The HS:19 serostrain of Campylobacter jejuni has a hyaluronic acid-type capsular polysaccharide with a nonstoichiometric sorbose branch and O-methyl phosphoramidate group David J McNally, Harold C Jarrell, Nam H Khieu, Jianjun Li, Evgeny Vinogradov, Dennis M Whitfield, Christine M Szymanski and Jean-Robert Brisson Institute for Biological Sciences, National Research Council of Canada, Ottawa Ontario, Canada Keywords Campylobacter jejuni; capsular polysaccharide; high-resolution magic angle spinning (HR-MAS) NMR; phosphoramidate; sorbose Correspondence J.-R Brisson, Institute for Biological Sciences, National Research Council of Canada, 100 Sussex Drive, Ottawa Ontario, Canada, K1A 0R6 Fax: +1 613 9529092 Tel: +1 613 9903244 E-mail: jean-robert.brisson@nrc-cnrc.gc.ca (Received April 2006, revised June 2006, accepted 29 June 2006) doi:10.1111/j.1742-4658.2006.05401.x A recent study that examined multiple strains of Campylobacter jejuni reported that HS:19, a serostrain that has been associated with the onset of ´ Guillain–Barre syndrome, had unidentified labile, capsular polysaccharide (CPS) structures In this study, we expand on this observation by using current glyco-analytical technologies to characterize these unknown groups Capillary electrophoresis electrospray ionization MS and NMR analysis with a cryogenically cooled probe (cold probe) of CPS purified using a gentle enzymatic method revealed a hyaluronic acid-type [-4)-b-d-GlcA6NGro(1–3)-b-d-GlcNAc-(1-]n repeating unit, where NGro is 2-aminoglycerol A labile a-sorbofuranose branch located at C2 of GlcA was determined to have the l configuration using a novel pyranose oxidase assay and is the first report of this sugar in a bacterial glycan A labile O-methyl phosphoramidate group, CH3OP(O)(NH2)(OR) (MeOPN), was found at C4 of GlcNAc Structural heterogeneity of the CPS was due to nonstoichiometric glycosylation with sorbose at C2 of GlcA and the nonstoichiometric, variably methylated phosphoramidate group Examination of whole bacterial cells using high-resolution magic angle spinning NMR revealed that the MeOPN group is a prominent feature on the cell surface for this serostrain These results are reminiscent of those in the 11168 and HS:1 strains and suggest that decoration of CPS with nonstoichiometric elements such as keto sugars and the phosphoramidate is a common mechanism used by this bacterium to produce a structurally complex surface glycan from a limited number of genes The findings of this work with the HS:19 serostrain now present a means to explore the role of CPS as a virulence factor in C jejuni Campylobacter jejuni is one of the leading causes of human gastroenteritis and surpasses Salmonella, Shigella and Escherichia in some regions as the primary cause of gastrointestinal disease [1–3] There is also a convincing body of evidence linking C jejuni infections ´ to the onset of Guillain–Barre syndrome [4–11] Although of relatively rare occurrence, this syndrome is the most common cause of acute neuromuscular paralysis since the eradication of polio It is characterized by weakness in the limbs and respiratory muscles, with paralysis generally occurring 1–3 weeks after infection [12,13] Penner’s passive haemagglutination Abbreviations CPS, capsular polysaccharide; CE-ESI-MS, capillary electrophoresis electrospray ionization mass spectrometry; HMBC, heteronuclear multiple-bond correlation; HR-MAS NMR, high-resolution magic angle spinning nuclear magnetic resonance spectroscopy; HSQC, heteronuclear single-quantum coherence; MeOPN, O-methyl phosphoramidate CH3OP(O)(NH2)(OR) FEBS Journal 273 (2006) 3975–3989 ª 2006 The Authors Journal compilation ª 2006 FEBS 3975 Campylobacter jejuni HS: 19 CPS D J McNally et al assay [14] was used to show that 81% of C jejuni iso´ lates from patients with Guillain–Barre syndrome in Japan belonged to Penner’s HS:19 serotype In the United States, 33% of C jejuni isolates from such patients were classified as HS:19 [11,15,16] It is now widely accepted that the major antigenic component of Penner’s serotyping system for C jejuni is capsular polysaccharide (CPS) [17] This was not always the case, as lipopolysaccharide was for many years thought to be the basis for Penner’s classification system [8] Because CPS is the outermost structure on the bacterial cell, it plays a key role in the interaction between the pathogen, host, and environment [18] and is generally thought to be important for bacterial survival and persistence in the environment [19] By mimicking host cell antigens and through structural variation, CPSs also convey evasion from host immune responses and are therefore considered important virulence factors CPS production in C jejuni remained unnoticed until the genome sequencing of C jejuni NCTC11168 in 2000 and the identification of genes implicated in CPS biosynthesis [20] Since this discovery, genetic, biochemical and microscopy studies have demonstrated CPS production in different strains of C jejuni [17,21,22] Our laboratory has shown that it is possible to study CPS directly on the surface of intact C jejuni cells using high-resolution magic angle spinning NMR spectroscopy (HR-MAS NMR) [18,23,24] On the basis of its role in epithelial cell invasion, diarrhoeal disease, serum resistance and maintenance of bacterial cell surface hydrophilicity [17,22], CPS is thought to play a critical role in pathogenesis for C jejuni The precise mechanisms by which CPS conveys virulence to C jejuni are poorly understood; however, structural variation of this surface glycan is emerging as a possible mechanism [23,24] The CPSs produced by C jejuni are structurally diverse and there are more than 60 serostrains described for this bacterium, excluding nontypeable strains, each having a different CPS structure [25] For each serostrain, there are often phase-variable structural modifications such as the incorporation of methyl, ethanolamine and aminoglycerol groups on CPS sugars [18,23,24] The most unusual of these modifications is the O-methyl phosphoramidate CH3OP(O)(NH2)(OR) (MeOPN), which is a highly labile phosphorylated structure that was first described on the GalfNAc CPS sugar for the 11168 strain [18] By using mild extraction conditions to purify CPS and HR-MAS NMR to study CPS in vivo, it was recently shown that the HS:1 serostrain also expresses this phosphorylated modification on two labile fructofuranose branches By placing variable MeOPN groups on labile branches, the HS:1 strain 3976 was shown to produce a structurally variable and therefore highly complex CPS despite having a small CPS biosynthetic locus containing 11 genes [23,25] Sequencing the CPS biosynthetic regions for several strains of C jejuni uncovered evidence for multiple mechanisms of CPS variation, including exchange of capsular genes and entire clusters by horizontal transfer, gene duplication, deletion, fusion and contingency gene variation [25] Of particular interest, the CPS locus for the HS:19 serostrain was shown to contain only 13 genes, including a udg homologue responsible for producing b-d-GlcA6NGro [25] This finding correlated well with the CPS structure reported for the HS:19 serostrain, which was shown to consist of a [-4)b-d-GlcANGro-(1–3)-b-d-GlcNAc-(1-]n repeating unit [5,6,12] The CPS locus of the HS:19 strain was also shown to contain HS19.07, a homologue of cj1421 in the 11168 strain, which is speculated to be a MeOPN transferase responsible for adding MeOPN to CPS sugars [24] By examining a partially purified CPS sample prepared from HS:19 cells, the latter study observed unidentified labile groups and speculated that one of these was a MeOPN modification similar to the one reported for other strains of C jejuni [18,23–25] In this study, we thoroughly investigate the complete CPS structure for the HS:19 serostrain of C jejuni by using the latest glyco-analytical technologies to characterize these unknown labile groups Initially, HR-MAS NMR was used to examine CPS directly on the surface of whole HS:19 cells To study the structure of the CPS in greater detail, we isolated it using a mild enzymatic extraction method that preserves labile groups [23] In previous studies [5,6,12], the classic hot water ⁄ phenol extraction method [26] was used, as this structure was originally thought to be a high-molecular-mass lipopolysaccharide High-resolution NMR at 600 MHz (1H) with an ultra-sensitive cryogenically cooled probe (cold probe), and capillary electrophoresis electrospray ionization mass spectrometry (CE-ESI-MS) with in-source collision-induced dissociation [27] were then used to determine the structure of purified CPS Herein we report the complete structure for the CPS of the HS:19 serostrain of C jejuni and discuss the biological significance of these new structural findings for this organism Results The results generated by HR-MAS NMR and highresolution NMR, CE-ESI-MS and chemical ⁄ enzymatic analyses revealed a hyaluronic acid-type CPS with a [-4)-b-d-GlcA6NGro-(1–3)-b-d-GlcNAc-(1-]n repeating unit (Fig 1) This finding is in agreement with FEBS Journal 273 (2006) 3975–3989 ª 2006 The Authors Journal compilation ª 2006 FEBS D J McNally et al Campylobacter jejuni HS: 19 CPS located at C2 of b-d-GlcA and a variably methylated nonstoichiometric MeOPN group at C4 of b-d-GlcNAc A -D-GlcA6NGro H HOH2C D CH2OH MeOPN O NH H2N O H O O O H H HO HOH2C O H O H H O H -D-GlcNAc CH2OH H HR-MAS NMR spectroscopy of whole cells B CH3 O P O NH H H O O H H CH2OH CH3 H HO OH C -L-Sorf Fig The complete structure of the repeating unit for the CPS of the HS:19 serostrain of C jejuni The repeating unit of the CPS consists of [-4)-b-D-GlcA6NGro-(1–3)-b-D-GlcNAc-(1-]n with an a-L-sorbofuranose branch located at C2 of GlcA and an MeOPN group at C4 of GlcNAc Structural heterogeneity is due to the nonstoichiometric sorbofuranose branch and the variably methylated nonstoichiometric MeOPN group Residue A represents b-D-glucuronic acid-6-N-glycerol, B is 2-acetamido-2-deoxy-b-D-glucose, C is a-L-sorbofuranose, and D is MeOPN previous studies that examined CPS in the HS:19 serostrain of C jejuni [5,6,12] However, the complete structure of the CPS was found to be more complex because of a nonstoichiometric a-l-sorbose branch Examination of CPS on the surface of whole HS:19 cells with HR-MAS NMR revealed multiple signals originating from the cell surface (Fig 2) The 1H HR-MAS NMR spectrum exhibited broad lines, however; a doublet characteristic of a MeOPN group at dH 3.77 p.p.m was observed to protrude above the broad CPS signals (Fig 2A) A scalar coupling of 12.0 Hz was measured for this doublet, which is in good agreement with 3JH,P couplings determined for the MeOPN in the 11168 and HS:1 serostrains [18,23,24] A 1D 31P heteronuclear single-quantum coherence (HSQC) experiment that specifically selects for the MeOPN group (31P decoupled, MeOPN-filtered 31P HSQC) confirmed that this doublet originated from a MeOPN group (Fig 2B) The chemical shift of the MeOPN signal at dP 14.7 p.p.m., determined using a 2D 31PHSQC experiment (Fig 2C), is highly unique to a phosphoramidate bond and is consistent with MeOPN signals observed in other strains of C jejuni, which range from dP 13.1 p.p.m to 14.7 p.p.m [18,23,24] For HR-MAS NMR of whole C jejuni cells, we typically observe only the 1H-31P correlation between the phosphorus and the sharp methyl group resonance of the MeOPN The correlation between phosphorus of the MeOPN and the ring protons of pyranose sugars is not observed [24], probably because of short T2 transverse A B C Fig HR-MAS NMR spectroscopy of intact C jejuni HS:19 cells (A) 1H-HR-MAS NMR spectrum (256 transients) showing a doublet originating from a MeOPN group (B) 1D 31 P-HSQC ‘MeOPN-filtered’ HR-MAS NMR spectrum (31P-decoupled, 256 transients, JH,P ¼ 10 Hz) showing a broad signal originating from a MeOPN group (C) 2D 31 P-HSQC HR-MAS NMR spectrum showing two MeOPNs (256 transients, 128 increments, 1JH,P ¼ 10 Hz) FEBS Journal 273 (2006) 3975–3989 ª 2006 The Authors Journal compilation ª 2006 FEBS 3977 Campylobacter jejuni HS: 19 CPS D J McNally et al relaxation times and the lower intensity of the broad GlcNAc H4 resonance due to homonuclear couplings and structural heterogeneity of the CPS A second minor MeOPN signal observed at dP 14.3 p.p.m indicated structural heterogeneity for CPS on the cell surface Spectral lines originating from CPS on the surface of HS:19 cells were too broad to draw additional conclusions regarding its structure, necessitating its isolation for further study Isolation of CPS Residue Position dH dC b-D-GlcA6NGro (A) A1 A2 A3 A4 A5 A6 A7 A8 ⁄ A8¢ B1 B2 B3 B4 B5 B6 ⁄ B6¢ B7 B8 C1 ⁄ 1¢ C2 C3 C4 C5 C6 ⁄ C6¢ D1 4.65 3.71 3.71 3.90 3.94 170.7 4.07 3.65 ⁄ 3.75 4.62 3.97 4.24 4.26 3.61 3.74 ⁄ 3.93 175.0 2.11 3.64 ⁄ 3.73 104.3 4.17 4.41 4.39 3.69 ⁄ 3.79 3.77 101.0 73.8 73.8 79.0 75.1 b-D-GlcNAc (B) Previous studies that have examined CPS in the HS:19 serostrain used Westphal’s classic hot water ⁄ phenol extraction method to isolate the polysaccharide [5,6,12,26] Our results using this method closely agreed with these studies in terms of the quantity and purity of CPS obtained However, CE-ESI-MS and NMR analyses of hot water ⁄ phenol-purified CPS revealed that labile groups such as the MeOPN and sorbose branch were mostly absent (not shown) On the basis of these observations, we concluded that the hot water ⁄ phenol method was responsible for removing labile CPS groups, and that removal of these labile modifications by this method resulted in them being overlooked by previous studies These findings support a recent study that found that the hot water ⁄ phenol method hydrolyzed labile groups that are present on the CPS of the HS:1 serostrain of C jejuni [23] With a less harsh enzymatic purification method,  mg CPS was obtained from a 6-L culture of HS:19 cells (7 g cells, wet pellet mass) CPS isolated with this gentler technique contained a moderately higher concentration of nucleic acid and protein impurities However, this enzymatic extraction method preserved the sought after labile groups, facilitating the characterization of the complete CPS structure High-resolution NMR spectroscopy of purified CPS Examination of purified CPS with NMR at 600 MHz (1H) with a cold probe revealed a [-4)-b-d-GlcA6NGro-(1–3)-b-d-GlcNAc-(1-]n backbone and showed the unknown labile CPS structures to be a MeOPN group and an a-sorbofuranose branch (Table 1, Fig 3) The proton spectrum of purified CPS closely resembled CPS on the cell surface in that a doublet originating from a MeOPN group (dH 3.77 p.p.m., JH,P ¼ 12.0 Hz) was observed to protrude above broad CPS signals (Fig 3A) Selective 1D TOCSY and 1D NOESY experiments were used to assign the proton resonances for GlcA, GlcNAc and sorbose 1D 3978 Table NMR proton and carbon chemical shifts d (p.p.m.) for CPS purified from HS:19 serostrain of C jejuni a-L-Sorf (C) MeOPN (D) 53.9 61.5 100.7 56.1 75.8 74.2 75.5 61.3 23.5 61.5 79.2 76.1 79.2 62.9 54.8 TOCSY of the sorbose H3 resonance revealed overlapping H4 and H5 signals (Fig 3B), and simultaneous excitation of the H4 and H5 resonances showed signals corresponding to H6 ⁄ H6¢ (Fig 3C) As C2 of sorbose does not have a proton, a 1D NOESY experiment of the sorbose H3 resonance was used to assign H1 ⁄ H1¢ resonances (Fig 3D) A 1H-31P correlation observed between the MeOPN OCH3 group and H4 of GlcNAc at dP 14.7 p.p.m indicated the location of the MeOPN at C4 of GlcNAc (Fig 3E) Carbon assignments were determined from 13C-1H correlations observed using a 13C-HMQC experiment (Fig 3F) With the exception of sorbose resonances, signals within the 13C-HMQC spectrum for purified HS:19 CPS were generally broad and therefore weak In particular, 13C-1H correlations for C3 and C4 of GlcNAc and C1 of GlcA and GlcNAc were visible only at higher temperature (40 °C) and at 600 MHz (1H) with a cryogenically cooled probe Proton and carbon resonances determined from 13 C-HMQC and heteronuclear multiple-bond correlation (HMBC) experiments were consistent with those reported for b-GlcA6NGro and b-GlcNAc [5,6,12], a MeOPN group [18,23,24] and a-sorbofuranose [28–30] Structural heterogeneity generated by the sorbose branch and MeOPN group was indicated by two sets of 13C-1H correlations for C2 and C3 of GlcA, as well FEBS Journal 273 (2006) 3975–3989 ª 2006 The Authors Journal compilation ª 2006 FEBS D J McNally et al Campylobacter jejuni HS: 19 CPS Fig High-resolution NMR spectroscopy of CPS purified from the HS:19 serostrain of C jejuni (A) 1H NMR spectrum (1024 transients) showing a doublet originating from the MeOPN group (B) 1D TOCSY (30 ms) of Sor H3 (C) 1D TOCSY (30 ms) of Sor H4 and H5 (D) 1D NOESY (400 ms) of Sor H3 (E) 31P-HSQC spectrum (64 transients, 32 increments, 1JH,P ¼ Hz) (F) 13C-HMQC spectrum (128 transients, 128 increments, JC,H ¼ 150 Hz) For selective 1D experiments, excited resonances are underlined Residues with asterisks correspond to structural heterogeneity generated by the nonstoichiometric MeOPN group and a-L-sorbofuranose branch Annotations for residues are the same as Fig and sm represents sorbose monosaccharide as for C4 and C5 of GlcNAc (Fig 3F, indicated with asterisks) The chemical shifts of these extra resonances were in excellent agreement with those reported for nonphosphoramidated, nonsorbosylated CPS [5,6,12] Compared with nonphosphoramidated CPS, our results indicated that the MeOPN caused a downfield shift in the H4 resonance of GlcNAc (0.71 p.p.m.) This observation is consistent with the effects of phosphoramidation reported for the 11168 and HS:1 strains, where the presence of the MeOPN group caused the signals for neighbouring protons to shift by 0.6–0.8 p.p.m [18,23,24] analyses and NMR spectroscopy (Fig 4A, Table 2) Interestingly, ions observed at m ⁄ z 514.4, 532.4, 694.5 and 984.8 indicated the presence of an O-phosphoramidate group OP(O)(NH2)(OR) without O-methylation, suggesting that the nonstoichiometric MeOPN group is also variably methylated for the HS:19 serostrain CE-ESI-MS ⁄ MS analysis of m ⁄ z 708.5, corresponding to one complete repeat of the CPS, revealed fragment ions at m ⁄ z 297.0 and m ⁄ z 412.3, confirming the location of the MeOPN group on GlcNAc and the presence of a single sorbose branch on GlcA (Fig 4B) MS analysis of purified CPS Determination of absolute configuration for CPS sugars Because of the large molecular mass of the HS:19 CPS, a high orifice voltage (+ 200 V) was used to promote in-source collision-induced dissociation [27] to facilitate its analysis with CE-ESI-MS (Fig 4) CE-ESI-MS analysis of purified CPS corroborated the hyaluronic acid-like structure deduced from chemical By comparing the GC retention times of the R- and S-butyl glycosides of authentic standards with the R-butyl glycosides prepared from a purified CPS sample, b-GlcA and b-GlcNAc were shown to have the d configuration (not shown) As the chiral alcohol FEBS Journal 273 (2006) 3975–3989 ª 2006 The Authors Journal compilation ª 2006 FEBS 3979 Campylobacter jejuni HS: 19 CPS D J McNally et al Fig MS analysis of CPS that was purified from the HS:19 serostrain of C jejuni (A) CE-ESI-MS total ion spectrum (positive ion mode, orifice voltage +200 V) (B) CE-ESIMS ⁄ MS analysis of m ⁄ z 708.5, which corresponds to one complete repeat of the CPS method cannot be used to determine the absolute configuration of keto sugars [31], we report for the first time the use of pyranose oxidase for this purpose Pyranose oxidase from the white rot fungus Trametes multicolor is reported to oxidize l-sorbose at the C5 position forming 5-keto-d-fructose and hydrogen peroxide [32–34] However, it was not known if this enzyme was specific to the l isoform By incubating pyranose oxidase with d-sorbose and l-sorbose standards, we established that this enzyme oxidizes l-sorbose but not d-sorbose (Figs 5A–D) Incubating hydrolyzed purified HS:19 CPS with pyranose oxidase resulted in the disappearance of signals associated with the sorbose monosaccharide and the production of the same oxidation product as observed for the l-sorbose standard (Figs 5E,F) On the basis of these results, 3980 sorbose was concluded to have the l absolute configuration Branching pattern for sorbose The results of an HMBC experiment confirmed the glycosidic linkages within the [-4)-b-d-GlcA6NGro-(1– 3)-b-d-GlcNAc-(1-]n repeating unit of the purified CPS and showed that the a-l-Sor branch was located at C2 or C3 of GlcA (not shown) However, we were unable to determine the precise location of the a-l-Sor branch using the HMBC experiment because of spectral overlap for the resonances originating from positions and of GlcA which have identical 13C and 1H chemical shifts (Fig 3F, Table 1) The results of methylation analysis were inconclusive because of the poor solubil- FEBS Journal 273 (2006) 3975–3989 ª 2006 The Authors Journal compilation ª 2006 FEBS D J McNally et al Campylobacter jejuni HS: 19 CPS Table Positive ion CE-ESI-MS data for CPS isolated from the HS:19 serostrain of C jejuni Isotope-averaged masses of residues were used for calculation of total molecular masses based on the following proposed compositions: Hex (a-L-Sor), 162.1; HexNAc (b-D-GlcNAc), 203.2; HexANGro (b-D-GlcA6NGro), 249.2; MeOPN [O-methyl phosphoramidate, CH3OP(O)(NH2)], 93.2; NGro (N-glycerol), 73.1; OPN (O-phosphoramidate), 79.0; H2O, 18.0 For these gas-phase (IS-CID) degradation products, no H2O molecule is added to the residue unless specifically indicated Molecular mass (m ⁄ z) Observed Calculated Difference Structure 74.0 149.8 168.2 186.0 204.3 221.5 231.5 243.3 250.0 279.3 297.0 412.3 453.3 514.4 528.3 532.3 546.3 615.4 656.5 690.5 694.2 708.5 749.4 795.4 842.4 887.5 905.8 957.8 984.5 998.7 1059.8 1078.0 1091.8 1108.5 1253.0 1357.5 1681.5 1844.0 74.1 150.1 168.2 186.2 204.2 221.2 231.2 243.2 250.2 279.2 297.2 412.4 453.4 514.4 528.4 532.4 546.4 615.6 656.6 690.8 694.5 708.6 749.6 795.7 842.7 887.8 905.8 957.8 984.8 998.8 1059.8 1077.8 1091.9 1108.8 1253.0 1357.2 1681.5 1843.7 0.1 0.3 0.0 0.2 0.1 0.3 0.3 0.1 0.2 0.1 0.2 0.1 0.1 0.0 0.1 0.1 0.1 0.2 0.1 0.3 0.3 0.1 0.2 0.3 0.3 0.3 0.0 0.0 0.3 0.1 0.0 0.2 0.1 0.3 0.0 0.3 0.0 0.3 NGro – H2O HexNAc – (H2O)4 HexNAc – (H2O)3 HexNAc – (H2O)2 HexNAc – H2O HexNAc HexANGro – (H2O)2 HexNAcMeOPN – (H2O)3 HexANGro – H2O HexNAcMeOPN – (H2O)2 HexNAcMeOPN – H2O HexANGro + Hex – H2O HexANGro + HexNAc – H2O HexANGro + HexNAcOPN – (H2O)2 HexANGro + HexNAcMeOPN – (H2O)2 HexANGro + HexNAcOPN – H2O HexANGro + HexNAcMeOPN – H2O HexANGro + HexNAc + Hex – H2O HexANGro + (HexNAc)2 – H2O HexANGro + HexNAcMeOPN + Hex – (H2O)2 HexANGro + HexNAcOPN + Hex – H2O HexANGro + HexNAcMeOPN + Hex – H2O HexANGro + HexNAcMeOPN + HexNAc – H2O (HexANGro)2 + HexNAcMeOPN – H2O HexANGro + (HexNAcMeOPN)2 – H2O (HexANGro)2 + (HexNAc)2 – (H2O)2 (HexANGro)2 + (HexNAc)2 – H2O (HexANGro)2 + HexNAcMeOPN + Hex – H2O (HexANGro)2 + HexNAcOPN + HexNAc – H2O (HexANGro)2 + HexNAcMeOPN + HexNAc – H2O (HexANGro)2 + HexNAcMeOPN + HexNAcOPN – (H2O)2 (HexANGro)2 + HexNAcMeOPN + HexNAcOPN – H2O (HexANGro)2 + (HexNAcMeOPN)2 – H2O (HexANGro)2 + (HexNAcMeOPN)2 (HexANGro)2 + (HexNAcMeOPN)2 + Hex – H2O (HexANGro)3 + (HexNAc)3 – H2O (HexANGro)3 + (HexNAc)3 + (Hex)2 – H2O (HexANGro)3 + (HexNAc)3 + (Hex)3 – H2O ity of the CPS in organic solvent, the labile Sor glycosidic bond, and the heterogeneous substitution of Sor within the CPS Furthermore, because signals for CPS protons were broad and several positions for Sor, GlcA and GlcNAc have overlapping proton signals, NOEs could not be used to resolve the location of the Sor branch Similar problems were reported by a study that discovered a nonstoichiometric fructose branch in the lipopolysaccharide of Vibrio cholerae 0139 Bengal [35] Nevertheless, two lines of evidence point to the location of the Sor branch at C2 of GlcA Comparison of 13C chemical shifts for intact and acid-hydrolyzed CPS revealed that substitution with Sor causes an upfield shift in the C3 signal for GlcA (0.35 p.p.m.) and a downfield shift in the C2 signal of GlcA (0.73 p.p.m.) Although we were unable to locate data for sorbose in the literature, substitution with fructose was shown to cause a small downfield shift in the signals originating from the carbon atoms FEBS Journal 273 (2006) 3975–3989 ª 2006 The Authors Journal compilation ª 2006 FEBS 3981 Campylobacter jejuni HS: 19 CPS D J McNally et al A B C D E F 3.95 3.85 3.75 3.65 3.55 H (p.p.m.) of pyranose rings [36–38] Furthermore, compared with acid-hydrolyzed CPS, we observed substantial upfield shifts for the C1 resonance of GlcA (2.8 p.p.m.) and for the C3 resonance of GlcNAc (7.5 p.p.m.) As substitution with a keto sugar or a phosphoramidate group causes a relatively small change (< p.p.m.) [3,18,23], the magnitude of these differences for the C1 signal of GlcA (A) and C3 signal of GlcNAc (B) indicated a change about the GlcA-(1–3)-GlcNAc (A–B) glycosidic bond To understand the effects of various substitutions on the conformation of the glycosidic bonds in the CPS, we modelled the B–A–B unit [GlcNAc-(1–4)GlcA6NGro-(1–3)-GlcNAc] with and without residues C (a-l-Sor) and D (MeOPN) We would expect major changes in the distribution of u (O5–C1–O1–Cx) and w (C1–O1–Cx–Cx)1), with x being the aglyconic linkage site, to be accompanied by a change in 13C chemical shifts for C1 and Cx [39–41] Compared with acid-hydrolyzed CPS (B–A–B), we observed large changes in the 13C chemical shifts for C1 of A and C3 of B (A–B) on substitution with C and D, but not for C1 of B or C4 of A (B–A) Modelling of the B–A–B unit with residue D on both B residues was performed to ascertain whether the presence of the MeOPN group by itself caused significant changes about the glycosidic bonds in the B–A–B unit As can be observed by comparing (u w) maps in Fig 6A–D, no significant changes were observed Then, by modelling the DB–A–BD unit with residue C at C3 of 3982 3.45 Fig Determination of absolute configuration for sorbose in the HS:19 CPS with pyranose oxidase (A) 1H-NMR spectrum of D-sorbose (B) H-NMR spectrum of the same D-sorbose sample incubated with pyranose oxidase (C) 1H-NMR spectrum of L-sorbose (D) H-NMR spectrum of L-sorbose incubated with pyranose oxidase (E) 1H-NMR spectrum of CPS that was purified from the HS:19 serostrain of C jejuni and treated with mild acid to liberate the sorbose monosaccharide (F) 1H-NMR spectrum of the same CPS sample incubated with pyranose oxidase residue A (C-3 A) or at C2 of residue A (C-2A), it was observed that a major change in the distribution of (u w) occurred for the B–A linkage for C-3A (Fig 6F) and for the A–B linkage for C-2A (Fig 6G) Hence, the NMR and molecular modelling results indicate the location of the a-l-Sor branch to be at C2 of GlcA A model of a minimum energy conformer for the B–A–B unit representing the [-4)-b-d-GlcA6NGro-(1– 3)-b-d-GlcNAc-(1-]n repeating unit with MeOPN groups positioned at C4 of two GlcNAc residues and an a-l-sorbofuranose branch at C2 of GlcA is shown in Fig As can be observed, the sorbose branch at C2 of GlcA influences the conformation about the GlcA-(1–3)-b-d-GlcNAc glycosidic linkage because of steric hindrance between sorbose and the N-acetyl group of the GlcNAc residue Based on the positions of the sorbose branch and MeOPN group that extend away from the repeating unit, it is expected that they are prominent structural features on the surface of HS:19 cells Discussion The HS:19 serostrain of C jejuni is an important Penner type, as it is commonly associated with gastrointestinal infections and has been linked to the onset of ´ Guillain–Barre syndrome in the United States and Japan [11,15,16,42] A recent study [25] that examined a partially purified CPS sample for this serostrain FEBS Journal 273 (2006) 3975–3989 ª 2006 The Authors Journal compilation ª 2006 FEBS D J McNally et al Campylobacter jejuni HS: 19 CPS A Fig Molecular modelling of glycosidic torsional angles u and w for the GlcA6NGro(1–3)-GlcNAc (A–B) and GlcNAc-(1–4)GlcA6NGro (B–A) glycosidic linkages For each linkage map (A–B or B–A), the unit modelled is indicated above For (c–h), residue D was modelled at C4 of residue B Glycosidic torsional angles are defined as u ¼ O5–C1–O1–Cx and w ¼ C1–O1–Cx– Cx)1, with x being the aglyconic linkage site Annotations for residues are the same as Fig C E G B D F H observed labile unidentified CPS structures that were not reported in previous studies [5,6,8,12] In this study, we have characterized the complete CPS structure for the HS:19 serostrain and have shown that these labile groups are an a-l-sorbofuranose branch attached at C2 of b-d-GlcA and a MeOPN modification located at C4 of b-d-GlcNAc There are very few reports for sorbose in the literature and it is an unusual sugar to find in a bacterial polysaccharide Although this sugar is found in the plant kingdom [30], this is the first report of sorbose in a bacterial glycan Multiple 13C-1H correlations observed for the C2 position of the b-d-GlcA residue indicated that the sorbose branch is nonstoichiometric and is therefore a source of structural heterogeneity Fig Molecular model for the repeating unit of the CPS of the HS:19 serostrain of C jejuni The CPS consists of a [-4)-b-D-GlcA6NGro-(1–3)-b-D-GlcNAc-(1-]n repeating unit with an a-L-sorbofuranose branch located at C2 of GlcA and a MeOPN group at C4 of GlcNAc OH groups have been removed to simplify the appearance of the model for the CPS The complete CPS structure for HS:19 resembles that produced by the HS:1 serostrain, which was shown to have two nonstoichiometric fructose branches [23] Although the biological role of these modifications is not clear, these similarities suggest that decoration of CPS with nonstoichiometric keto sugar branches is a common structural feature in C jejuni Genetic determinants for the biosynthesis of these keto sugars were not readily detected within the CPS biosynthetic locus of either strain [25] This discrepancy points to the novelty of these modifications and the lack of other bacterial homologues in the current databases Alternatively, the biosynthesis genes may reside elsewhere in the genome Studies that have examined CPS biosynthesis in Escherichia coli K4, which has chondroitin-type [-4)-b-d-GlcA-(1–3)-b-dGalNAc-(1-]n CPS with a fructose branch at C3 of GlcA, showed that the Fru residue is added after CPS chain elongation is complete [43,44] Because the Fru and Sor branches in HS:1 and HS:19 are nonstoichiometric, addition of these keto sugars to CPS might be achieved using the mechanism described for E coli K4 Our results indicate that the sorbose branch is a prominent feature on the cell surface and that sorbose induces a conformational change in the structure of the CPS One might speculate that CPS, with and without sorbose, presents different epitopes, which has implications for how the HS:19 serostrain is perceived by its hosts and also in the success of CPSbased vaccine development The MeOPN modification is found on the CPSs of several strains of C jejuni, including 11168, HS:1, 81-176, HS:36 [18,23,24] and now HS:19, suggesting that the MeOPN is a common feature in this bacterium On the basis of the location of the MeOPN on both pyranose and furanose sugars, it was speculated FEBS Journal 273 (2006) 3975–3989 ª 2006 The Authors Journal compilation ª 2006 FEBS 3983 Campylobacter jejuni HS: 19 CPS D J McNally et al that transfer of the MeOPN to CPS sugars requires more than one gene product [24] cj1422c, a putative glycosyl transferase in 11168, was singled out as one of the genes [25], and cj1421c was speculated to be the other Our results support cj1421c as a possible MeOPN transferase, as the CPS biosynthetic locus in HS:19 contains only the cj1421 homologue HS19.07 [25] Multiple 13C-1H correlations observed for C4 of GlcNAc indicated that the MeOPN group is nonstoichiometric and is therefore an additional source of structural variation Moreover, our MS results showed that the MeOPN group in HS:19 is variably methylated Because fragment ions corresponding to nonmethylated MeOPN were not observed for the CPS of 11168 or HS:1 [23,24], those observed for HS:19 were concluded to be real and not an artefact of the MS analyses These observations suggest that the MeOPN is synthesized in a nonmethylated form and is methylated at a later point in time Despite having a small CPS locus with only 13 genes [25], the HS:19 serostrain produces a structurally heterogeneous CPS through the addition of nonstoichiometric elements such as a sorbose branch and MeOPN modification These findings support the incorporation of these modifications as a mechanism to produce a structurally complex surface glycan from a limited number of genes As hyaluronic acid is a common constituent of connective tissue and biological fluids in mammals, it is believed that several pathogenic bacteria produce a hyaluronic acid-type CPS to evade the host immune response Among these bacteria, Streptococcus pyogenes (group A streptococcus) has been studied the most [45] By examining CPS-deficient mutants, several studies have shown that the hyaluronic acid CPS produced by S pyogenes is an important virulence factor and a prerequisite for infection For instance, the CPS was shown to offer protection from phagocytes and epithelial cells [46–50], induce apoptosis in host cells [51], increase invasiveness and adherence [52], be important in mucoidy and biofilm formation [48,53], and was found to be responsible for subcutaneous spread of the bacterium [54] The CPS in S pyogenes was also shown to act as a universal adhesin by binding to CD44, a hyaluronic acid-binding glycoprotein found on pharyngeal and epidermal keratinocytes in humans [47] On the basis of these virulence roles that have been demonstrated for the hyaluronic acid-type CPS produced by S pyogenes, the importance of the structurally similar CPS produced by the HS:19 serostrain of C jejuni merits investigation The HS:19 serostrain of C jejuni is known to produce oligosaccharide structures in the outer core of its 3984 lipo-oligosaccharide that are identical with peripheral nerve gangliosides As a result, patients with gastroenteritis resulting from infection with C jejuni occasionally develop antiganglioside antibodies This molecular mimicry was proposed to result in antibody-mediated nerve damage to Schwann cells expressing these gangliosides thereby explaining paralysis in patients with ´ Guillain–Barre syndrome [11,42] Interestingly, there are also an increasing number of population studies that indicate a link between C jejuni infections and acute reactive arthritis [55–66] In the light of the prevalence of the HS:19 serostrain and the similarities shared by its CPS structure and the connective tissues of mammals, a link between HS:19 infections and reactive arthritis cannot be ruled out Conclusions The discovery of labile groups such as the nonstoichiometric a-l-sorbose branch and a variably methylated MeOPN modification in the HS:19 serostrain reinforces the importance of using mild analytical methods for examining the CPSs produced by C jejuni We believe that the analytical methods described here will be useful for solving the structures of complex CPSs in other bacteria that contain labile components Although CPS is recognized as a major virulence factor in C jejuni, the precise mechanisms explaining how CPS conveys virulence and the importance of the extensive phase-variable modifications decorating the CPSs are not known Because production of a hyaluronic acid-type CPS is a prerequisite for infection in other pathogens, most notably group A streptococcus, the role of the CPS in the infection process of HS:19 merits investigation Based on the wealth of knowledge that is available for the genetics, regulation and biosynthesis of hyaluronic acid-type CPSs in other organisms and the findings of the current study, the HS:19 serostrain now presents an interesting model to explore CPS as a virulence factor in C jejuni Experimental procedures Solvents and reagents Unless otherwise stated, solvents and reagents were purchased from Sigma Biochemicals and Reagents (Oakville, ON, Canada) Media and growth conditions The HS:19 strain (ATCC 43446, designation MK104 [14]) of C jejuni was routinely maintained on Mueller Hinton FEBS Journal 273 (2006) 3975–3989 ª 2006 The Authors Journal compilation ª 2006 FEBS D J McNally et al (MH) agar (Difco, Kansas City, MO, USA) plates under microaerophilic conditions (10% CO2, 5% O2, 85% N2) at 37 °C For large-scale extraction of CPS, L C jejuni HS:19 was grown in brain heart infusion (BHI) broth (Difco) under microaerophilic conditions at 37 °C for 24 h with agitation at 100 r.p.m Bacterial cells were then harvested by centrifugation (9000 g, 20 min) and placed in 70% ethanol Cells were removed from the ethanol solution by centrifugation (9000 g, 20 min), and the bacterial pellet was refrigerated until extraction Isolation of CPS Campylobacter jejuni HS: 19 CPS HS:19 serostrain of C jejuni was suspended in 150 lL ammonium bicarbonate-buffered 99% D2O (50 mm NH4HCO3, pD 8.2; Cambridge Isotopes Laboratories Inc, Andover, MA, USA) and placed in a mm NMR tube (Wilmad, Buena, NJ, USA) NMR experiments were performed at 600 MHz (1H) using a Varian mm, Z-gradient, triple-resonance, cryogenically cooled probe, and at 500 MHz (1H) with a Varian Inova spectrometer equipped with a Varian mm, Z-gradient, triple-resonance (1H, 13C, 31 P) probe (Varian, Palo Alto, CA, USA) 1D 31P spectra were acquired using a Varian Mercury 200 MHz (1H) spectrometer and a Nalorac mm four nuclei probe Standard homonuclear and heteronuclear correlated 1D and 2D pulse sequences from Varian were used for general assignments, and selective 1D TOCSY and NOESY experiments with a Z-filter were used for complete residue assignment and characterization of individual spin systems [67,68] NMR experiments were typically performed at 25 °C, with suppression of the HOD resonance at 4.78 p.p.m The methyl resonance of acetone was used as an internal reference (dH 2.225 p.p.m and dC 31.07 p.p.m.), and 31P NMR spectra were referenced to an external 85% phosphoric acid standard (dP p.p.m.) A gentle enzymatic method was used to isolate CPS from bacterial cells in order to preserve labile groups [23] Briefly, cells harvested from L BHI broth were suspended in NaCl ⁄ Pi buffer (pH 7.4) Lysozyme was then added to a final concentration of mgỈmL)1 before the addition of mutanolysin to a final concentration of 67 mL)1 The bacterial cell suspension was then incubated for 24 h at 37 °C with agitation at 100 r.p.m The mixture was then emulsiflexed twice (144 789.75 kPa; 21 000 p.s.i.) to lyse cells, and DNAse I and RNAse (130 lgỈmL)1 DNAse I and RNAse) was added before being incubated for h at 37 °C with agitation at 100 r.p.m Pronase E (type XIV from Streptomyces griseus, EC 3.4.24.31, 5.2 mg)1) and protease (type XXIV from Bacillus licheniformis, EC 3.4.21.62, 8.8 mg)1) were both added to a final concentration of 200 lgỈmL)1 followed by incubation at 37 °C overnight with agitation at 100 r.p.m The crude CPS extract was then dialyzed against running water for 72 h (molecular mass cut-off 12 kDa), ultracentrifuged for h (140 000 g, 15 °C), and the supernatant was lyophilized Crude CPS was resuspended in water and purified using a SephadexÒ superfine G-50 column equipped with a Waters differential refractometer (model R403; Waters, Mississauga, ON, Canada) Fractions containing CPS were combined and lyophilized Semi-purified CPS was then resuspended in water and purified using a Gilson liquid chromatograph (model 306 and 302 pumps, 811 dynamic mixer, 802B manometric module; Gilson, Middleton, WI, USA) with a Gilson UV detector (220 nm; model UV ⁄ Vis-151 detector; Gilson) equipped with a tandem QHP HiTrapÔ ion-exchange column (Amersham Biosciences, Piscataway, NJ, USA) Fractions containing CPS were combined and lyophilized Purified bacterial CPS was then desalted using a SephadexÒ superfine G-15 column, lyophilized, and stored at )20 °C until further analysis CPS isolated from the HS:19 serostrain of C jejuni was mass-analyzed using in-source collision-induced dissociation CE-ESI ⁄ MS [27] with a Crystal model 310 capillary electrophoresis instrument (ATI Unicam, Boston, MA, USA) coupled to an API 3000 mass spectrometer (Applied Biosystems ⁄ Sciex, Concord, ON, Canada) via a microIon spray interface A sheath solution (propan-2-ol ⁄ methanol, : 1, v ⁄ v) was delivered at a flow rate of lLỈmin)1 Separations were achieved on  90 cm of bare fused-silica capillary (190 lm outside diameter · 50 lm internal diameter; Polymicro Technologies, Phoenix, AZ, USA) using 15 mm ammonium acetate ⁄ ammonium hydroxide in deionized water (pH 9.0) containing 5% methanol as separation buffer A voltage of 20 kV was typically applied during CE separation, and +5 kV was used as electrospray voltage Mass spectra were acquired with dwell times of 3.0 ms per step of 0.1 m ⁄ z unit in full-mass scan mode 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 All samples were analyzed in positive ion mode using an orifice voltage of +200 V HR-MAS NMR and high-resolution NMR spectroscopy Determination of absolute configuration for CPS sugars HR-MAS NMR analysis of intact bacterial cells was performed as described by McNally et al [23] For high-resolution NMR spectroscopy, mg CPS isolated from the The absolute configuration (d or l) of GlcA and GlcNAc CPS sugars isolated from the HS:19 serostrain of C jejuni was assigned by characterization of their R-butyl glyco- MS analysis of purified CPS FEBS Journal 273 (2006) 3975–3989 ª 2006 The Authors Journal compilation ª 2006 FEBS 3985 Campylobacter jejuni HS: 19 CPS D J McNally et al sides using GC as described by McNally et al [23] The absolute configuration of sorbofuranose was assigned enzymatically using pyranose oxidase (EC 1.1.3.10; oxygen 2-oxidoreductase) from Coriolus sp Although l-sorbose has been reported to be a preferred substrate of this enzyme [32–34], its specificity for the l-isoform was not known To investigate the specificity of this enzyme, mg l-sorbose or d-sorbose was incubated with mg pyranose oxidase (27 mL)1) overnight at 37 °C in 200 lL D2O phosphate buffer (100 mm KH2PO4, 100 mm NaCl, pD 5.6), and the resulting oxidation products were analyzed with NMR To determine the absolute configuration of sorbose within the HS:19 CPS, mg pure CPS was first hydrolyzed overnight by mild acid treatment (addition of dilute HCl, pH 3.0) to liberate sorbose monosaccharide The hydrolyzed CPS sample was then neutralized, lyophilized, and resuspended in 200 lL D2O phosphate buffer (100 mm KH2PO4, 100 mm NaCl, pD 5.6) The hydrolyzed CPS sample containing the sorbose monosaccharide was then incubated overnight at 37 °C with mg pyranose oxidase (27 mL)1), and the reaction products were analyzed by NMR Molecular modelling and molecular dynamics simulations Molecular dynamics simulations were performed for one repeat unit of the CPS [b-d-GlcNAc-(1–4)-b-d-GlcA6NGro-(1–3)-b-d-GlcNAc] without and with the MeOPN at C4 of GlcNAc, and without a-l-sorbose and with a-l-sorbose at C2 or C3 of GlcA Molecular structures were constructed using the Biopolymer Module running on the Insight II environment (Accelrys Inc., San Diego, CA, USA) Glycosidic torsional angles, defined as u ¼ O5–C1– O1–Cx and w ¼ C1–O1–Cx–Cx)1, with x being the aglyconic linkage site, were chosen from potential energy maps generated for CPS constituent disaccharides All molecular structures were subjected to 1000-step energy minimization using a conjugate gradient method Potential energy calculations, energy minimizations and dynamics simulations were performed using the Discover-3 program (Accelrys Inc.) Atomic potentials and charges were assigned using an Amber force field supported by Discover-3 A group˚ based nonbond method with a cut-off distance of 9.5 A and a distance-dependent dielectric value of were used for all calculations Energy-minimized structures were then subjected to a 500-ps dynamics simulation in vacuum with the first 100 ps being discarded A Verlet algorithm with a 1-fs time step was used for simulations, with trajectory frames being saved every 0.25 ps During simulations, the initial chair conformations for six-membered sugar rings were conserved using a periodic cosine function with a force constant of 100 kcalỈmol)1Ỉrad)2 to restrain three of the possible six ring torsions The conformations of sixmember rings, such as the pyran rings of sugars, are well 3986 studied from a conformational perspective [69] If torsion angles are used to describe ring puckering and if ring closure and redundancy conditions are taken into consideration, then it is possible to define 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labile groups are an a- l-sorbofuranose branch attached at C2 of b-d-GlcA and a MeOPN modification located at C4 of b-d-GlcNAc There are very... the disappearance of signals associated with the sorbose monosaccharide and the production of the same oxidation product as observed for the l -sorbose standard (Figs 5E,F) On the basis of these... a- L-sorbofuranose branch located at C2 of GlcA and an MeOPN group at C4 of GlcNAc Structural heterogeneity is due to the nonstoichiometric sorbofuranose branch and the variably methylated nonstoichiometric