Báo cáo khoa học: The HS:19 serostrain of Campylobacter jejuni has a hyaluronic acid-type capsular polysaccharide with a nonstoichiometric sorbose branch and O-methyl phosphoramidate group

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 3 April 2006, revised 2 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 gen- tle 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 phosphor- amidate group, CH3OP(O)(NH2)(OR) (MeOPN), was found at C4 of Glc- NAc. Structural heterogeneity of the CPS was due to nonstoichiometric glycosylation with sorbose at C2 of GlcA and the nonstoichiometric, vari- ably 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 sero- strain. 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 lim- ited 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.

Although of relatively rare occurrence, this syndrome is the most common cause of acute neuromuscular paralysis since the eradication of polio. It is character- ized by weakness in the limbs and respiratory muscles, with paralysis generally occurring 1–3 weeks after infection [12,13]. Penner’s passive haemagglutination

Campylobacter jejuni is one of the leading causes of human gastroenteritis and surpasses Salmonella, Shig- ella 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´ [4–11].

syndrome

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).

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was shown to produce a structurally variable and therefore highly complex CPS despite having a small CPS biosynthetic locus containing 11 genes [23,25].

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].

latter

the

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 trans- fer, 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 corre- lated 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 sam- ple prepared from HS:19 cells, 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].

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 survi- val and persistence in the environment [19]. By mimick- ing host cell antigens and through structural variation, CPSs also convey evasion from host immune responses and are therefore considered important virulence fac- tors. 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, bio- chemical and microscopy studies have demonstrated strains of C. jejuni CPS production in different [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 sur- face hydrophilicity [17,22], CPS is thought to play a critical role in pathogenesis for C. jejuni.

studies

[5,6,12],

In previous

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 charac- terize 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 enzy- matic extraction method that preserves labile groups [23]. the classic hot water ⁄ phenol extraction method [26] was used, as this structure was originally thought to be a high-molecu- lar-mass lipopolysaccharide. High-resolution NMR at 600 MHz (1H) with an ultra-sensitive cryogenically cooled probe (cold probe), and capillary electro- phoresis electrospray ionization mass spectrometry (CE-ESI-MS) with in-source collision-induced dissoci- ation [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 struc- tural ﬁndings for this organism.

Results

The results generated by HR-MAS NMR and high- resolution 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 in agreement with unit

(Fig. 1). This ﬁnding is

The precise mechanisms by which CPS conveys viru- lence 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, exclu- ding 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 amino- glycerol groups on CPS sugars [18,23,24]. The most unusual of these modiﬁcations is the O-methyl phos- phoramidate 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

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A - -GlcA NGro D

located at C2 of b-d-GlcA and a variably methylated nonstoichiometric MeOPN group at C4 of b-d-Glc- NAc.

D MeO NP

8 HOH2C

CH2OH

CH3

HR-MAS NMR spectroscopy of whole cells

B - -GlcNAc D

NH2

6 CH2OH

6 HOH2C

CH3

1 CH2OH

3 H

single-quantum coherence

C f - -Sor L

Fig. 1. 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 non- stoichiometric sorbofuranose branch and the variably methylated nonstoichiometric MeOPN group. Residue A represents b-D-glucu- ronic acid-6-N-glycerol, B is 2-acetamido-2-deoxy-b-D-glucose, C is a-L-sorbofuranose, and D is MeOPN.

[5,6,12]. However,

previous studies that examined CPS in the HS:19 sero- strain of C. jejuni 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, how- ever; 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 agree- ment with 3JH,P couplings determined for the MeOPN in the 11168 and HS:1 serostrains [18,23,24]. A 1D 31P (HSQC) heteronuclear experiment that speciﬁcally selects for the MeOPN group (31P decoupled, MeOPN-ﬁltered 31P HSQC) con- ﬁrmed 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 31P- HSQC 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 phos- phorus 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. 2. 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 31P-HSQC ‘MeOPN-ﬁltered’ HR-MAS NMR spectrum (31P-decoupled, 256 transients, 1JH,P ¼ 10 Hz) showing a broad signal ori- ginating from a MeOPN group. (C) 2D 31P-HSQC HR-MAS NMR spectrum showing two MeOPNs (256 transients, 128 incre- ments, 1JH,P ¼ 10 Hz).

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Table 1. NMR proton and carbon chemical shifts d (p.p.m.) for CPS puriﬁed from HS:19 serostrain of C. jejuni.

Residue

Position

b-D-GlcA6NGro (A)

4.65 3.71 3.71 3.90 3.94

101.0 73.8 73.8 79.0 75.1

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. indi- cated structural heterogeneity for CPS on the cell sur- face. Spectral lines originating from CPS on the surface of HS:19 cells were too broad to draw additional con- clusions regarding its structure, necessitating its isola- tion for further study.

170.7

Isolation of CPS

b-D-GlcNAc (B)

53.9 61.5 100.7 56.1 75.8 74.2 75.5 61.3

4.07 3.65 ⁄ 3.75 4.62 3.97 4.24 4.26 3.61 3.74 ⁄ 3.93

175.0

23.5 61.5

a-L-Sorf (C)

2.11 3.64 ⁄ 3.73

104.3

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

4.17 4.41 4.39 3.69 ⁄ 3.79 3.77

79.2 76.1 79.2 62.9 54.8

MeOPN (D)

TOCSY of the sorbose H3 resonance revealed overlap- ping 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).

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 remov- ing 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, (cid:2) 5 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

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 Glc- NAc (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 (cid:2)C) and at 600 MHz (1H) with a cryogenically cooled probe. Proton and carbon resonances determined from 13C-HMQC and heteronuclear multiple-bond correla- tion (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

Examination of puriﬁed CPS with NMR at 600 MHz (1H) with a cold probe revealed a [-4)-b-d-GlcA6N- Gro-(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., 3JH,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 pro- ton resonances for GlcA, GlcNAc and sorbose. 1D

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Fig. 3. High-resolution NMR spectroscopy of CPS puriﬁed from the HS:19 serostrain of C. jejuni. (A) 1H NMR spectrum (1024 tran- sients) 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 7 Hz). (F) 13C-HMQC spectrum (128 transients, 128 increments, 1JC,H ¼ 150 Hz). For selective 1D experi- ments, excited resonances are underlined. Residues with asterisks correspond to struc- tural heterogeneity generated by the non- stoichiometric MeOPN group and a-L-sorbofuranose branch. Annotations for residues are the same as Fig. 1 and sm represents sorbose monosaccharide.

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-phosphor- amidate group OP(O)(NH2)(OR) without O-methyla- tion, suggesting that the nonstoichiometric MeOPN group is also variably methylated for the HS:19 sero- strain. CE-ESI-MS ⁄ MS analysis of m ⁄ z 708.5, corres- ponding 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).

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 phos- the 11168 and HS:1 phoramidation reported for the MeOPN group strains, where the presence of caused the signals for neighbouring protons to shift by 0.6–0.8 p.p.m. [18,23,24].

MS analysis of puriﬁed CPS

Determination of absolute conﬁguration for CPS sugars

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 sam- ple, b-GlcA and b-GlcNAc were shown to have the d conﬁguration (not shown). As the chiral alcohol

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

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Fig. 4. 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-ESI- MS ⁄ MS analysis of m ⁄ z 708.5, which cor- responds to one complete repeat of the CPS.

concluded to have

the l absolute

sorbose was conﬁguration.

Branching pattern for sorbose

it was not known if

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 over- lap for the resonances originating from positions 2 and 3 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-

method cannot be used to determine the absolute con- ﬁguration of keto sugars [31], we report for the ﬁrst time the use of pyranose oxidase for this purpose. Pyr- anose 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 per- oxide [32–34]. However, 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,

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Table 2. 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

Nevertheless,

two lines of evidence point

signal

to the location of the Sor branch at C2 of GlcA. Compar- ison of 13C chemical shifts for intact and acid-hydro- lyzed CPS revealed that substitution with Sor causes an upﬁeld shift for GlcA in the C3 (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

ity of the CPS in organic solvent, the labile Sor glycos- idic 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].

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A

B

C

D

E

F

3.95

3.85

3.75

3.65

3.55

3.45

1H (p.p.m.)

Fig. 5. Determination of absolute conﬁgur- ation for sorbose in the HS:19 CPS with pyr- anose oxidase. (A) 1H-NMR spectrum of D-sorbose. (B) 1H-NMR spectrum of the same D-sorbose sample incubated with pyranose oxidase. (C) 1H-NMR spectrum of L-sorbose. (D) 1H-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 (u w) occurred for the B–A linkage for C-3A of (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.

of pyranose rings [36–38]. Furthermore, compared with acid-hydrolyzed CPS, we observed substantial up- ﬁeld 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 (< 1 p.p.m.) [3,18,23], the magnitude of these differences for the C1 signal of GlcA (A) and C3 signal of GlcNAc (B) indi- cated a change about the GlcA-(1–3)-GlcNAc (A–B) glycosidic bond.

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. 7. 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 Pen- ner type, as it is commonly associated with gastrointes- infections and has been linked to the onset of tinal 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

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 link- age site, to be accompanied by a change in 13C chem- ical 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 model- ling the DB–A–BD unit with residue C at C3 of

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A

C

E

G

B

D

F

H

Fig. 6. 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. 1.

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 struc- ture 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 modiﬁca- tion located at C4 of b-d-GlcNAc.

There are very few reports for sorbose in the litera- ture 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

the mechanism described indicate that results

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 these similarities suggest modiﬁcations is not clear, 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 dis- crepancy 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-d- GalNAc-(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 nonstoichio- metric, addition of these keto sugars to CPS might be achieved for using E. coli K4. Our 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 CPS- based 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 bacter- ium. On the basis of the location of the MeOPN on both pyranose and furanose sugars, it was speculated

Fig. 7. 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-GlcA6N- Gro-(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.

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results

lipo-oligosaccharide that are identical with peripheral nerve gangliosides. As a result, patients with gastroen- teritis resulting from infection with C. jejuni occasion- ally develop antiganglioside antibodies. This molecular mimicry was proposed to result in antibody-mediated nerve damage to Schwann cells expressing these gan- gliosides 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 pre- valence 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

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 support cj1421c as a possible other. Our 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 nonstoi- chiometric and is therefore an additional source of structural variation. Moreover, our MS results showed that the MeOPN group in HS:19 is variably methyla- ted. Because fragment ions corresponding to nonmeth- ylated 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 methyla- ted at a later point in time. Despite having a small CPS locus with only 13 genes [25], the HS:19 sero- strain 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 mod- iﬁcations as a mechanism to produce a structurally complex surface glycan from a limited number of genes.

these

bacteria,

The discovery of labile groups such as the nonstoichio- metric a-l-sorbose branch and a variably methylated MeOPN modiﬁcation in the HS:19 serostrain reinfor- ces 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 fac- tor 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 hyalu- ronic 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 bio- synthesis of hyaluronic acid-type CPSs in other organ- isms 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 pur- chased from Sigma Biochemicals and Reagents (Oakville, ON, Canada).

Media and growth conditions

As hyaluronic acid is a common constituent of con- nective tissue and biological ﬂuids in mammals, it is believed that several pathogenic bacteria produce a hyaluronic acid-type CPS to evade the host immune Streptococcus response. Among pyogenes (group A streptococcus) has been studied the most [45]. By examining CPS-deﬁcient mutants, several studies have shown that the hyaluronic acid CPS pro- duced 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 bind- ing 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 sero- strain of C. jejuni merits investigation.

The HS:19 strain (ATCC 43446, designation MK104 [14]) of C. jejuni was routinely maintained on Mueller Hinton

The HS:19 serostrain of C. jejuni is known to pro- duce oligosaccharide structures in the outer core of its

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cryogenically cooled probe,

(MH) agar (Difco, Kansas City, MO, USA) plates under microaerophilic conditions (10% CO2, 5% O2, 85% N2) at 37 (cid:2)C. For large-scale extraction of CPS, 6 L C. jejuni HS:19 was grown in brain heart infusion (BHI) broth (Difco) under microaerophilic conditions at 37 (cid:2)C for 24 h with agitation at 100 r.p.m. Bacterial cells were then har- vested 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

HS:19 serostrain of C. jejuni was suspended in 150 lL 99% D2O (50 mm ammonium bicarbonate-buffered NH4HCO3, pD 8.2; Cambridge Isotopes Laboratories Inc, Andover, MA, USA) and placed in a 3 mm NMR tube (Wilmad, Buena, NJ, USA). NMR experiments were per- formed at 600 MHz (1H) using a Varian 5 mm, Z-gradient, triple-resonance, and at 500 MHz (1H) with a Varian Inova spectrometer equipped with a Varian 3 mm, Z-gradient, triple-resonance (1H, 13C, 31P) probe (Varian, Palo Alto, CA, USA). 1D 31P spectra were acquired using a Varian Mercury 200 MHz (1H) spec- trometer and a Nalorac 5 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 (cid:2)C, with sup- pression 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 stand- ard (dP 0 p.p.m.).

MS analysis of puriﬁed CPS

(type XXIV from Bacillus

instrument

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 (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, 2 : 1, v ⁄ v) was delivered at a ﬂow rate of 1 lLÆmin)1. Sepa- rations were achieved on (cid:2) 90 cm of bare fused-silica capil- lary (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 buf- fer. 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 sam- ples were analyzed in positive ion mode using an oriﬁce voltage of +200 V. A gentle enzymatic method was used to isolate CPS from bacterial cells in order to preserve labile groups [23]. Brieﬂy, cells harvested from 6 L BHI broth were suspended in NaCl ⁄ Pi buffer (pH 7.4). Lysozyme was then added to a ﬁnal concentration of 1 mgÆmL)1 before the addition of mutanolysin to a ﬁnal concentration of 67 UÆmL)1. The bacterial cell suspension was then incubated for 24 h at 37 (cid:2)C with agitation at 100 r.p.m. The mixture was then to lyse emulsiﬂexed twice (144 789.75 kPa; 21 000 p.s.i.) cells, and DNAse I and RNAse (130 lgÆmL)1 DNAse I and RNAse) was added before being incubated for 4 h at 37 (cid:2)C with agitation at 100 r.p.m. Pronase E (type XIV from Streptomyces griseus, EC 3.4.24.31, 5.2 UÆmg)1) and licheniformis, EC protease 3.4.21.62, 8.8 UÆmg)1) were both added to a ﬁnal concen- tration of 200 lgÆmL)1 followed by incubation at 37 (cid:2)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 2 h (140 000 g, 15 (cid:2)C), and the supernatant was lyophilized. Crude CPS was resuspended in water and puriﬁed using a Sephadex(cid:3) superﬁne G-50 column equipped with a Waters differential refractometer (model R403; Waters, Mississ- auga, ON, Canada). Fractions containing CPS were com- bined 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 HiTrap(cid:4) ion-exchange column (Amersham Biosciences, Piscataway, NJ, USA). Fractions containing CPS were combined and lyophilized. Puriﬁed bacterial CPS was then desalted using a Sephadex(cid:3) superﬁne G-15 column, lyophi- lized, and stored at )20 (cid:2)C until further analysis.

HR-MAS NMR and high-resolution NMR spectroscopy

Determination of absolute conﬁguration for CPS sugars

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HR-MAS NMR analysis of intact bacterial cells was per- formed as described by McNally et al. [23]. For high-reso- lution NMR spectroscopy, 3 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-

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Campylobacter jejuni HS: 19 CPS

studied from a conformational perspective [69]. If torsion angles are used to describe ring puckering and if ring clo- sure and redundancy conditions are taken into considera- tion, then it is possible to deﬁne the conformation of any three torsion six-membered ring using a minimum of angles [70]. For this reason, it was possible to conserve the initial chair conformations of the six-membered sugar rings during molecular dynamics simulations by restraining three torsion angles. Scatter plots for the glycosidic torsion angles u and w shown in Fig. 6 were generated using the Analysis Module running on Insight II. The molecular model for the CPS presented in Fig. 7 was drawn using Visual Molecular Dynamics [71].

Acknowledgements

We are grateful to Dr Michel Gilbert for the HS:19 serostrain, Dr Malcolm Perry for insightful discus- sions, Dr Dietmar Haltrich for helpful discussions per- taining to pyranose oxidase and to Marc Lamoureux for assistance with bacterial growths.

sides using GC as described by McNally et al. [23]. The absolute conﬁguration of sorbofuranose was assigned en- zymatically using pyranose oxidase (EC 1.1.3.10; oxygen from Coriolus sp. Although l-sorbose 2-oxidoreductase) this has been reported to be a preferred substrate of its speciﬁcity for the l-isoform was not enzyme [32–34], known. To investigate the speciﬁcity of this enzyme, 1 mg l-sorbose or d-sorbose was incubated with 2 mg pyranose oxidase (27 UÆmL)1) overnight at 37 (cid:2)C in 200 lL D2O 100 mm NaCl, (100 mm KH2PO4, phosphate buffer pD 5.6), and the resulting oxidation products were ana- lyzed with NMR. To determine the absolute conﬁguration of sorbose within the HS:19 CPS, 1 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, lyophi- lized, and resuspended in 200 lL D2O phosphate buffer (100 mm KH2PO4, 100 mm NaCl, pD 5.6). The hydro- lyzed CPS sample containing the sorbose monosaccharide was then incubated overnight at 37 (cid:2)C with 2 mg pyranose oxidase (27 UÆmL)1), and the reaction products were ana- lyzed by NMR.

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