Báo cáo khoa học: The HS:1 serostrain of Campylobacter jejuni has a complex teichoic acid-like capsular polysaccharide with nonstoichiometric fructofuranose branches and O-methyl phosphoramidate groups pot

The HS:1 serostrain of Campylobacter jejuni has a complex teichoic acid-like capsular polysaccharide with nonstoichiometric fructofuranose branches and O-methyl phosphoramidate groups David J McNally, Harold C Jarrell, Jianjun Li, Nam H Khieu, Evgeny Vinogradov, Christine M Szymanski and Jean-Robert Brisson Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario, Canada Keywords Campylobacter jejuni; capsular polysaccharide; CE-ESI-MS; HR-MAS NMR; phosphoramidate Correspondence J.-R Brisson, Institute for Biological Sciences, National Research Council of Canada, Ottawa, Canada, K1A 0R6 Fax: +1 613 9529092 Tel: +1 613 9903244 E-mail: jean-robert.brisson@nrc-cnrc.gc.ca (Received 11 May 2005, revised July 2005, accepted 11 July 2005) doi:10.1111/j.1742-4658.2005.04856.x Recently, the CPS biosynthetic loci for several strains of Campylobacter jejuni were sequenced and revealed evidence for multiple mechanisms of structural variation In this study, the CPS structure for the HS:1 serostrain of C jejuni was determined using mass spectrometry and NMR at 600 MHz equipped with an ultra-sensitive cryogenically cooled probe Analysis of CPS purified using a mild enzymatic method revealed a teichoic acid-like [-4)-a-d-Galp-(1–2)-(R)-Gro-(1-P]n, repeating unit, where Gro is glycerol Two branches at C-2 and C-3 of galactose were identified as b-d-fructofuranoses substituted at C-3 with CH3OP(O)(NH2)(OR) groups Structural heterogeneity was due to nonstoichiometric glycosylation at C-3 of galactose and variable phosphoramidate groups Identical structural features were found for cell-bound CPS on intact cells using proton homonuclear and 31P heteronuclear two-dimensional HR-MAS NMR at 500 MHz In contrast, spectroscopic data acquired for hot water ⁄ phenol purified CPS was complicated by the hydrolysis and subsequent loss of labile groups during extraction Collectively, the results of this study established the importance of using sensitive isolation techniques and HR-MAS NMR to examine CPS structures in vivo when labile groups are present This study uncovered how incorporation of variable O-methyl phosphoramidate groups on nonstoichiometric fructose branches is used in C jejuni HS:1 as a strategy to produce a highly complex polysaccharide from its small CPS biosynthetic locus and a limited number of sugars The Gram-negative, spiral-shaped bacterium Campylobacter jejuni is one of the leading causative agents of human enteritis and surpasses Salmonella, Shigella and Escherichia in some regions as the primary cause of gastrointestinal disease [1,2] Furthermore, there is a growing body of evidence suggesting that infection with C jejuni is linked to the development of Guillain´ Barre and Miller-Fisher neuropathies [3–5] Although the bacteria now recognized as members of the genus Campylobacter were first described at the beginning of the 20th century, public awareness remains limited and much of the biology of Campylobacters and the mechanisms by which they cause disease are still relatively poorly understood [1] In recent years, the health and economic burden associated with Campylobacter infection has fueled interest for this genus and in 2000, the genome sequence for C jejuni NCTC 11168 (HS:2) was reported [6] Abbreviations CPS, capsular polysaccharide; CE-ESI-MS, capillary electrophoresis-electrospray ionization-mass spectrometry; HMBC, heteronuclear multiple bond coherence; HMW LPS, high-molecular-weight lipopolysaccharides; HR-MAS NMR, high resolution magic angle spinning nuclear magnetic resonance; HSQC, heteronuclear single quantum coherence; MeOPN, O-methyl phosphoramidate CH3OP(O)(NH2)(OR) FEBS Journal 272 (2005) 4407–4422 ª 2005 FEBS 4407 Campylobacter jejuni HS:1 serostrain CPS Analysis of the genome sequence revealed that this strain possesses four gene clusters necessary for carbohydrate biosynthesis [6] Among these was the first description of a type II ⁄ III capsule locus similar to that found in encapsulated organisms, such as those found in Escherichia coli K1 and Neisseria meningitidis group B [6,7] In particular, identification of kps genes, responsible for transferring the CPS repeat to the outer membrane, prompted a systematic genetic analysis of the corresponding locus and resulted in the identification of CPS in several strains of C jejuni [8] The CPS product of this locus was subsequently shown to be the major serodeterminant in the heat-stable typing scheme first described by Penner and Hennessy [9–11], and alcian blue staining led to the visualization of capsule in C jejuni by electron microscopy [12] Because of these pioneering studies, C jejuni is now widely accepted as a species that produces a CPS and what was previously reported in the literature as high-molecular-weight lipopolysaccharides (HMW LPSs) for C jejuni, are now considered to be CPSs [12–14] Because capsular polysaccharide is the outermost structure on the bacterial cell, it plays a key role in the interaction between the pathogen, host, and environment [15] Generally, CPS is thought to be important for bacterial survival and persistence in the environment and often contributes to pathogenesis [16] For C jejuni, CPS is considered to be an important virulence factor based on its involvement in epithelial cell invasion, diarrhoeal disease, serum resistance and maintenance of bacterial cell surface hydrophilicity [11] Furthermore, the CPSs produced by different strains of C jejuni are structurally complex and highly variable For instance, there are presently more than 60 serostrains described for C jejuni, not including nontypeable strains, each with a unique CPS structure [14] Moreover, for each serogroup it is possible to have phase-variable CPS modifications such as the addition of methyl, ethanolamine and aminoglycerol groups reported for C jejuni NCTC 11168 CPS [15] In a recent study, the CPS biosynthetic regions for selected strains of C jejuni were sequenced including: serostrain HS:41 (176.83), 81–176 (HS:23 ⁄ 26), serostrain HS:36 (ATCC 43456), serostrain HS:23 (CCUG 10954), serostrain HS:19 (NCTC 12517) and G1 (HS:1) [14] Comparison of the determined cps sequences of the HS:19, HS:41 and HS:1 strains with the genome sequenced NCTC 11168 (HS:2) strain provided 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 [14] The study also demonstrated for the first time that strains 4408 D J McNally et al belonging to the same serogroup (e.g 81–176 (HS:23 ⁄ 36), HS:23 and HS:36) contain capsule loci with the same gene complement In contrast to C jejuni NCTC 11168 and HS:19, the biosynthetic region of the HS:1 strain was the smallest and was shown to contain only 11 genes (Fig 1a) Of importance, the cps locus of the HS:1 strain was shown to contain a tagD homologue encoding a glycerol-3-phosphate cytidylyltransferase necessary for the biosynthesis of CDP-glycerol Moreover, it was shown that the (a) (b) Fig Predicted capsule gene schematic and determined structures for the defructosylated repeating unit (CPS-1) and complete CPS structure (CPS-2) of the C jejuni HS:1 serostrain (a) Carbohydrate biosynthetic genes located between the genes encoding the capsule transport system are shown from the sequenced locus of the HS:1 strain, G1 [14] The phase variable genes in G1 which could be involved in the structural heterogeneity described in this report are indicated by grey arrows (b) For CPS-2, the repeating unit is [-4)-a-D-Galp-(1–2)-(R)-Gro-(1-P-]n with MeOPN-3-b-D-fructofuranose branches at C-2 and C-3 of Gal Structural heterogeneity is due to variable phosphoramidate groups on nonstoichiometric fructose branches Residue A is a-D-Galp, a-D-galactopyranose, residue B is GroP, glycerol-phosphate, residue C is b-D-Fruf, b-D-fructofuranose; and MeOPN is O-methyl phosphoramidate, CH3OP(O)(NH2)(OR) FEBS Journal 272 (2005) 4407–4422 ª 2005 FEBS D J McNally et al HS:1 strain encodes a tagF homologue responsible for transferring glycerol-phosphate residues from CDPglycerol These genetic findings for the cps loci of this HS:1 strain corroborated the structures reported for the C jejuni HS:1 serostrain HMW LPS (CPS), where the repeat unit was [-4)-a-d-Gal-(1–2)-Gro-(3-P-]n [17,18] We did however, observe important discrepancies between these structures reported for HS:1 HMW LPS and preliminary NMR data obtained for the partially purified CPS of G1 (HS:1) and the HS:1 serostrain of C jejuni [14] For instance, we detected the presence of at least two acid-labile groups and provided evidence showing that one of these was likely an MeOPN CH3OP(O)(NH2)(OR) modification similar to the one identified on the CPS structure of the genomesequenced strain of C jejuni, NCTC 11168 [14,15] In the current study, we thoroughly investigated the chemical structure of CPS for the HS:1 serostrain of C jejuni Initially, CPS was isolated from bacterial cells using a traditional hot water ⁄ phenol method [19]; however, due to the extent of structural degradation observed for CPS purified using this method, a gentler procedure for isolating CPS was required to preserve the labile constituents of HS:1 CPS Accordingly, the methods of Darveau and Hancock [20], Huebner et al [21] and Hsieh et al [22] were combined and used to isolate CPS from this strain of C jejuni High resolution NMR at 600 MHz with an ultra-sensitive cryogenically cooled probe was then used to elucidate the structure of purified CPS, and HR-MAS NMR at 500 MHz was used to examine native CPS directly on the surface of whole bacterial cells Concurrently, CE-ESI-MS and in-source collision-induced dissociation [23] was used to analyze the structure of purified HS:1 CPS, corroborate NMR findings and characterize the extent of heterogeneity for HS:1 CPS In this study, we present the advantages and importance of using sensitive techniques for examining CPS in C jejuni, report the complete structure of C jejuni HS:1 CPS and discuss the biological significance of these new structural findings for this strain of the bacterium Results The results generated by HR-MAS and high resolution NMR, CE-ESI-MS and chemical ⁄ enzymatic analyses provided strong evidence showing that the backbone of C jejuni HS:1 CPS resembles teichoic acid and consists of a [-4)-a-d-Galp-(1–2)-(R)-Gro-(1-P-]n repeating unit (Fig 1b) The complete CPS structure for HS:1 is complex due to the presence of a nonstoichiometric fructose branch at C-3 of galactose, and variable MeOPN groups at C-3 of both fructose branches at FEBS Journal 272 (2005) 4407–4422 ª 2005 FEBS Campylobacter jejuni HS:1 serostrain CPS C-2 and C-3 of galactose (Fig 1b) Most importantly, it was established that this structural heterogeneity was not an artifact of the isolation procedure, but reflects that which is maintained in vivo Isolation of CPS and 1H NMR spectroscopy Using the hot water ⁄ phenol extraction method, 7.3 mg of pure CPS was obtained from g (wet pellet mass) of bacterial cells, while the enzymatic method afforded 6.8 mg of pure CPS from the same mass of bacterial cells By suspending an enzyme purified sample of HS:1 CPS in nonbuffered D2O (pD 2.2), the autohydrolyzed defructosylated repeating unit was obtained (CPS-1), as well as other hydrolysis fragments The 1H NMR spectrum of this auto-hydrolyzed CPS sample showed sharp spectral lines and one major anomeric signal for Gal H-1 (Fig 2A) Signals originating from the methyl group of the MeOPN modification, normally present at 3.78 p.p.m., were absent [15] The 1H NMR spectrum of a hot water ⁄ phenol purified sample of HS:1 CPS showed two broad anomeric signals for Gal H-1 and resonances originating from the MeOPN modification were weak and therefore difficult to observe (Fig 2B) In contrast, the spectrum for the enzyme isolated CPS sample (CPS-2) showed one signal for Gal H-1 and signals originating from the methyl group of the MeOPN modification were sharp and clearly discernable (Fig 2C) HR-MAS NMR of HS:1 cells provided valuable insight into the nature of cell-bound CPS on the surface of bacterial cells The HR-MAS 1H NMR spectrum of HS:1 cells (Fig 2D) closely resembled the proton spectrum obtained for the enzyme purified CPS sample in that one anomeric signal was observed for Gal H-1, and signals arising from the MeOPN modification were sharp and clearly visible In light of the degradation observed for the hot water ⁄ phenol purified CPS sample, and because an enzyme purified CPS sample most closely resembled CPS on the surface of HS:1 cells; chemical analyses, high resolution NMR analyses and mass spectrometry analyses were performed using enzyme purified CPS Sugar composition analysis of enzyme purified CPS By comparing the GC retention times of alditol acetate derivatives for common aldo sugar standards with those prepared from an enzyme purified HS:1 CPS sample, galactose and the reduction products of fructose, mannose and glucose, were unambiguously identified (data not shown) 4409 Campylobacter jejuni HS:1 serostrain CPS D J McNally et al Fig NMR analysis of purified and cellbound C jejuni HS:1 CPS (A) 1H NMR spectrum of an auto-hydrolyzed enzyme purified CPS sample (B) 1H NMR spectrum of a hot water ⁄ phenol purified CPS sample (C) 1H NMR spectrum of an enzyme purified CPS sample (D) HR-MAS 1H NMR spectrum (10 °C) of cell-bound CPS N-linked glycan anomeric resonances are indicated with asterisks (E) 1D-NOESY spectrum (400 ms) of Gal H-1 for an enzyme purified CPS sample (F) HR-MAS NOESY (23 °C, 100 ms) showing the trace of Gal H-1 for cell-bound CPS (G) 1D-NOESY HR-MAS spectrum (10 °C, 200 ms) of Gal H-4a and H-4b for cell-bound CPS (H) HR-MAS 31P HSQC spectrum (10 °C, 512 transients, 64 increments, 1JP,H ¼ 10 Hz) for cell-bound CPS (I) HR-MAS 31P HSQC spectrum (23 °C, 512 transients, 64 increments, 1JP,H ¼ 10 Hz) for cell-bound CPS For the selective 1D experiments, excited resonances are underlined Determination of absolute configuration for enzyme purified CPS By comparing the GC retention times of the R- and S-butyl glycosides of an authentic d-galactose standard to the R-butyl glycosides of an enzyme purified HS:1 CPS sample, galactose was shown to have the D configuration (data not shown) Furthermore, an intense increase in adsorption at 340 nm following treatment with a hexokinase-phosphoglucoisomerase-glucose-6dehydrogenase-NADP fructose assay kit (Sigma, Oakville, Canada) indicated that fructose also had the D configuration (data not shown) The chirality of naturally occurring glycerols can be determined chemically, enzymatically or can be deduced from the biosynthetic pathway responsible for their production [24] When CDP-glycerol is used as a precursor to incorporate glycerol in the growing repeating chain 4410 of teichoic acids, the resulting glycerol-1-phosphate unit has the D, or R configuration [24] Alternatively, when glycerophosphate is biosynthetically derived from phosphatidylglycerol, the resulting product is L- or S-glycerol-1-phosphate [24] Based on previous work where we reported that the CPS biosynthetic locus of a HS:1 strain of C jejuni contains a tagF homologue responsible for transferring glycerol-phosphate residues from CDP-glycerol [14], the glycerol-1-phosphate residue was concluded to have the R configuration High resolution NMR analysis of auto-hydrolyzed enzyme purified CPS (CPS-1) Due to the complexity of the NMR spectrum of the native CPS, the backbone structure was first determined Examination of an auto-hydrolyzed defructosylated enzyme purified HS:1 CPS sample revealed a FEBS Journal 272 (2005) 4407–4422 ª 2005 FEBS D J McNally et al Campylobacter jejuni HS:1 serostrain CPS Fig NMR analysis of an auto-hydrolyzed defructosylated sample of C jejuni HS:1 CPS, CPS-1 (A) 1D-TOCSY (80 ms) of Gal H-1 (B) 1D-NOESY (800 ms) of Gal H-4 (C) 1D-TOCSY (60 ms) of Gal H-5 (D) 1D-NOESY-TOCSY of Gal H-1 (800 ms) and Gro H-2 (60 ms) (E) 31P HSQC with 1JP,H ¼ 10 Hz, 64 transients and 240 increments (F) 13C HSQC with 1JC,H 140 Hz, transients and 256 increments For the selective 1D experiments, excited resonances are underlined Ff and Fp represent the fructofuranose and fructopyranose monosaccharides, respectively [-4)-a-d-Galp-(1–2)-(R)-Gro-(1-P]n repeating unit (CPS-1) as well as other hydrolysis products (Fig 3) The 1D-TOCSY of Gal H-1 revealed J-correlated peaks for Gal H-2, H-3 and H-4 (Fig 3A) The 1D-NOESY of Gal H-4 revealed NOEs for Gal H-3 and H-5 (Fig 3B), and the 1D-TOCSY of Gal H-5 identified the Gal H-6 resonances (Fig 3C) A 1D-NOESY-TOCSY experiment with selective excitation of Gal H-1 ⁄ Gro H-2 was used to identify the glycerol resonances (Fig 3D) The Gal H-1 ⁄ Gro C-2 HMBC correlation confirmed the Gal-(1–2)-Gro linkage The 31P HSQC spectrum (Fig 3E) showed that Gal H-4 and Gro H-1 ⁄ 1¢ were linked by a phosphorus atom with a chemical shift characteristic of a monophosphate diester bond [25,26] The 13C HSQC spectrum (Fig 3G) and HMBC spectrum were used to assign the 13C resonances (Table 1) and signals consistent with those reported for fructofuranose and fructopyranose monosaccharides were observed [27–29] High resolution NMR analysis of intact enzyme purified CPS (CPS-2) Analysis of an intact enzyme-purified sample of HS:1 CPS using NMR at 600 MHz revealed fructose FEBS Journal 272 (2005) 4407–4422 ª 2005 FEBS Table NMR proton and carbon chemical shifts d (p.p.m) for an auto-hydrolyzed enzyme purified sample of C jejuni HS:1 CPS (CPS-1) and corresponding hydrolysis products The 31P chemical shift for the monophosphate diester linkage was dP 0.49 p.p.m CPS-1 Atom Type dH dC A1 A2 A3 A4 A5 A6 ⁄ A6¢ B1 ⁄ B1¢ B2 B3 ⁄ B3¢ Fruf1 ⁄ 1¢ Fruf2 Fruf3 Fruf4 Fruf5 Fruf6 ⁄ 6¢ Frup1 ⁄ 1¢ Frup2 Frup3 Frup4 Frup5 Frup6 ⁄ 6¢ CH CH CH CH CH CH2 CH2 CH CH2 CH2 C CH CH CH CH2 CH2 C CH CH CH CH2 5.20 3.87 3.98 4.54 4.17 3.74 ⁄ 3.74 4.11 ⁄ 4.05 3.97 3.76 ⁄ 3.76 3.56 ⁄ 3.64 – 4.10 4.10 3.82 3.67 ⁄ 3.79 3.55 ⁄ 3.70 – 3.79 3.88 4.02 3.70 ⁄ 4.02 98.9 70.4 69.9 75.5 71.5 61.6 65.2 77.9 62.1 63.4 105.1 76.1 75.2 81.4 63.2 64.5 98.8 68.3 69.3 69.4 64.1 4411 Campylobacter jejuni HS:1 serostrain CPS branches located at C-2 and C-3 of Gal and MeOPN groups on C-3 of the fructoses Due to the instability of HS:1 CPS, a cryogenically cooled probe was used as it permitted the acquisition of 1H and 13C NMR experiments in a relatively short period of time The 1D-TOCSY of Gal H-1 revealed two separate resonances for Gal H-2, H-3 and H4 labeled A2a, A2b, A3a, A3b, A4a and A4b, respectively (Fig 4A) The 1D-NOESY of Gal H-4a showed NOEs for Gal H-2a, Gal H-3a, Gal H-5 and Gal H-6 ⁄ 6¢, as well as for Fru H-4 and Fru H-6 ⁄ 6¢ (Fig 4B) Conversely, excitation of Gal H-4b revealed NOE enhancements for Gal H-2b, Gal H-3b, Gal H-5 and H-6 ⁄ 6¢ as well as for Fru H-6 ⁄ 6¢ (Fig 4C) A 1D-NOESY ⁄ TOCSY experiment with selective excitation of Gal H-1 and Gro H-1 ⁄ 1¢ permitted the assignment of Gro H-2 and Gro H-3 ⁄ 3¢ (Fig 4D) The HMBC experiment revealed three-bond correlations between Gal H-2, Gal H-3b and Fru C-2 indicating that two fructose branches were present for the CPS of C jejuni HS:1 (data not shown) The 1D-NOESY of Fru H-3 revealed enhancements for Fru H-1 ⁄ 1¢, H-4 D J McNally et al and H-5 (Fig 4E) while the 1D-TOCSY of Fru H-4 showed correlations to Fru H-3 and H-5 (Fig 4F) The 31P HSQC experiment revealed monophosphate diester linkages between Gal C-4a, C-4b and Gro C-1 with different chemical shifts at dP 0.40 p.p.m and 0.49 p.p.m., respectively (Fig 4G) A proton-phosphorus correlation at dP 14.67 p.p.m observed between the methyl group of the MeOPN and H-3 of Fru indicated that this CPS modification was located at C-3 of the b-d-fructofuranoside residues (Fig 4G) The phosphorus chemical shift of the MeOPN was consistent with those reported for phosphoramidates in the literature [25,26,30] Comparison of carbon chemical shifts for defructosylated CPS-1 and intact CPS-2 indicated that fructose branches were located at C-2 and C-3 of Gal (Fig 4H, Table 2) The small upfield shift changes caused by fructosylation of Gal at C-2 and C-3 of 2.1 p.p.m and 1.2 p.p.m., respectively, were consistent with those reported for the CPSs of Escherichia coli strains 04:K52:H– and O13:K11:H11 (Tables and 2) [28,29] The 13C HSQC spectrum also showed minor contaminating signals similar to those reported for Fig NMR analysis of an enzyme purified sample of C jejuni HS:1 CPS, CPS-2 (A) 1D-TOCSY (80 ms) of Gal H-1 (B) 1D-NOESY (400 ms) of Gal H-4a (C) 1D-NOESY (400 ms) of Gal H-4b (D) 1D-NOESY-TOCSY of Gal H-1 (400 ms) and Gro H-1 ⁄ 1¢ (50 ms) (E) 1D-NOESY (400 ms) of Fru H-3 (F) 1D-TOCSY (80 ms) of Fru H-4 (G) 31P HSQC with 1JP,H ¼ 20 Hz, transients and 32 increments (H) 13C HSQC with 1JC,H ¼ 150 Hz, 80 transients and 256 increments For the selective 1D experiments, excited resonances are underlined Residue C represents Fru with MeOPN present and residue *C, Fru with no MeOPN 4412 FEBS Journal 272 (2005) 4407–4422 ª 2005 FEBS D J McNally et al Campylobacter jejuni HS:1 serostrain CPS Table NMR proton and carbon chemical shifts d (p.p.m) for an intact enzyme purified sample of C jejuni HS:1 CPS (CPS-2) The 31 P chemical shifts for the monophosphate diester linkages of Gal H-4a and b were dP 0.40 p.p.m and 0.49 p.p.m., respectively The 31 P chemical shift for the MeOPN groups was 14.67 p.p.m., and a scalar coupling 3JP,H of 11.1 Hz was observed CPS-2 Atom Type dH dC A1 A2a A2b A3a A3b A4a A4b A5 A6 ⁄ A6¢ B1 ⁄ B1¢ B2 B3 ⁄ B3¢ C1 ⁄ C1¢ C2 C3 C4 C5 C6 ⁄ C6¢ MeOPN C1 ⁄ C1¢a C2a C3a C4a C5a C6 ⁄ C6¢a CH CH CH CH CH CH CH CH CH2 CH2 CH CH2 CH2 C CH CH CH CH2 CH3 CH2 C CH CH CH CH2 5.40 4.29 4.28 4.33 4.40 4.74 4.69 4.16 3.76 ⁄ 3.76 4.15 ⁄ 4.11 4.02 3.84 ⁄ 3.76 3.78 ⁄ 3.63 – 4.84 4.52 3.85 3.86 ⁄ 3.77 3.81 3.78 ⁄ 3.63 – 4.12 4.12 3.75 3.86 ⁄ 3.77 98.8 68.5 68.3 69.4 68.7 77.2 77.3 72.0 61.6 64.5 77.1 61.6 62.4 104.1 79.7 73.2 81.2 62.5 54.9 62.4 104.1 77.0 76.6 81.5 62.5 a Chemical shift data d (p.p.m) for unsubstituted b-D-fructofuranoside (MeOPN is absent) C jejuni HS:1 LPS (CPS) [17,31] as well as for peptides and nucleic acids Furthermore, signals belonging to nonsubstituted b-fructofuranoside indicated that fructose branches were variably substituted with MeOPN groups [27–29] Mass spectrometry analysis CE-ESI-MS analysis corroborated the structure proposed for HS:1 CPS and clearly established that two branches are present in the repeating unit with various degrees of heterogeneity (Fig and Table 3) Low orifice voltage ()110 V) CE-ESI-MS analysis of an auto-hydrolyzed enzyme purified sample of HS:1 CPS (CPS-1) revealed a mixture of negatively charged ions originating from the backbone (Fig 5A) In particular, ions observed at m ⁄ z 315.0, 407.1 and 631.2, corresponding to the masses of Hex + GroP, Hex + FEBS Journal 272 (2005) 4407–4422 ª 2005 FEBS GroP + Gro + H2O and (Hex)2 + (GroP)2, respectively, confirmed that the natural acidity of HS:1 CPS (pD 2.2) had hydrolyzed both fructofuranose branches and confirmed the structure of the backbone repeating unit as [-4)-a-d-Galp-(1–2)-(R)-Gro-(1-P-]n Due to the high molecular mass of HS:1 CPS, a high negative orifice voltage ()400 V) was used to promote in-source collision-induced dissociation [23] for an intact enzyme purified sample of HS:1 CPS (CPS-2) to facilitate its analysis by CE-ESI-MS (Fig 5B) In addition to observing ions originating from the repeating unit, ions at m ⁄ z 639.4 and 801.6 corresponding to (Hex)3 + GroP and (Hex)4 + GroP, respectively, confirmed the attachment of both fructose branches on galactose Furthermore, ions observed at m ⁄ z 671.4, 894.6, 905.5 and 987.7 corresponding to (Hex)3 + (MeOPN)2, (Hex)4 + GroP + MeOPN, (Hex)3 + GroP + (MeOPN)2 + P, and (Hex)4 + GroP + (MeOPN)2, respectively, supported that MeOPN groups were located on both fructose branches Of particular importance, CE-ESI-MS ⁄ MS analysis of m ⁄ z 732.5, corresponding to one full repeat of HS:1 CPS, showed an ion at m ⁄ z 658.2, corresponding to (Hex)3 + MeOPN + P, and corroborated the findings of NMR analysis by demonstrating that Fru branches in HS:1 CPS are variably substituted with MeOPN groups (Fig 5C) Branching pattern of CPS-2 Two unique spin systems, a and b, were identified for Gal indicative of structural heterogeneity due to two different forms of the repeating unit For the enzyme purified CPS sample and whole cells, only one Gal H-1 resonance at 5.40 p.p.m was detected in their corresponding 1H spectra (Fig 2C,D) Because loss of the fructose branch at Gal C-2 would have caused an upfield shift of Gal H-1 similar to that observed for the hot water ⁄ phenol purified CPS sample or the autohydrolyzed CPS sample (Fig 2A,B), the Gal C-2 fructosyl branch was the dominant form present in the native CPS Hence, these different spin systems arose from two forms of CPS due to nonstoichiometric branching at C-3 of Gal The larger carbon chemical shift difference observed for Gal C-3a and b (0.9 p.p.m) compared to the one for Gal C-2a and b (0.2 p.p.m) also indicated that variable glycosydation occurred at C-3 of Gal Based on the Gal H-3b and Fru C-2 HMBC correlation, spin system b was attributed to the form where both fructose branches were simultaneously present at C-2 and C-3 of Gal, while spin system a represents the form where the fructose branch at Gal C-3 was absent 4413 Campylobacter jejuni HS:1 serostrain CPS D J McNally et al Fig Mass spectrometry analysis of C jejuni HS:1 CPS (A) CE-ESI-MS analysis of an auto-hydrolyzed defructosylated sample of HS:1 CPS (CPS-1) (negative ion mode, orifice voltage )110 V) (B) CE-ESI-MS analysis of an intact enzyme purified sample of HS:1 CPS (CPS-2) (negative ion mode, orifice voltage )400 V) (C) CE-ESI-MS ⁄ MS analysis for an intact enzyme purified sample of HS:1 CPS (CPS-2) m/z 732.2 (negative ion mode, orifice voltage )400 V) Collision energy was ramped from )35 to )55 V for the scan range of m/z 100–800 HR-MAS NMR spectroscopy of cell-bound CPS In order to characterize the heterogeneity of the CPS in its native state, HR-MAS NMR studies were performed on intact cells As observed for the purified CPS, two signals arising from Gal H-4, H-4a and H-4b, were detected on the surface of HS:1 cells and appeared to be present in equal proportions (Fig 2) The 1D NOESY of Gal H-1 for an enzyme purified 4414 CPS sample showed NOEs for Gal H-2, Gro H-1 ⁄ 1¢, Gro H-2 and Gro H-3 ⁄ 3¢ (Fig 2E) Likewise, in the HR-MAS NOESY trace of Gal H-1 (Fig 2F) for HS:1 cells, the same NOE pattern was observed The 1D HR-MAS NOESY for the Gal H-4a and H-4b resonances (Fig 2G) of cell-bound CPS revealed NOEs for Gal H-3b, H-2a and b, H-5, H-6 ⁄ 6¢ as well as for fructose H-6 ⁄ 6¢, similar to those observed for the purified CPS (Fig 4) The 31P HSQC HR-MAS FEBS Journal 272 (2005) 4407–4422 ª 2005 FEBS D J McNally et al Campylobacter jejuni HS:1 serostrain CPS Table Negative ion CE-ESI-MS data ()400 V orifice voltage), calculated masses and proposed fragments for auto-hydrolyzed (CPS1) and intact (CPS-2) samples of HS:1 CPS Isotope-averaged masses of residues were used for calculation of total molecular masses based on the following proposed compositions: Gro (glycerol), 74.1; Hex (a-D-galactopyranoside), 162.1; MeOPN (O-methyl phosphoramidate CH3OP(O)(NH2)), 93.2; P (phosphate), 80.0; H2O, 18.0 For these gas-phase (IS-CID) degradation products, no H2O molecule is added to the residues unless specifically indicated Molecular mass (m ⁄ z) Observed Calculated Difference Structure 153.1 171.3 223.3 254.8 259.0 297.3 315.3 333.5 377.5 385.3 395.3 398.3 407.5 416.8 453.3 459.3 469.3 477.3 487.3 490.8 495.5 509.6 539.3 551.5 557.5 570.3 578.8 613.5 621.5 631.3 639.3 652.3 658.2 667.5 671.8 701.5 723.3 732.3 751.4 775.5 793.5 793.3 153.1 171.1 223.1 254.2 259.1 297.2 315.2 333.2 377.2 385.2 395.2 398.2 407.3 416.3 453.3 459.5 469.3 477.3 487.3 490.4 495.4 509.4 539.3 551.4 557.3 570.4 578.4 613.4 621.5 631.4 639.5 652.5 658.4 667.4 671.5 701.4 723.5 732.5 751.4 775.5 793.5 793.5 0.0 0.2 0.2 0.6 0.1 0.1 0.1 0.3 0.3 0.1 0.1 0.1 0.2 0.5 0.0 0.2 0.0 0.0 0.0 0.4 0.1 0.2 0.0 0.1 0.2 0.1 0.4 0.1 0.0 0.1 0.2 0.2 0.2 0.1 0.3 0.1 0.2 0.2 0.0 0.0 0.0 0.2 801.6 829.5 855.3 801.6 829.6 855.5 0.0 0.1 0.2 GroP GroP + H2O Hex + P ) (H2O)2 Hex + MeOPN Hex + P + H2O Hex + GroP ) H2O Hex + GroP Hex + GroP + H2O Hex + GroP + P ) H2O (Hex)2 + P ) H2O Hex + GroP + P (Hex)2 + MeOPN ) H2O Hex + GroP + Gro + H2O (Hex)2 + MeOPN (Hex)2 + MeOPN + (H2O)2 (Hex)2 + GroP ) H2O Hex + (GroP)2 ) H2O (Hex)2 + GroP Hex + (GroP)2 + H2O (Hex)2 + Gro + MeOPN (Hex)2 + GroP + H2O (Hex)2 + Gro + MeOPN (Hex)2 + GroP + P ) H2O (Hex)2 + GroP + Gro (Hex)2 + GroP + P (Hex)2 + GroP + MeOPN (Hex)3 + MeOPN (Hex)2 + (GroP)2 ) H2O (Hex)2 + GroP ) H2O (Hex)2 + (GroP)2 (Hex)3 + GroP (Hex)3 + Gro + MeOPN (Hex)3 + MeOPN + P (Hex)2 + (GroP)2 + (H2O)2 (Hex)3 + (MeOPN)2 (Hex)3 + GroP + P ) H2O (Hex)2 + (GroP)2 + Gro + H2O (Hex)3 + GroP + MeOPN (Hex)3 + (MeOPN)2 + P (Hex)3 + (GroP)2 ) H2O (Hex)3 + (GroP)2 (Hex)2 + GroP + MeOPN + P ) H2O (Hex)4 + GroP (Hex)3 + (GroP)2 + (H2O)2 (Hex)3 + (GroP)2 + P ) H2O FEBS Journal 272 (2005) 4407–4422 ª 2005 FEBS Table (Continued) Molecular mass (m ⁄ z) Observed Calculated Difference Structure 886.8 894.5 905.5 929.8 947.5 987.5 1048.5 1109.5 886.6 894.6 905.5 929.6 947.6 987.7 1048.7 1109.7 0.2 0.1 0.0 0.2 0.1 0.2 0.2 0.2 (Hex)3 (Hex)4 (Hex)3 (Hex)3 (Hex)3 (Hex)4 (Hex)4 (Hex)4 + + + + + + + + (GroP)2 GroP + GroP + (GroP)3 (GroP)3 GroP + (GroP)2 (GroP)3 + MeOPN MeOPN (MeOPN)2 + P ) H2O (MeOPN)2 + MeOPN spectrum of whole HS:1 cells showed proton-phosphorus correlations for Gal H-4a and b with at dP 0.33 p.p.m and dP 0.49 p.p.m., respectively (Fig 2H) The correlation between MeOPN at dP 14.67 p.p.m with H-3 of fructofuranose was also observed Hence, the NOEs and 31P HSQC indicated that structural heterogeneity due to different branching patterns on the Gal residue was also present for intact cells Molecular dynamics simulations Three models were constructed for the [-4)-a-d-Galp(1–2)-(R)-Gro-(1-P-]n repeating unit of HS:1 CPS representing different substitution patterns for the fructose branches located at C-2 and C-3 of a-d-Galp (present ⁄ absent, absent ⁄ present and present ⁄ present) These models were then used to verify NOEs observed during NMR analysis A minimum energy conformer generated using a Metropolis Monte-Carlo calculation for HS:1 CPS with both MeOPN-substituted fructose branches (in the same plane as the page) attached to the repeating unit (out of plane, with P closest to the reader) is shown in Fig Molecular dynamics simulations showed that regardless of the substitution pattern of Gal, the average interproton distance between Gal ˚ ˚ H-1 and Gro H-2 was approximately 2.6 A ± 0.2 A and therefore confirmed the strong NOE observed between these two residues Steric hindrance between both fructofuranose branches was found to be minimal as reflected in the mobility of these groups Interestingly, the fructose branch at C-3 of Gal was shown to be substantially more flexible than its Gal C-2 substituted counterpart Molecular dynamics simulations indicated that the weak interresidue NOE observed between Gal H-4a and fructose H-4 for an intact enzyme purified CPS sample (Fig 4B) could have originated from either fructose branch as interproton distances were comparable for both branches and ranged ˚ from to A The results of molecular dynamics 4415 Campylobacter jejuni HS:1 serostrain CPS Fig Molecular model for the C jejuni HS:1 CPS The [-4)-a-DGalp-(1–2)-(R)-Gro-(1-P-]n repeating unit with both MeOPN-substituted b-D-fructofuranose branches at C-2 and C-3 of Gal An additional phosphate group is added at C-4 of Gal OH groups have been removed to simplify the appearance of the model simulations also suggested that NOEs observed at 3.86 p.p.m in the NOESY spectra of Gal H-4a and Gal H-4b (Fig 4B,C) likely arose from Fru H-6 ⁄ 6¢ as the minimum interproton distance for these protons ˚ was approximately A In contrast, interproton distances calculated for Gal H-4 ⁄ Fru H-5, and Gal ˚ H-4 ⁄ Gro H-3 were on the order of 5–7 A thereby negating the likelihood of observing these interresidue NOEs Discussion We previously demonstrated that HR-MAS NMR can be used to rapidly compare C jejuni CPS structures from intact cells and provided the first structural evidence that CPS is associated with Penner serotype [14,15] In this study, we investigated the CPS structure for the representative HS:1 serostrain of C jejuni to complement data recently reported for CPS biosynthesis in strain G1 (HS:1) [14], and to determine the structure of labile CPS constituents not detected by previous studies examining HMW LPS (CPS) for the HS:1 serostrain [17,18] Together, different analytical methods showed that the HS:1type CPS of C jejuni is complex and has a teichoic acid-like [-4)-a-d-Galp-(1–2)-(R)-Gro-(1-P-]n repeating unit with a b-d-fructofuranose branch at C-2 of Gal, a nonstoichiometric fructose branch at C-3 of Gal and variable MeOPN modifications on C-3 of both fructose sugars By using a conventional hot water ⁄ phenol CPS isolation method [19] and a more sensitive enzymatic 4416 D J McNally et al approach [20–22], we demonstrated that the method used to isolate CPS was an important factor that influenced the structure of the purified polysaccharide thereby establishing the importance of using mild isolation conditions to examine CPS structures For instance, due to the hydrolysis of the labile fructose branches during extraction, two a-d-Galp anomeric signals were observed for hot water ⁄ phenol purified CPS: one at 5.40 p.p.m when the fructose branch at Gal C-2 was present and; another at 5.20 p.p.m when it was absent These structural artifacts complicated NMR and mass spectrometry data and as a result, hindered the identification of these labile branches and MeOPN groups In contrast, spectroscopic data acquired for an enzyme purified CPS sample was comparatively simple due to the preservation of both fructose branches, as was indicated by the appearance of only one anomeric signal for a-d-Galp at 5.40 p.p.m Most importantly, HR-MAS NMR analysis confirmed that the enzyme purified CPS sample was biologically more representative of native cell-bound CPS on the surface of HS:1 cells For this study, use of this gentle enzymatic method coupled with HR-MAS NMR proved pivotal in determining the structure and location of the fructose branches and MeOPN groups as both are labile structures that are easily hydrolyzed by high temperature and moderately acidic conditions [15,28,29] Because CPS is considered to be an important virulence factor for C jejuni [1,15], sensitive analytical techniques that facilitate the study of its fragile CPS structures are fundamental in increasing our understanding of host–pathogen interactions, mechanisms of infectivity and to guide the development of effective therapeutics for this bacterium This latter point is illustrated by the fact that although fructose has been reported for only a few bacterial CPSs, it was found to be the immunodominant sugar of the capsular K11 antigen of Escherichia coli O13:K11:H11 [28,29,32,33] NMR and mass spectrometry analyses of an autohydrolyzed defructosylated sample of enzyme purified HS:1 CPS showed that it resembled teichoic acid, and consisted of a [-4)-a-d-Galp-(1–2)-(R)-Gro-(1-P-]n repeating unit (CPS-1) Carbon and proton chemical shifts were identical to those of the capsular antigen of Neisseria meningitidis that has the same backbone [34] Moreover, these findings supported those reported for HMW LPS (CPS) isolated from this strain of C jejuni by McDonald [17], who showed it to consist of a [-4)a-d-Gal-(1–2)-Gro-(3-P-]n repeating unit However, the presence of fructose or MeOPN modifications was not reported The extraction and purification methods used by the previous work probably resulted in the FEBS Journal 272 (2005) 4407–4422 ª 2005 FEBS D J McNally et al hydrolysis of these labile constituents Crude extracts prepared using a hot water ⁄ phenol method were treated with acid to liberate a glycan polymer believed to be HMW LPS In the review by Moran et al [18], the structure is reported as [-4)-a-d-Gal-(1–3)-Gro-(1-P-]n which is probably a typographical error for the GalGro linkage as it refers to the original work by McDonald [17] Also, in our case, as the absolute configuration of glycerol was determined from genetic analysis, the glycerol-phosphate linkage is reported as Gro-(1-P instead of Gro-(3-P [17] The identification of MeOPN-substituted and unsubstituted fructose branches suggested that this modification could be expressed in a phase-variable manner in C jejuni HS:1 as found for C jejuni NCTC 11168 [15] Phosphoramidate structures are quite rare in nature and have not, to our knowledge, been shown to exist on CPS for any other bacterium and therefore appear to be unique to C jejuni Previous work examining synthetic phosphoramidate molecules have shown that they are high energy, labile structures with large standard free energies of hydrolysis and greater phospho donor potential than ATP [35–37] Although very little is known about their biological role in vivo, because of their reactive nature and high phosphodonor capabilities, phosphoramidates are thought to interact nonspecifically with accessible amino acids of proteins [38] Furthermore, there is a growing body of evidence suggesting that natural phosphoramidates, such as phosphohistidine, play an important role in two-component and phosphorelay signal transduction pathways in bacteria that mediate responses such as sporulation, chemotaxis, mucoidy, and flagellar movement to environmental stimuli [39–45] Accordingly, a range of small-molecular-weight phosphoramidate molecules have been identified that are able to elicit similar responses from bacteria and are therefore thought to mimic these naturally occurring phosphoramidate messengers [38–40,44,46] Although a two component system regulating growth and colonization in response to environmental temperature was reported for C jejuni [47], the relationship between the biological roles reported for phosphoramidates in other bacteria and the MeOPN CPS modification in C jejuni is not clear Thus, the biological role of this capsular modification in C jejuni, much like the biosynthetic pathway responsible for its production, is unknown at this time In conclusion, in this study we determined the complete structure of the CPS for the C jejuni HS:1 serostrain In doing so, we established the importance of using mild isolation methods and noninvasive analytical techniques for examining CPS in this bacterium FEBS Journal 272 (2005) 4407–4422 ª 2005 FEBS Campylobacter jejuni HS:1 serostrain CPS due to the presence of highly labile constituents that are easily overlooked using conventional methods Because the hot water ⁄ phenol method of extraction and treatment of CPS with acid are still commonplace, it is conceivable that such labile groups as these are more widely distributed in bacteria than is currently acknowledged Further, one might speculate that such discrepancies may be influential in the success of CPSbased vaccine development As a result of using HR-MAS NMR to examine CPS directly on the surface of bacterial cells we showed that the HS:1-type CPS of C jejuni consists of a [-4)-a-d-Galp-(1–2)-(R)-Gro-(1-P-]n repeating unit with two labile fructofuranoside branches and variable MeOPN modifications Hence, this strain of C jejuni can achieve a structurally variable and complex CPS from its relatively small CPS biosynthetic locus [14] This structural heterogeneity may be a mechanism to convey antigenic variation and protection from host defenses [14] Alternatively, CPS heterogeneity may be due to incorporation of incomplete glycan blocks or differences in the activity of the enzymes involved in the biosynthesis of the CPS repeats Future work will focus on elucidating the biosynthetic pathway responsible for MeOPN production; establishing the biological role of the MeOPN in C jejuni and; determining the structure of CPS in other strains of C jejuni to establish the commonality of this CPS modification within this species The results generated by these future initiatives will ultimately determine the potential of the MeOPN modification as a useful marker and therapeutic target for this mucosal pathogen Experimental procedures Solvents and reagents Unless otherwise stated, all solvents and reagents were purchased from Sigma Biochemicals and Reagents (Oakville, Canada) Media and growth conditions The C jejuni HS:1 serostrain (ATCC 43429, designation MK5-S7630) was routinely maintained on Mueller Hinton (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 of C jejuni HS:1 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 for 20 min) and placed in 4417 Campylobacter jejuni HS:1 serostrain CPS 70% (v ⁄ v) ethanol Cells were removed from the ethanol solution by centrifugation (9000 g for 20 min) and the bacterial pellet was refrigerated until extraction Hot water/phenol isolation of CPS Bacterial CPS was extracted using the hot water ⁄ phenol method according to Westphal and Jann [19] Briefly, bacterial cells harvested from L of BHI broth were blended in 90% phenol at 96 °C for 15 min, allowed to cool for 30 and then dialyzed (MWCO 12 KDa, Sigma) against running water for 72 h The volume of the bacterial extract was then reduced to approximately 100 mL under vacuum (37 °C), ultracentrifuged (140 kG, 15 °C) for h and the supernatant, which contained crude CPS, was flash frozen in an acetone ⁄ dry ice bath and lyophilized to dryness Crude CPS was then resuspended in H2O and purified using a SephadexÒ superfine G-50 column (Sigma, Oakville, Canada) equipped with a Waters differential refractometer (model R403, Waters, Mississauga, Canada) 1H NMR at 400 MHz (Varian, Palo Alto, CA, USA) was then used to screen fractions and those found to contain CPS were combined, flash frozen in an acetone ⁄ dry ice bath and lyophilized to dryness Semi-purified CPS was then re-suspended in H2O and purified using a Gilson liquid chromatograph (model 306 and 302 pumps, 811 dynamic mixer, 802B manometric module, with a Gilson UV detector (220 nm) (model UV ⁄ Vis-151 detector, Gilson, Middleton, WI, USA) equipped with a tandem QHP HiTrapTM ion exchange column (Amersham Biosciences, Piscataway, NJ, USA) Fractions containing CPS were combined, flash frozen in an acetone ⁄ dry ice bath and lyophilized to dryness Purified bacterial CPS was then de-salted using a SephadexÒ superfine G-15 column (Sigma) and fractions found to contain CPS were combined, flash frozen in an acetone ⁄ dry ice bath, lyophilized to dryness and stored at )20 °C until further analysis D J McNally et al 200 lgỈmL)1 (Sigma) before being incubated at 37 °C overnight with agitation at 100 r.p.m The crude CPS extract was then dialyzed against running water for 72 h (MWCO 12 kDa, Sigma), ultracentrifuged for h (140 kG, 15 °C) and the supernatant, containing crude CPS, was lyophilized to dryness CPS was then purified using the same chromatographic protocol described above Sugar composition analysis of enzyme purified CPS The composition of an enzyme purified sample of C jejuni HS:1 CPS was determined using the alditol acetate method adapted from Sawardeker et al [48] A mg sample of CPS was hydrolyzed by adding 0.5 mL of m trifluoroacetic acid and heating at 100 °C for h Hydrolyzed CPS was then dried under a nitrogen stream at room temperature prior to reduction with mg of NaBH4 in 300 lL of H2O The reaction was allowed to proceed for h at room temperature and was stopped by the addition of 0.5 mL of HOAc Reduced CPS sugars were then dried under a nitrogen stream at room temperature prior to the addition of three volumes of MeOH (3 · mL), with a drying step performed between each volume of MeOH Acetylation was achieved by the addition of 0.5 mL of acetic anhydride and heating at 85 °C for 30 prior to being dried at room temperature under a nitrogen stream Alditol acetate derived CPS sugars were then suspended in 1.5 mL of CH2Cl2 and analyzed using an Agilent 6850 series GC system, equipped with an Agilent 19091 L-433E 50% phenyl siloxane capillary column (30 m · 250 lm · 0.25 lm) (170 °C to 250 °C, 2.8 °CỈmin)1) (Agilent Technologies, Palo Alto, CA, USA) Alditol acetate derivatives of authentic standards for common keto and aldo sugars (Sigma) were then prepared using the same protocol outlined above The composition of C jejuni HS:1 CPS was then unambiguously determined by comparing the retention times of CPS alditol acetate derivatives to those of authentic standards Enzymatic isolation of CPS An enzymatic method of isolating CPS from C jejuni HS:1 cells was developed based on the methodologies of [20], Huebner et al [21] and Hsieh et al [22] Bacterial cells harvested from L of BHI broth were suspended in NaCl ⁄ Pi buffer (pH 7.4) Lysozyme was then added to a final concentration of mgỈmL)1 (Sigma) prior to the addition of mutanolysin to a final concentration of 67 mL)1 (Sigma) 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 (21 000 psi) to lyse cells, and DNAse I and RNAse (130 lgỈmL)1 DNAse I and RNAse, Sigma) was added prior to being incubated for h at 37 °C with agitation at 100 r.p.m Following digestion with nucleases, pronase and protease was added to a final concentration of 4418 Determination of absolute configuration for enzyme purified CPS The absolute configuration (d or l) of galactose within an enzyme purified sample of HS:1 CPS was assigned by characterization of its R-butyl glycoside using GC according to Loentein et al [49] Approximately 300 lL of R-butanol and 30 lL of acetyl chloride (Sigma) was added to mg of enzyme purified CPS The mixture was then heated at 85 °C for h prior to being dried under a nitrogen stream at room temperature Following the addition of 500 lL of acetic anhydride and pyridine, the mixture was heated at 85 °C for h before being dried a second time The R-butyl glycoside of galactose was then suspended in 1.5 mL of FEBS Journal 272 (2005) 4407–4422 ª 2005 FEBS D J McNally et al CH2Cl2 and analyzed using an Agilent 6850 series GC system, equipped with an Agilent 19091 L-433E 50% phenyl siloxane capillary column (30 m · 250 lm · 0.25 lm) (170 °C to 250 °C, 2.8 °CỈmin)1) (Agilent Technologies) The absolute configuration of galactose in the CPS sample was then unambiguously determined by comparing the retention time of its R-butyl glycoside to the R- and S-butyl glycosides of an authentic d-galactose standard prepared using the same method (Sigma) In light of the complications reported for producing the butyl-glycosides of keto sugars [50], the absolute configuration of fructose in HS:1 CPS was assigned enzymatically using a fructose assay kit (Sigma) and a mg sample of hydrolyzed enzyme purified HS:1 CPS according to Rodriguez et al [29] As recommended by the manufacturer, the CPS sample was first treated overnight with b-d-glucose oxidase (100 lgỈmL)1, 37 °C, Sigma) to eliminate traces of d-glucose HR-MAS NMR spectroscopy of cell-bound CPS For HR-MAS analysis, C jejuni HS:1 cells were prepared as according to Szymanski et al [15] Overnight growth from one MH agar plate was harvested and placed in mL of 10 mm potassium-buffered 98% D2O (pD 7.0) (Cambridge Isotopes Laboratories Inc, Andover, MA, USA) containing 10% sodium azide (w ⁄ v) for h at room temperature to kill cells Cells were then pelleted by centrifugation (8900 g for min), and washed once with 10 mm potassium-buffered D2O Approximately 10 lL of 1% (w ⁄ v) TSP was then added as an internal standard (0 p.p.m) to the cell suspension prior to being loaded into a 40 lL nano-NMR tube (Varian, Palo Alto, CA, USA) using a long tipped pipette cut diagonally approximately cm from the end HR-MAS experiments were performed using a Varian Inova 500 MHz spectrometer equipped with a Varian mm indirect detection gradient nano-NMR probe with a broadband decoupling coil (Varian) as previously described [2,15,51] Spectra from 40 lL cell samples were spun at kHz and recorded in ambient temperature (23 °C), or at 10 °C to shift the HOD signal, and all experiments were performed with suppression of the HOD signal H NMR spectra of bacterial cells were acquired using the Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence (90-(s180-s)n-acquisition) [52] to remove broad signals originating from lipids and solid-like materials, and the total duration of the CPMG pulse (n*2 s) was 10 ms with s set to (1 ⁄ MAS spin rate) 1H NMR spectra for cell-bound CPS on bacterial cells were typically obtained using 256 transients (11 min) The 2D-NOESY spectrum for cell-bound CPS was acquired using 16 transients ⁄ 256 increments and a mixing time of 100 ms (3 h), and the 1D-NOESY spectrum was acquired using 8100 transients ⁄ 64 increments and a mixing time of 200 ms (14 h) 31P-decoupled 31P HSQC spectra were acquired using 512 transients ⁄ 64 increments and a coupling constant of 10 Hz (33 h) FEBS Journal 272 (2005) 4407–4422 ª 2005 FEBS Campylobacter jejuni HS:1 serostrain CPS High resolution NMR spectroscopy To obtain the hydrolyzed defructosylated repeating unit of HS:1 CPS (CPS-1), a mg sample of enzyme purified CPS was suspended in 150 lL of nonbuffered 99% D2O (pD 2.2) (Cambridge Isotopes Laboratories Inc) and placed in a mm NMR tube (Wilmad, Buena, NJ, USA) The hydrolysis reaction, achieved using the natural acidity of HS:1 CPS, was then surveyed periodically over the course of four days using NMR analysis at 600 MHz with an ultra-sensitive, cryogenically cooled probe Analysis of the repeating unit and hydrolysis products over time was facilitated by the high sensitivity of the cryoprobe as 13C HSQC spectra were typically acquired in approximately h For analysis of hot water ⁄ phenol purified CPS and enzyme purified CPS samples (CPS-2), a mg sample of each was suspended in 150 lL of NH4HCO3 buffered 99% D2O (54 mm, pD 8.6), placed in mm NMR tubes and analyzed by NMR For all CPS samples, 1H NMR, 13C HSQC, HMBC, HMQCTOCSY, COSY, TOCSY, NOESY and selective one-dimensional TOCSY, NOESY and NOESY-TOCSY NMR experiments were performed at 600 MHz (1H) using a Varian mm, Z-gradient triple resonance cryogenically cooled probe (Varian) The methyl resonance of acetone was used as an internal reference (dH 2.225 p.p.m and dC 31.07 p.p.m.) The 31P HSQC experiments were performed using a Varian Inova 500 MHz spectrometer equipped with a Varian Z-gradient mm triple resonance (1H, 13C, 31P) probe The 1D 31P spectra were acquired using a Varian Mercury 200 MHz (1H) spectrometer and a Nalorac mm four nuclei probe For all 31P experiments, spectra were referenced to an external 85% (v ⁄ v) phosphoric acid standard (dP p.p.m.) NMR experiments were typically performed at 25 °C with suppression of the deuterated HOD resonance at 4.78 p.p.m Standard homo- and heteronuclear correlated two-dimensional pulse sequences from Varian were used for general assignments, and selective one-dimensional TOCSY and NOESY experiments with a Z-filter were used for complete residue assignment and characterization of individual spin systems [53,54] Mass spectrometry analysis CE-ESI-MS and CE-ESI-MS ⁄ MS analysis was performed using a Crystal Model 310 Capillary Electrophoresis instrument (ATI Unicam, Boston, MA, USA) coupled to a 4000 QTRAP mass spectrometer (Applied Biosystems ⁄ Sciex, Concord, Canada) via a Turbo ‘V’ CE-MS probe A sheath solution (isopropanol ⁄ methanol, : 1, v ⁄ v) was delivered at a flow rate of lLỈmin)1 Separations were achieved on approximately 90 cm of bare fused-silica capillary (360 lm outside diameter · 50 lm i.d., Polymicro Technologies, Phoenix, AZ, USA) and 15 mm ammonium acetate ⁄ ammonium hydroxide in deionized water (pH 9.0) containing 5% (v ⁄ v) MeOH as mobile phase A voltage of 20 kV was 4419 Campylobacter jejuni HS:1 serostrain CPS typically applied during CE separation and )5 kV was used as electrospray voltage Mass spectra were acquired with dwell times of 5.5 ms per step of 0.1 m ⁄ z)1 unit in Q1 scan mode Tandem mass spectra were acquired in the enhanced product ion scan (EPI) mode, using nitrogen as collision gas Fill time of the trap (Q3) was set to 20 ms and the LIT scan rate was adjusted to 4000 ams)1 Molecular dynamics simulations Molecular dynamics modeling was used to verify NOEs observed for C jejuni HS:1 CPS during NMR analyses CPS molecular models were constructed using the Biopolymer module of the insight ii software package (Accelrys Inc, San Diego, CA, USA), and then subjected to a 3000step energy minimization using a conjugate gradient method Atomic potentials were assigned automatically using an extensible systematic forcefield, and glycosidic tortions of energy-minimized structures were compared to a potential energy map constructed using the same forcefield, nonbond cutoff distance and dielectric value used for molecular dynamics simulations Molecular dynamics simulations were then performed in vacuum for 500 ps, using the discover-3 software running on an Insight II environment (Accelrys Inc) and data generated during the first 100 ps was discarded to allow the systems to reach equilibrium A Verlet algorithm with a fs timestep, extensible systematic forcefield, group-based nonbond method with a ˚ cutoff distance of 9.5 A and a distance-dependent dielectric value of was 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microbial polysaccharides by high resolution NMR spectroscopy In NMR in Microbiology: Theory and Applications (Barbotin JN & Portais JC, eds), pp 165– 190 Horizon Scientific Press, Wymondham, UK 55 Humphrey W, Dalke A & Schulten K (1996) VMD: visual molecular dynamics J Mol Graph 14, 33–38 FEBS Journal 272 (2005) 4407–4422 ª 2005 FEBS ... structural artifacts complicated NMR and mass spectrometry data and as a result, hindered the identification of these labile branches and MeOPN groups In contrast, spectroscopic data acquired for an... 4411 Campylobacter jejuni HS:1 serostrain CPS branches located at C-2 and C-3 of Gal and MeOPN groups on C-3 of the fructoses Due to the instability of HS:1 CPS, a cryogenically cooled probe was... different analytical methods showed that the HS:1type CPS of C jejuni is complex and has a teichoic acid-like [-4) -a- d-Galp-(1–2)-(R)-Gro-(1-P-]n repeating unit with a b-d -fructofuranose branch at C-2