (shown below) backbone: Two unresolved anomeric resonances, characteristic of sugars with the mannose configuration, were observed at d 5.207 and d 5.07 in the 1 H nuclear magnetic resonance spectrum of the Smith degradation product. The same spectrum also showed one b anomeric resonance at 4.55 (J 1,2 7.2 Hz). The 1 H NMR data just described indicated that the Man ( LD -Hep) units possessed an a anomeric configuration, whereas the 2,3-disubstituted D -Gal had a b anomeric configuration. Therefore, at this time, the follow- ing structure for the core OS region, where Hex represents Glc or Gal, was proposed: The sugar linkage analysis (Table 1) performed on the core OS suggested the presence of slightly more than one unit of terminal Gal and two units of terminal Glc. Given the fact that the hexose at the nonreducing terminus was only present in trace amounts, as observed by linkage analysis (traces of 3-substituted GalNAc), ESI-MS and FAB-MS, the three side-branches hexoses could be assigned to two units of Glc and one unit of Gal. The nonstoichiometric hexose present in trace amounts that is connected to the O-3 position of GalNAc was a Gal residue. The 1 H NMR spectrum, in combination with a 1 H– 1 H TOCSY experiment, of the core OS yielded three a anomeric protons, at d 5.67 (J 1,2 3Hz)thatwasassignedto an a- D -Gal residue [H-2 d 3.79 (J 2,3 9.5 Hz) and H-3 d 3.93 (J 3,4 3Hz)],andatd 5.40 (unresolved doublet) [H-2 d 4.39 (J 2,3 2Hz) and H-3 d 4.28 (J 3,4 3 Hz)], and at 5.10 (unresolved doublet) [H-2 d 4.17 (J 2,3 2 Hz)], typical of a- D -Man configurations and were thus assigned to the L -a- D -Hep residues, as were observed in the Smith degradation 1 H NMR product described above. All other anomeric resonances detected possessed b anomeric con- figuration and could be seen at d 4.99 (J 1,2 )7Hz),d 4.88 (J 1,2 )7Hz),d 4.69 (J 1,2 )7Hz),d 4.66 (J 1,2 )7Hz)andd 4.62 (J 1,2 )7 Hz). To situate the side-branch hexoses, a 2D 1 H– 1 H NOESY experiment was performed and conclusive evidence, an inter-NOE between H-1 (d 5.67) of the a-Gal and H-2 (d 3.98) of the residue with the b anomeric H-1 (d 4.99), was obtained that placed the sole a- D -Gal side- branch at O-2 of the unit with the anomeric at d 4.99, which has to belong to the sole 2-substituted unit, that being the 2,3-disubstituted Gal in the backbone. The other side-branch hexoses, two Glc units, must then have the b anomeric configuration and be attached to the L -a- D -Hep residues (b- D -Glc-(1fi2)- L -a- D -Hep) and (b- D -Glc-(1fi4)- L -a- D -Hep). The GM2 and GM1a ganglioside mimics in C. jejuni NCTC 11168 LOSs were covalently attached to the inner core region (Fig. 3) composed of basal core OS units, Gal, LD -Hep and Glc. The GM2 and GM1a epitopes completed a core OS similar to that present in C. jejuni serogroup HS:1 [6]. In addition, the innermost LD -Hep was phos- phorylated by a monoester phosphate or by a 2-amino ethyl phosphate. Structure of the capsule polysaccharide The polysaccharide, obtained from the aqueous phase after ultracentrifugation, was purified on a Bio-Gel P-2 (PS-1). Alditol acetate analysis revealed the presence of D -Glc, Table 2. Negative ion ESI-MS data and proposed compositions for C. jejuni NCTC 11168 core OS and de-O-acylated LOS (masses include the addition of water) a . Core OS Observed molecular mass (Da) Proposed structure 1635 ()18) HexNAcÆHex 5 ÆHep 2 ÆKdo 1714 ()18) HexNAcÆHex 5 ÆHep 2 ÆPÆKdo 1758 ()18) HexNAcÆHex 5 ÆHep 2 ÆPEtnÆKdo 1876 HexNAcÆHex 6 ÆHep 2 ÆPÆKdo 1920 HexNAcÆHex 6 ÆHep 2 ÆPEtnÆKdo 2005 ()18) Neu5AcÆHexNAcÆHex 5 ÆHep 2 ÆPÆKdo 2049 ()18) Neu5AcÆHexNAcÆHex 5 ÆHep 2 ÆPEtnÆKdo a Residues used and their molecular mass: Neu5Ac, 291; HexNAc, 203; Hex, 162; Hep, 192; PEtn, 123; P, 79; Kdo, 220. P/PEtn fl [ D -Hex-(1fi3)]±- D -GalNAc-(1fi4)- D -Gal-(1fi3)-b- D -Gal-(1fi3)- L -a- D -Hep-(1fi3)-L-a- D -Hep-(1fi5)-Kdo 32 2 4 ›› › › 21 1 1 Neu5Ac D -Hex D -Hex D -Hex Major Smith degradation product [ D -GalNAc-(1fi4)- D -Gal-(1fi3)]±- D -Gal-(1fi3)- D -Man*-(1fi3)- D -Man*-(1fi)-3-d-hexitol**** 3 › 2 [Neu5Ac*]± 5124 F. St. Michael et al. (Eur. J. Biochem. 269) Ó FEBS 2002 D -Gal, LD -Hep (from the core region) and D -GlcNAc as minor components, and as major units D -ribose ( D -Rib), 6-O-methyl-heptose (6-O-Me-Hep), and N-acetyl- D -gal- actosamine ( D -GalNAc). From the alditol acetate analysis it was observed that PS-1 was slightly contaminated with LOS, additional efforts at purification were not successful. The methylation linkage analysis revealed the presence of 2-substituted D -Rib, 4-substituted D -GalNAc, and a terminal heptose unit. No substituted heptose was detected, and thus the 6-O-Me-Hep was present as a side chain residue. For further characterization of the structure of PS-1, the sample was acid hydrolyzed under mild conditions (1 M HCl, 100 °C for 5 min) and the resultant hydrolysate was purified on a Bio-Gel P-2 column. The sample was then analyzed by CE-ESI-MS (Fig. 4a) and gave rise to two components having a mass of 791 as the major product and a minor mass of 762. The MS/MS spectra of m/z 791 (Fig. 4b) revealed fragments m/z 588 and 585. This showed the loss of either 6-O-Me-Hep or GalNAc, respectively; thus illustrating that they were terminal units in this OS from acid hydrolysis of PS-1. This finding was consistent with the previous observation in the linkage analysis that the 6-O-Me-Hep was a terminal unit. The fragment ion at 382 arose from the loss of both the 6-O-Me-Hep and GalNAc, whichthenleadtothelossofRibwithafinalmassof 250 Da. The MS/MS spectra of m/z 762 (Fig. 4c), as with m/z 791, showed the loss GalNAc (m/z 558) and 6-O-Me-Hep (m/z 555) as terminal residues. It can also be observed, as in the previous example, that after the loss of the 6-O-Me-Hep and the GalNAc leading to fragment ion m/z 352, Rib is also lost, which furnished a final mass of 220 Da. Therefore, from this CE-MS/MS analysis it was suspected that there might be two polysaccharide chains present or that one of the components in the capsule can be modified. The 1 H NMR spectrum of PS-1 (Fig. 5a,b) revealed the presence of four anomerics. COSY, NOESY, and TOCSY NMR experiments were performed on PS-1, but the heterogeneity lead to broad lines, making interpretation difficult. This lead to the use of HR-MAS NMR to examine capsular polysaccharide resonances on intact Campylobacter cells without the need for extensive growth and purification [34]. HR-MAS spectra of wild-type and capsule mutants were used for the initial screening and selection of a mutant that lacked the 6-O-Me-Hep, as this sugar was suspected to be a side chain. The disappearance of one anomeric and the loss of the OMe resonance at 3.56 p.p.m. (Fig. 5c,d) were used to ascertain this. Once an appropriate mutant was generated (Cj1428c mutant; see below), its polysaccharide was purified (the absence of the 6-O-Me-Hep was also verified by alditol acetate analysis, results not shown). The polysaccharide (isolated as described above) of the Cj1428c mutant was denoted as PS-2 (Fig. 6). The proton spectrum of PS-2 was more homogeneous and had sharper lines than the proton spectrum of PS-1. As the spectrum of PS-2 was less complex than the spectrum of the native PS-1, its structural determination was first undertaken. NMR methods, as outlined before [33,38,39], were used for the structural determination of polysaccharides 1 and Fig. 2. FAB-MS spectra of the methylated C. jejuni NCTC 11168 core OS. Ó FEBS 2002 LOS and capsule structures of C. jejuni NCTC 11168 (Eur. J. Biochem. 269) 5125 Table 3. Interpretation of m/z ions in the FAB-MS spectrum of the methylated core OS from C. j ejuni NCTC 11168. Primary m/z ion Secondary m/z ion Double cleavage m/z ion Proposed structure 260 228 (260–32) GlcNAc + 376 344 (376–32) Neu5Ac + 464 432 (464–32) Gal-(1fi3)-GalNAc + 547 Glc-(1fi4)-Hep + › P 668 GalNAc-(1fi4)-Gal-(1fi3)-Gal + 826 GalNAc-(1fi4)-Gal + 3 › 2 Neu5Ac 872 GalNAc-(1fi4)-Gal-(1fi3)-Gal + 3 › 2 Neu5Ac 1029 Gal-(1fi3)-GalNAc-(1fi4)-Gal + 3 › 2 Neu5Ac 1120 GalNAc-(1fi4)-Gal-(1fi3)-Gal-(1fi3)-Hep + 2 › 1 Gal 1233 GalNAc-(1fi4)-Gal-(1fi3)-Gal + 32 ›› 21 Neu5Ac Gal 1324 GalNAc-(1fi4)-Gal-(1fi3)-Gal-(1fi3)-Hep + 22 ›› 11 Gal Glc 1407 P fl Gal-(1fi3)-Hep-(1fi3)-Hep + 22 4 ›› › 11 1 Gal Glc Glc 1477 PEtn fl Gal-(1fi3)-Hep-(1fi3)-Hep + 224 ›› › 111 GalGlc Glc 1685 GalNAc-(1fi4)-Gal-(1fi3)-Gal-(1fi3)-Hep + 322 ››› 211 Neu5Ac Gal Glc 5126 F. St. Michael et al. (Eur. J. Biochem. 269) Ó FEBS 2002 2 obtained from C. jejuni. 1D selective NMR methods were also used to characterize individual components [33]. 1D-TOCSY experiments were used to detect the coupled spin systems and spin simulation was used to obtain accurate coupling constants (Figs 7a–d). HMQC was used to assign CH, CH 2 and CH 3 carbon resonances (Fig. 7f). The assignments were verified by means of an HMQC- TOCSY experiment. HMBC was used to locate the C¼O resonances (Fig. 7g). A correlation was also observed between C-7C and H-8C for the NAc group of residue C (results not shown). Location of nitrogen-bearing groups was carried out by performing experiments in 90% H 2 Oin order to detect the NH resonances (Fig. 7i). A COSY experiment (results not shown) was used to detect the NH- C-H correlation and assign the NH resonances. The NOESY experiment was used to detect NOEs between the pendant groups and the ring protons. Finally the HMBC experiment was used to detect the multiple bond correlation between the C¼O and NH resonances (Fig. 7i). Table 3. (Continued). 1857 P fl GalNAc-(1fi4)-Gal-(1fi3)-Gal-(1fi3)-Hep-(1fi3)-Hep + 322 4 ››› › 211 1 Neu5Ac Gal Glc Glc 1928 PEtn fl GalNAc-(1fi4)-Gal-(1fi3)-Gal-(1fi3)-Hep-(1fi3)-Hep + 3224 ››› › 211 1 Neu5Ac Gal Glc Glc Atomic mass units of the residues discussed above: Glc/Gal GalNAc Hep Neu5Ac P PEtn Terminal 219 260 263 376 94 166 Monosubstituted 204 245 248 Disubstituted 189 233 Fig. 3. The complete structure of C. jejuni NCTC 11168 LOS. Fig. 4. CE-ESI-MS and CE-MS/MS analysis. CE-ESI-MS of PS-1 after acid hydrolysis (1 M HCl, 100 °C for 5 min) and Bio-Gel P-2 purification(a),CE-MS/MSofm/z 791 (b), CE-MS/MS of m/z 762 (c). Fig. 5. Proton spectra of C. jejuni whole cells and isolated polysaccha- rides. HR-MAS spectrum of C. jejuni whole cells (a) and its isolated polysaccharide (b). HR-MAS spectrum of C. jejuni Cj1428c mutant whole cells (c) and its isolated polysaccharide (d). The anomeric resonances are labeled according to the structures shown in Fig. 6. Ó FEBS 2002 LOS and capsule structures of C. jejuni NCTC 11168 (Eur. J. Biochem. 269) 5127 As the absolute configuration of the sugars was known from the chemical analysis, from a comparison of chemical shifts and coupling constants with those of monosaccharides, residue A was assigned as b- D -Ribf, residue B as the amide of a- D -GlcpA with -NH-CH 2 -CH 2 OH at C-6, and residue C as b- D -GalfNAc. The sequence of sugars was established by an HMBC experiment (Fig. 7h). The (H-1 A, C-5C) (H-1C, C-4B) and (H-1B, C-2 A) HMBC correlations established the (-A-C-B-) n polymeric sequence (Fig. 6). The NMR data areshowninTable4. The linkage analysis of the Cj1428c mutant, PS-2, by the methylation method [22] revealed the same 2-substituted Rib and 4-substituted GalpNAc,whichinthiscase,as observed by NMR, is a 5-substituted GalfNAc. As expected, this PS-2 lacked the terminal 6-O-Me-Hep. Confirmation of the repeat established by NMR was observed in the FAB- MS and MALDI-MS (Fig. 8) of the methylated PS-2. The MALDI-MS spectra shows a series of A-type primary glycosyl oxonium ions and secondary ions of defined compositions at m/z 260fi228 (GalNAc) + , m/z 420 (Gal- NAc, Rib) + , m/z 695 (GalNAc, Rib, GlcA), m/z 941 (GalNAc 2 ,Rib,GlcA) + , m/z 1101 (GalNAc 2 ,Rib 2 , GlcA) + , m/z 1620 (GalNAc 3 ,Rib 2 ,GlcA 2 ) + , m/z 1781 Fig. 6. Structure of polysaccharides PS-1 and PS-2 from C. jejuni,and labeling of the residues and atoms. Residue A is b- D -Ribf,residueBis the amide of a- D -GlcpA with ethanolamine at C-6 for PS-2 and with 2-amino-2-deoxyglycerol at C-6 for PS-1, residue C is b- D -GalfNAc, and residue D is D -glycero-a- L -gluco-heptopyranose. Fig. 7. NMR spectrum of PS-2. Spin simula- ted spectra for residue A (a), residue B (b), residue C (c) and the substituent at C-6 of residue B (d), along with the resolution enhanced proton spectrum (e). In (f) is the HMQC spectrum of the ring protons. In (g) is the HMBC spectrum showing the C¼O region for assignment of C-6B and C-7C. The HMBC spectrum in (h) shows the intergly- cosidic 3 J(C,H) correlations. In (i) the proton spectrum, NOESY and HMBC correlations for the NH resonances obtained in 90% H 2 O are shown. 5128 F. St. Michael et al. (Eur. J. Biochem. 269) Ó FEBS 2002 [...]... that of C jejuni In summary, we have presented the complete structures of the LOS and capsule polysaccharide of the genome sequenced strain C jejuni NCTC 11168 The outer core LOS of NCTC 11168 has structural homology with the human gangliosides, GM2 and GM1a As demonstrated previously in NCTC 11168 [16,17,48] and in 81–176 [12], C jejuni can exhibit variable ganglioside mimics due to variation of the. .. to find L-glycero-D-mannoheptopyranose in the LOS core and 6-O-Me-Hep in the capsule polysaccharide in this study We are currently characterizing the heptose biosynthetic pathways in NCTC 11168 The sugar composition of the NCTC 11168 polysaccharides was examined in earlier studies by Naess and Hofstad [47] The authors actually noted the presence of ribose in their preparations but attributed the finding... Cj1428c in Campylobacter polysaccharide biosynthesis is currently unknown There is no evidence for the presence of fucose in any carbohydrate structures of NCTC 11168 identified to date, but we are currently investigating this possibility Alternatively, this enzyme may be involved in the epimerization reaction necessary for the biosynthesis of the capsular heptopyranose as alditol acetate analysis (results... replication [17] The authors predominantly observed the in-frame version of wlaN while our laboratory observes Ó FEBS 2002 LOS and capsule structures of C jejuni NCTC 11168 (Eur J Biochem 269) 5133 mostly the truncated version of the gene (results not shown) Examination of LOS cores isolated from growth of single colonies of our NCTC 11168 by deoxycholate-PAGE and MS yielded structures with and without the terminal... Analysis of the genome sequence of NCTC 11168 demonstrates that the strain contains two gene clusters involved in heptose biosynthesis [8] One cluster, located in the LOS gene cluster, is similar to that found in other Gramnegative bacteria and is necessary for the biosynthesis of the L-glycero-D-manno-heptopyranoses commonly found in LPS of many Gram-negative bacteria The second cluster, found in the. .. involved in capsule biosynthesis being flanked by the kps genes involved in the transport of the sugar polymer (Fig 11a) [9–11] Class II/III capsules have been shown to be attached to the membrane by glycerophosphate anchors [9] However, we were unable to conclusively demonstrate the linkage of the capsule polysaccharide to its membrane anchor and are currently trying to determine the nature of the membrane... in the capsular polysaccharide The genes necessary for ribose biosynthesis are not obvious in the capsule gene cluster, thus we speculate that the sugar is synthesized during normal cell metabolism and that the genes required for biosynthesis are located elsewhere on the chromosome As already mentioned, both N-linked glycerol and ethanol modifications were found on the glucuronosyl residue of wild-type... portion of the LPS containing 2,3,4-tri-O-methylfucosyl caps [57] Methyl capping of terminal perosamines of LPS has also been described in Klebsiella, E coli, Rhizobium and Vibrio as well as on the S-layer glycoprotein of Geobacillus stearothermophilus [51,57–59] In this report, we describe variable methyl, glycerol, and ethanol modifications on the capsule polysaccharides of C jejuni NCTC 11168 As... the complex capsular carbohydrate structure of wild-type NCTC 11168 with the simpler capsule of the Cj1428c mutant (Figs 5c and 11a) Cj1428c is homologous to fcl of E coli whose product is involved in the conversion of GDP-4-keto-6-deoxy-D-mannose to GDP-4-keto-6-Ldeoxygalactose to GDP-L-fucose in the formation of fucose in the colanic acid extracellular polysaccharide [43] The role of Cj1428c in Campylobacter. .. LOS core The mimicry of human cellsurface glycolipids and glycoproteins appears to be a general trend of mucosal pathogens such as Haemophilus, Neisseria, Helicobacter and Campylobacter (for reviews see [7,61]) The peculiar property of producing structures analogous to cell-surface molecules of the host and having the ability to vary these structures plays an important role in pathogenesis and survival . The structures of the lipooligosaccharide and capsule polysaccharide of Campylobacter jejuni genome sequenced strain NCTC 11168 Frank St that of C. jejuni. In summary, we have presented the complete structures of the LOS and capsule polysaccharide of the genome sequenced strain C. jejuni NCTC