CA2518317A1 - Phosphoramide and uses thereof - Google Patents

Description

PHOSPHORAMIDE AND USES THEREOF
FIELD OF THE INVENTION
The invention relates to cell surface structures useful in the identification and targeting of C. jejuni.
BACKGROUND
Campylobacter jejuni is the major bacterial cause of gastrointestinal disease in developed countries and infection can lead to the development of the neuropathy to known as Guillain-Barre syndrome.
Carbohydrates are implicated in a variety of functions in all domains of life.
There continues to be a growing demand for methodologies that can analyze carbohydrate structures with increasing levels of sensitivity and simplicity.
Genome sequencing of C. jejuni NCTC11168 demonstrated that the strain contained four gene clusters necessary for carbohydrate biosynthesis. The flagellar modification locus, adjacent to the flagellin structural genes flaA and flag, encodes enzymes involved in the biosynthesis of O-linked pseudaminic acid and its derivatives. The LOS and adjacent protein glycosylation loci encode enzymes involved in the formation of outer core ganglioside mimics and bacillosamine-containing N-linked heptasaccharide, respectively. While the capsular biosynthesis locus, containing a Kps transport system similar to that found in other encapsulated organisms, transfers a branched tetrasaccharide repeat to the outer membrane surface.
The CPS product of this locus has been demonstrated to be the major serodeterminant in the heat stabile typing scheme first described by Penner and Hennessy.
However, an inconsistency in the literature developed when only a limited number of C.
jejuni serotypes were believed to produce capsules based on detection by immunoblotting yet, all strains examined contained kps genes necessary for capsule transport.
It was then shown in G jejuni 81-176 that the high molecular weight CPS was antigenically variable but it remained to be determined whether the loss in CPS reactivity was due to the lack of CPS production or changes in its structure.
Since capsular polysaccharides are the outermost structure on the bacterial cell they play an important role in the interaction between the pathogen, host, and environment. In C. jejuni 81-176 the capsule is involved in INT407 cell invasion, virulence in ferrets, serum resistance and maintenance of bacterial cell surface hydrophilicity.
The C. jejuni pgl locus encodes enzymes necessary for the glycosylation of multiple proteins and disruption of this pathway by mutagenesis results in multiple pleiotrophic effects. The structure of the N linked glycan is GaINAc-a1,4-GaINAc a1,4-[Glc-(31,3-]GaINAc-a1,4-GaINAc-a1,4-GaINAc-a1,3-Bac. However, it was unknown in the literature whether the same N linked glycan was present in multiple campylobacter isolates or whether slight structural variations exist as is observed for the campylobacter O-linked flagellin glycan.
C. jejuni LOS have received much attention due to their unique mimicry of human ganglioside structures and their potential involvement in the induction of the autoimmune polyneuropathies, Guillain-Barre (GBS) and Miller Fisher syndromes.
C.
jejuni LOS have also recently been shown to be phase variable and important in virulence. However, in the elucidation of C. jejuni LOS structures there are two major problems, the need for a large amount of biomass and the time consuming effort to isolate and purify LOS.
Capsular polysaccharides (CPSs) are found on the surface of a large number of bacterial species. CPSs are known to play an important role in bacterial survival and persistence in the environment and often contribute to pathogenesis. In addition, 2o through stnzctural variation, the potential to mimic host cell antigens, and the ability to resist innate mechanisms such as phagocytosis and complement-mediated killing, bacterial CPSs play a role in evasion of host immune responses.
Assembly of these surface polysaccharides is remarkably conserved in bacteria. Nucleotide diphosphate sugars are synthesized in the cytoplasm and sequentially added by glycosyltransferases to an undecaprenyl pyrophosphate carrier anchored in the membrane. Many Gram-negative bacteria flip the assembled polysaccharide across the membrane using an ABC transporter consisting of the transmembrane channel, KpsM, and the ATPase, KpsT. These transporters form a complex with 4-5 additional Kps proteins to ensure proper translocation of the polysaccharide to the bacterial surface. The genetic organization of the capsule gene clusters is also conserved in bacteria with kp.r transporter genes flanking polysaccharide biosynthesis genes, an organization conducive to genetic recombination and reorganization.

2 Identification of kps genes potentially involved in capsule biosynthesis during sample sequencing of the shot-gun library of NCTC11168 prompted a systematic genetic analysis of the corresponding locus and resulted in identification of CPSs in a number of strains of C. jejuni. These molecules were found to be the major antigens in the Penner serotyping scheme. Similar experiments performed on C. jejuni 81-confirmed these findings and demonstrated a role for the capsule in serum resistance, epithelial cell invasion and diarrhoeal disease. Subsequent characterisation of the CPSs by Alcian blue staining led to the visualisation of capsule by electron microscopy. These experiments suggested that the previously described high molecular weight "lipopolysaccharides" (HMW LPSs) of G jejuni are in fact CPSs.
Recently, the CPS structure of NCTC 11168 was determined to contain 6-O-methyl-D-glycero-a-L-gluco-heptose, (3-D-glucuronic acid modified with 2-amino-deoxyglycerol, ~i-D-GalflVAc and (3-D-ribose. There are several notable features encoded by the cps locus of NCTC11168 that correlate well with the published structure: homologues of the GDP-D-glycero-D-ynannoheptose pathway (GmhA2, HddA and HddC); homologue of the UDP-glucose dehydrogenase, Udg, involved in the formation of UDP-glucuronic acid; and a homologue of the UDP-pyranose mutase, Glf, predicted to catalyse the reversible conversion of pyranoses to furanoses and shown to cause loss of CPS when mutated in NCTC11168 {St Michael, 2002 #}.
Early studies of the structural analysis of HMW LPSs (now realised as CPSs) of G jejuna, showed that these molecules are highly heterogeneous. Microarray hybridisation analysis also demonstrated some differences in the CPS-related genes between the strains of various serotypes. However, hybridisation analysis does not allow detailed investigation of gene content. Sequencing of the C. jejuni genome revealed that the GC content of the cps locus (cj1415-cj1442) is lower (26.5%o) in comparison to that for the entire genome (30.6%) suggesting that this locus was acquired through horizontal gene transfer. In addition, the biosynthetic region of the cps locus is also prone to phase variation due to the presence of six genes with homopolymeric tracts. It was subsequently shown that CPS from 81-undergoes antigenic variation at high frequency {Bacon, 2001 } and that CPS
from NCTC11168 can vary in structure. However, the genetic mechanisms underlying the structural heterogeneity and antigenic variation remain unknown.

3 SUMMARY OF THE INVENTION
There is disclosed herein phosphoramide structures found on the surface of a majority of Campylobacter jejuni isolates and uses thereof.
C. jejuni produces a capsular polysaccharide (CPS) that is the major antigenic component of the classical Penner serotyping system. High resolution magic angle spinning (HR-MAS) NMR was used to examine capsular polysaccharides directly from campylobacter cells and showed profiles similar to those observed for purified polysaccharides analysed by solution NMR. This method also exhibited the potential 1o for campylobacter serotyping, mutant verification, and preliminary sugar analysis.
HR-MAS NMR examination of growth from individual colonies of C. jejuni NCTC11168 indicated that the capsular glycan modifications are also phase variable.
These variants show different staining patterns on deoxycholate-PAGE and reactivity with immune sera. One of the identified modifications, that showed both reduced reactivity with silver staining and rabbit sera, was a novel -OP=O(NH2)OMe phosphoramide not observed previously in nature. This modification was attached to the 3-position of the CPS Gal, f NAc.
Biosynthetic cps regions were sequenced, ranging in size from 15 to 34 kb, from G jejuni strains of HS:1, HS:19, HS:23, HS:36, HS:23/36 and HS:41 serotypes and compared with the sequenced strain, NCTC11168 (HS:2). Extensive structural studies, including HR-MAS NMR, demonstrated polysaccharide heterogeneity in campylobacter CPS and demonstrated the presence of additional CPS
modifications and the commonality of the recently described phosphoramide.
Development of a novel HRMAS filtering method has allowed investigation of multiple isolates of C. jejuni from various clinical presentations and geographical locations and revealed that the phosphoramide is common to approximately 70%
of all strains examined. This modification appears specific to C. jejuni and was not observed in the closely related Campylobacter coli. Structural analysis of the HS:1 and HS:19 strains demonstrated that the phosphoramide can be attached to different sugars in different linkages. Multiple phosphoramide signals are observed during HRMAS analysis suggesting that the modification is attached to varying capsule backbones, attached to alternate structures and/or being detected as biosynthetic intermediates.

4 Sequential inactivation of the cps biosynthetic genes in C. jejuna NCTC11168 followed by phosphoramide filter analysis has allowed identification of multiple genes encoding enzymes involved in the biosynthesis of phosphoramide: cj1416c, cj1417c, cj1418c and cj1421 c (and potentially the duplicated gene, cj1422c).
All or most of these genes are missing in other Campylobacter species and genome sequenced strains belonging to the epsilon proteobacteria confirming their inability to synthesize phosphoramide. Preliminary examination of the C. jejuna mutants in human cell culture assays has demonstrated that the phosphoramide is required for efficient adherence but is not necessary for cell invasion. Furthermore, the expression l0 of this modifications renders the bacteria more sensitive to human sera.
In an embodiment of the invention there is provided use of the phosporamide OP=O(NH2)OMe or an immunologically active derivative thereof in the identification of Campylobacter jejuna. In some instances the phosphoramide is used as a target for a binder.
In some instances, the phosphoramide further includes an alkyl group attached to the O which is attached to the P of the methyl amidophosphate group, wherein the alkyl group is a sugar producable in Campylobacter jejuna. The sugar may be a naturally occurring sugax, an enantiomer, or other variant, or a non-natuarally-occurring sugax.
2o In an embodiment of the invention there is provided use of alkyl methyl amidophosphate or an immunologically active derivative thereof in the identification of Canzpylobacter jejuna, wherein the alkyl group is a structure attached to a sugar producable in Campylobacter jejuna. The phosphoramides discussed above may be used as vaccines in mammal to compute or reduce the severity of C. jejuna infection.
In an embodiment of the invention there is provided a method of modulating the adhesion of C. jejuna cells to a surface, the method comprising modulating the concentration of binders (to a phosphorarnide disclosed herein) in surrounding fluid.
In an embodiment of the invention there is provided a pharmaceutical composition comprising one or more the phosphoramide described herein and a physiologically 3o acceptable carrier. Pharmaceutical compositions of interest include those containing immunogenic conjugates and/or immunostimulants capable of enhancing immune response in a mammal (by way of non-limiting example, E. cola labile toxin).

5 In an embodiment of the invention there is provided at least one of the phosphoramides described above which is linked through the O which is attached to the P of the methyl amidophosphate group, to an amino acid or alkyl group wherein the alkyl group is a sugar producable in Campylobacter jejuni and the amino acid is an amino acid producablein Campylobacter jejuni.
In an embodiment of the invention there is provided a kit comprising:
a) the binder as described herein; and b) instructions for carrying out the method of claim 1 or 13.
In an embodiment of the invention there is provided use of an isolated nucleic acid sequence encoding Campylobacter jejuni Cj 1421c or Cj 1422c or a portion or variant thereof in producing a polypeptide sequence having wild-type transferase activity.
In an embodiment of the invention there is provided use of an isolated nucleic acid sequence encoding Campylobacter jejuni Cj I421c or Cj 1422c or a portion or variant thereof in producing an amino acid sequence useful in producing non-natually occurring antigenic compounds comprising the phosphoramide disclosed herein.
In some cases the nucleic acid sequence encodes an amino acid sequence at least 90% identical to the wild type Cj 1421 c or Cj 1422c sequence.
In an embodiment of the invention there is provided use of a nucleic acid sequence encoding Campylobacter jejuni Cj 1421 c or Cj 1422c or a portion or variant thereof having wild-type transferase activity in producing an amino acid sequence useful in producing non-natually occurring antigenic compounds comprising a phosphoramide described herein. 1n some instances the amino acid sequence encodes a variant at least 90% identical to the wild type Cj 1421 c or Cj 1422c sequence. The wild type sequence of Cj 1421c or Cj 1422c can be readily determined by reference to published sequences. It will be understood that some variability between sequences of different stxains may occur and such variant sequences are specifically comtemplated and will be variant sequences as recited in the claims so long as they exhibit transferase activity sufficient fox the production of the phosphoramide described herein. By way of non-limiting example, previously disclosed amino acid sequences of potential interest are listed in Table XV. A transferase encoded by a variant sequence will be considered to have transferase activity if it is capable of producing in vitro the phosphoramide described herein at at least 60% of the level observed for either a Cj 1421c or Cj 1422c wild-type transferase under the same conditions.
s BRIEF DESCRIPTION OF THE FIGURES OF PART A
FIGURE 1 Is a depiction of Proton NMR spectra of NCTC11168 and HS:2 serostrain. (a) 1H spectrum of NCTC11168 purified CPS with the structure of the major component shown above. The anomeric, OMe and NAc resonances are to labeled. HR-MAS proton NMR spectra with 10 ms CPMG filter of NCTC11168 (b) whole cells, (c) 1/100 dilution of whole cells, and (d) HS:2 serostrain. The Asp (aspartic acid) resonances are labeled in (d). The HOD resonance at 4.8 ppm was saturated and digitally filtered affecting the intensity of the anomeric resonance C in b) and c).
FIGURE 2 Is a depiction of comparison of individual colonies of NCTC11168.
(a) Silver-stained deoxycholate-PAGE: lane 1-NCTC11168 wild type population;
lane 2-NCTC11168 variant #1; lane 3-NCTC11168 variant #2; lane 4-NCTC11168 variant #3. (b) Western blot of same samples loaded in same order and immunodetected with HS:2 typing sera. HR-MAS NMR spectrum of (c) the wild type population, (d) variant #1 with arrow indicating presence of an ethanolamine resonance, (e) variant #2 with arrow indicating presence of the novel modification, and (fj variant #3 with arrow indicating loss of OMe resonance. The anomeric resonances are labeled A, B, C and D. Also note the movement of the anomeric peak for residue C in all variant spectra. The HOD resonance at 4.8 ppm was saturated and digitally filtered sometimes affecting the intensity of the anomeric resonance C.
FIGURE 3 Is a depiction of NMR experiments for the G jejuni NCTC1II68 variant 2 CPS. The structure of the CPS is shown above the spectra. (a) 1H
3o spectrum of the purified CPS. (b) Selective TOCSY (H-3C, 50 Hz, 80 ms) for assignments of the proton resonances of residue C. (c) Selective NOESY(H-4C, Hz, 200 ms) to detect inter-residue NOEs between residue C and D. (d) Trace from the 1H-31P HMQC for the 31P signal at 13.6 ppm. (e) 1H-13C HMQC spectrum showing assignments for residue C, the anomeric resonances and the POMe resonance.
FIGURE 4 Is a depiction of MS analysis for the C. jejuni NCTC11168 variant 2 CPS. (a) CE-MS (m/z 100-1600) with orifice voltage of 200 V. (b) MS/MS
spectrum of m/z 884 prompted by front-end collision induced dissociation.
FIGURE 5 Is a depiction of strategy for amplification of the cps regions. The primers corresponding to the conserved cps genes (shown in open arrows) were used l0 in combination with primers derived from conserved regions of kps genes (thick solid arrows) for long-range PCR as described in the section Experimental Procedures.
FIGURE 6 Is a depiction of graphical representation of the sequenced CPS
biosynthethic regions. In cases with high Ievel of similarity between putative gene products (usually with e-values below le-30), the genes were given names of counterparts found in other bacteria. When no such similarity was found, the genes were assigned the names of respective genes from strain NCTC11168. The genes with no similarity to either NCTCIII68 or other bacteria are given strain-specific systematic names. The cps clusters of serostrains HS:23 and HS:36 are almost 2o identical to that of strain 8I-176 and are not shown (see text).
FIGURE 7 Is a depiction of summary of the C. jejuni capsular polysaccharide structures described in this study. The CPS structures of the heat-stable (HS) Penner type strains HS:1, HS:19, HS:23 and HS:36 have been reviewed by Moran et al. { Moran, 2000 #217 } . The structures of NCTC 11168 CPS { St Michael, 2002 #212} and HS:41 CPS {Hanniffy, 1999 #175} have recently been described. Sugars are shown in pyranose configurations unless otherwise noted. P, phosphate;
Gal, galactose; Gro, glycerol; Me, methyl; Hep, heptose; Rib, ribose; GaINAc, N
acetylgalactosamine; GlcA6, glucuronic acid; NGro, aminoglycerol; GIcNAc, N
acetylglucosamine; Ara, arabinose; Alt, altrose; Fuc, fucose.
FIGURE 8 Is a depiction of proton NMR spectra of C. jejuni strains NCTC12500 (HS:l serostrain) and G1 (HS:1). a) HR-MAS spectrum of NCTC12500 (HS:1) whole cells at 21°C. b) HR-MAS spectrum of G1 whole cells at 21°C. c) NMR spectrum of partially purified G1 CPS at 40°C with acetone as the internal reference. The CPS
anomeric resonance corresponding to Gal and the methyl resonance from the common phosphoramide are labeled.
FIGURE 9 Is a depiction of proton NMR spectra of C, jejuni strains CCUG
10954 (HS:23 serostrain), ATCC 43456 (HS:36 serostrain) and 81-176 (HS:23/HS:36). a) NMR spectrum of partially purified CCUG 10954 CPS. b) NMR
spectrum of ATCC 43456. c) NMR spectrum of 8I-176. HR-MAS spectra were acquired at 21°C. NMR spectra were acquired at 40°C. For the CPS, acetone was used as the internal reference. Is also a depiction of the HMQC and TOCSY spectra of the C. jejuni strains CCUG 10954 (a), ATCC 43456 (b), and 81-176 (c). For the HMQC
spectra, the anomeric region is shown. For the TOCSY, the mixing time was 90ms.
Crosspeaks between signals in the anomeric region (4.7 to 5.5 ppm) and the sugar ring region (3.4 to 4.4 ppm) are shown.
FIGURE 10 Is a depiction of comparisons of the cps clusters of 81-176 (top) with NCTC 11168 (middle) and NCTC 12517 (bottom). Each of the genes are shown as boxes.
FIGURE 11 Is a depiction of regions of significant homology between the capsule locus (Cj1413c to Cj1448c) in C. jejuni NCTC11168 and C. jejuni RM1221, Helicobacter hepaticus ATCC51449, Helicobacter mustelae ATCC43772, and Wolinella succinogenes DMSZ 1740. No significant homology was seen with Helicobacter pylori strains 26695 or J99. Note that the genome sequences of C.
jejuni RM1221 and H. mustelae ATCC43772 are incomplete.
FIGURE 12 31P HMQC NMR analysis of purified capsule isolated from C. jejuni HS:1. The signal at 3.81 ppm (arrow) is characteristic of a phosphoramide 3o modification found on the capsule and originates from the CH3 methyl group of the phosphoramide. The signal at 4.84 ppm (arrow) is indicative of the capsular sugar to which the phosphoramide is attached. This sugar was not described in previous structural studies of HS:l and is currently under investigation.

FIGURE 13 31P HMQC NMR analysis of purified capsule isolated from C. jejuni HS:19. The signal at 3.76 ppm (arrow) is characteristic of a phosphoramide modification found on the capsule and originates from the CH3 methyl group of the phosphoramide. The signal at 4.26 ppm (arrow) is indicative of the capsular sugar to which the phosphoramide is attached (position 4 of (3-GIcNAc).
FIGURE 14 Whole cell 31P-IH filtered IH HR-MAS NMR spectra of several strains of C. jejuni. The number of peaks represent the number of phosphoramide to residues in different chemical( structural) environments. The amplitude of the peaks reflects the relative amounts of each residue.
FIGURE 15 Adherence and invasion results for phosphoramide mutants C. jejuni 1416-1, 1417-1, 1418-3 and their parent strain, NCTC11168H (UK-H) as well as the phosphoramide mutant C. jejuni 1421-3 and its parent strain, Variant 4 (V4).
Efficiency is defined as the number of bacteria that either adhered or invaded CaCo-2 cells divided by the total number of bacteria added and expressed as a percentage.
Results are presented as the means of at least three experiments ~ the standard error of the mean.
FIGURE 16 Results of motility assays for phosphoramide mutants C. jejuni 1416-1, 1417-1, 1418-3 and their parent strain, NCTC11168H (UK-H) as well as the phosphoramide mutant C. jejuni 1421-3 and its parent strain, Variant 4 (V4).
Results are presented as the mean of at least two experiments ~ the standard deviations.
FIGURE 17 Results of two serum sensitivity assays for the phosphoramide mutant C. jejuni 1416-1 and the parent strain, NCTC11168 (UK-H). Bars in grey represent colony counts without serum while bars in white represent counts in the presence of 100 ~.l of serum.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
While the invention is discussed with respect to particular examples and embodiments, it will be readily understood that it is not so limited, but in fact includes all variants and alternative embodiments thereof. While possible mechanisms and/or s modes of action may be discussed, it will be understood that the invention is not so limited.
The inclusion of a reference is not an admission or suggestion that it is relevant to the patentability of anything disclosed herein.
1 The abbreviations used are: Bac, bacillosamine, 2,4-diacetamido-2,4,6-trideoxy-D-glucopyranose; CE, capillary electrophoresis; CPMG, Carr-Purcell-Meiboom-Gill;
CPS, capsular polysaccharide; DIPSI-2, decoupling in the presence of scalar interactions; ESI-MS, electrospray ionization mass spectrometry; GBS, Guillain-Barre Syndrome; HR-MAS, high resolution magic angle spinning; LOS, lipooligosaccharides; LPS, lipopolysaccharide; MAS, magic angle spinning;
NOESY, nuclear Overhauser effect spectroscopy; PVDF, polyvinylidene difluoride;
TOCSY, total correlation spectroscopy; WURST-2, wideband, uniform rate, and smooth truncation; HMQC, heteronuclear multiple quantum correlation; HMBC, heteronuclear multiple bond correlation; NMR, nuclear magnetic resonance.
Examples Bacterial strains and growth conditions - Campylobacter jejuni NCTC11168 (HS:2) was isolated from a case of human enteritis and later sequenced by Parkhill et al.. C. jejuni serostrains: HS:1 (ATCC 43429), HS:2 (ATCC 43430), HS:3 (ATCC
43431), HS:4 (ATCC 43432), HS:10 (ATCC 43438), HS:19 (ATCC 43446), HS:36 (ATCC 43456) and HS:41 (ATCC 43460) were obtained from ATCC; C. jejuni HS:23 was obtained from Dr. Peggy Godschalk, Erasmus University Medical Center, Rotterdam; C. jejuni OH4382 and OH4384 were obtained from Health Canada; and C. coli HS:30 (NCTC 12532) was obtained from NCTC. All campylobacter strains were routinely grown on Mueller Hinton agar (Difco) under microaerophilic conditions at 37°C. C. jejuni NCTC11168 mutants were grown on Mueller Hinton agar with 30 ~g/mL kanamycin.
Spectroscopy - All CE-ESI-MS and CE-ESI-MS/MS experiments and structural analysis of the purified CPS by NMR were performed substantially as described (in St. Michael et al. Eur.J. Biochem 269:5119, (2002)). 31P NMR
experiments were acquired using a Varian Inova 500 MHz spectrometer equipped with a Z-gradient 3 mm triple resonance (1H, 13C, 3iP) probe substantially as described in Kneidinger et al. JBC 278, 3615 (2003). External 85% phosphoric acid was used at the chemical shift reference.
Preparation of cells for HR-MAS NMR - C. jejuni overnight growth from one agar plate 0101° cells) was harvested and suspended in 1 mL of 10 mM
potassium buffered saline (pH 7) made in DZO containing 10% sodium azide (w/v). The suspension was incubated for 1 h at room temperature to kill the bacteria. The cells were pelleted by centrifugation (7 500 X g fox 2 min) and washed once with 10 mM
potassium buffered saline in D20. The pellet was resuspended by adding 20 ~,L
of D20 and then 40 ~.L of the suspension was inserted into the rotor for analysis.
HR-MAS NMR spectroscopy - HR-MAS experiments were performed using a Varian Inova 600 MHz spectrometer equipped with a Varian nano-NMR probe substantially as described in St. Michael (2002), above, and Young et. al. JBC
277:42530 (2002). Spectra from 40 p.L samples were spun at 3 KHz and recorded at ambient temperature (21°C). The experiments were performed with suppression of the HOD signal at 4.8 ppm. Proton spectra of bacterial cells were acquired with the Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence [90-(~-180-i)ri acquisition]
to remove broad lines arising from lipids and solid-like material. The total duration of the CPMG pulse (n*2~) was 10 ms with ~ set to (1/MAS spin rate). One-dimensional selective TOCSY experiments with various spin-lock times from 30-150 ms and selective NOESY with mixing times from 100-400 ms were performed substantially as described in Uhrin and Brisson (2000) in NMR in Microbiology, p.165-210 Horizon Science Press, UK, and in Brisson et.al. (2002) in NMR spectroscopy of glycoconjugates p.59-93, Wiley-BCH, Weinheim. For use under MAS conditions, the TOCSY sequences were modified so that the DIPSI-2 mixing sequence was replaced with the adiabatic WLTRST-2 pulses. Selective experiments were described as EXP[selected spins, selective excitation bandwidth, mixing time] where EXP
is TOCSY or NOESY. Typically, proton spectra of bacterial cells could be obtained using 256 to 1024 transients (15 min to 1 hour). For the selective experiments on the N linked glycan resonances present as a minor component in the bacterial cells, the s time for each TOCSY and NOESY varied from 1 to 8 hours.
Deoxycholate-PAGE, silver-staining and immunoblotting of polysaccharides -Proteinase K treated whole cells of C jejuni wild type and phase variants were prepared and analyzed by deoxycholate-PAGE substantially as described in St Michael (2002) above. One portion of the gel was silver-stained while the other to portion of the gel was transferred to a PVDF membrane (Roche Molecular Biochemicals) and immunodetected with HS:2 antiserum (1:500 dilution). The immunoblot was then incubated with goat-anti-rabbit secondary antibody conjugated to alkaline phosphatase (1:2500 dilution, Sigma) and then developed with the vitro blue tetrazolium chloride / 5-bromo-4-chloro-3-indolyl phosphate detection system 15 (Roche Molecular Biochemicals).
Bacterial strains and growth conditions - Sequences of cps regions from a wide range of C jejuni strains have been investigated: NCTC11168 (HS:2, genome sequenced strain, enteritis isolate), 176.83 (HS:41 serostxain, enteritis isolate, NCTC12517 (HS:19 serostrain, enteritis isolate), G1 (HS:1, GBS isolate), 8I-20 (HS:23/36, enteritis isolate used in human challenge studies), CCUG 10954 (HS:23 serostrain, enteritis isolate) and ATCC 43456 (HS:36 serostrain, enteritis isolate).
Three of the strains examined in this study (NCTC11168, G1 and serostrain HS:19) and the additional strains used for comparative analysis of homopolymeric tracts in the cps region axe listed in Table IV. C. jejuni strains were grown in microaerophilic 25 conditions at 37°C on 7% blood agar plates for 2 days. The E. cola XL2 Blue MRF' strain (Stratagene), used in cloning experiments, was grown overnight at 37°C on LB
agar plates supplemented with 100 ug/ml ampicillin when necessary.
Sequencing of homopolymeric tracts in contingency genes - Genes cj1420 and cj1421 were amplified with primers ak149 (GAGTGCCACTGCTTACACGAGC
30 SEQ. ID. N0.1) and ak150 (GCTCAACCCAAATTCAGCCATAGAAAG SEQ. ID.
N0.2) and sequenced with primers ak152 (CACCTCCTTTATACCAATTCTGATAAGCC SEQ. ID. N0.3) and ak151 (GGCATAAAGGGAGTGGCGAAGAAACCTGC SEQ. ID. N0.4), respectively.

Gene cj1422 was amplified using primers ak153 (GATACGGCACAGTAAATGTTGATG SEQ. 117. N0.5) and ak147 (GCTACTATATCTGGACGATGTTGCTTG SEQ. ID. N0.6) and sequenced with primers ak150 and ak152. Genes cj1426 and cj1429 were amplified using primers akI44 (CTCATTCGACCTTTGGAATTGCCTTTG SEQ. ID. N0.7) and ak145 (CTGTTTCATAATTTCTGTCCGATACTGC SEQ. ID. N0.8) and sequenced using the same primers. Gene cj1437 was amplified with primers ak154 (CTCCTTATTTATCTATTCCACAC SEQ. ID. N0.9) and ak155 (CTTGTATTTTTTCAGCAACATAACTC SEQ. ID. NO.10) and sequenced with l0 primer ak156 (GCATTGGCGAGTTTTAGGATAGG SEQ. ID. NO.11). The single base run polymorphisms are detected directly from the sequencing chromatograms, from which the variable number Gs for a particular region can be estimated via comparison with the published consensus sequence.
The strategy of amplification and sequencing of C, jejuni CPS regions - In the preliminary experiments using PCR analyses, high sequence conservation of the CPS
transport and assembly genes (kps genes,) was found flanking the internal biosynthetic region of the CPS locus (cps cluster). In some cases such conservation extended into the adjacent internal cps genes. As all CPS-related genes in strain NCTC11168 are transcribed in the same direction, the strategy of PCR
amplification 2o was based on the assumption that this is the case in all other C. jejuni strains. In order to design primers suitable for long-range PCR, short sequences of kpsC and kpsF
genes using the SP-PCR procedure were derived. Sequence comparison allowed detection of highly conserved regions suitable for the design of universal PCR
primers.
Primers ak176 (CGGTTACCGCTTAACACATCAGGATGGGG SEQ. ID.
NO. 12) and ak177 (GTTAAACCCCAGCCCGCATAAAAAGGC SEQ. 117. N0.13) were used in SP-PCR sequencing of kpsC genes, and primers ak173 (GGGCGTTGCATAGTTAGTGGTATGGGTAAATCAGG SEQ. ID. N0.14) and ak174 (GGCGCTAAGATAGCAGCTACTTTAGCAAGCACAGG SEQ. ID. NO.
15) were used for SP-PCR sequencing of fragments of kpsF genes in. various strains of C. jejuni. Alignment of the derived sequences allowed the design of universal primers suitable for long PCR: ak186 for kpsF gene and ak188 for kpsC (see below).
For serostrain HS:19 a more optimal kpsC primer ak187 was designed.

Long-range PCR with kpsF and kpsC primers alone failed to produce any product with the reference strain NCTC11168. This could be due to a relatively large size of the amplicon (over 36 kb). However, it was possible to amplify the entire cps region as two long-range PCR products, when kpsC and kpsF primers were combined with primers derived from internal biosynthetic genes.
The same strategy was used for amplification of cps regions from other strains. A possibility of extension of the strategy to other strains was based on an assumption that various strains would share some genes in the internal cps clusters.
This was supported by preliminary hybridisation data. It was assumed that the conserved genes, when present, would be located in the same orientation. The sequencing of the cps regions of serostrains HS:23 and HS:36 was performed after the sequencing of strain 81-176 was complete. The identical gene content and high sequence identity between these three strains allowed complete sequencing of the cps regions of serostrains HS:23 and HS:36 using custom-made oligonucleotides.
The biosynthetic cps region of strain NCTC 11168 contains 28 genes. The conserved genes present in the biosynthetic regions of other strains were identified using PCR amplification with primers derived from the sequence of NCTC 11168 genome. When a product of expected size was present, the corresponding primers could be used in combination with the kpsC and kpsF primers for long-range PCR.
Primer pairs specific to the biosynthetic cps genes of strain NCTC11168 are indicated in the supplementary material. The primers designed for the genes found in internal cps regions of various strains were used in combination with the primers corresponding to the conserved flanking kpsC and kpsF genes to generate long PCR
products (Fig. 5). The long-range PCR resulted in overlapping products suitable for generation of complete sequences of the internal biosynthetic regions. Long-range PCR was performed using the Expand 20 kbPLUS pCR System (Roche) using conditions described by the manufacturer. KpsC specific primers ak188 (CCCCTAAAATCATCGAAGCATCATCTTCAACTTGAGC SEQ. ID. N0.16) and ak187 (CATGCTTTAAACCATTATACTTTGAAAAGCGGTTCTCAAG SEQ. ID.
3o N0.17), for serostrain HS:19) and kpsF specific primer ak186 (GAA.AAGGAAGCTTGTCCTTTGCAGCTTGC SEQ. ID. N0.18) were used in long-range PCR experiments.

The long-range PCR products were treated with polynucleotide kinase, sonicated, and blunt-ended with T4 DNA polymerase. Then, 1-2 kb fragments were gel extracted and cloned into alkaline phosphatase-treated pUC 18 (Promega) prior to sequencing. For closing gaps, primers corresponding to the ends of the contigs were designed and the regions were amplified and sequenced either directly or after cloning into pGEM-T-Easy vector (Promega) using the automatic sequencer. DNA
sequencing was performed on ABI 377 or ABI 3700 automatic sequencers using an ABI PRISM BigDye Terminator Cycle Sequencing Kit (Perkin-Elmer). The sequences generated via shot-gun sequencing were assembled and edited using l0 or GeneTool software (DoubleTwist.com), and were deposited at EMBL database with the following IDs and accession numbers:
Designation Strain ID Acc. Number HS41 176.83 CJ17683CPS BX545857 HS:36 ATCC 43456 CPS_036 AY332624 HS:23 CCUG 10954 CPS 023 AY332625 Multiple sequence alignment was performed using the ClustalW program (htt~://www2.ebi.ac.uk/clustalw/). The cps sequences were analysed using Artemis software and the extracted amino acid sequences were analysed by similarity searches with the BLASTp program against NCTC1I168 at http://www.sanger.ac.uk/Projects/C,jejuni/ and a non-redundant protein database at http://www.blast.genome.ad.jp/. The entire cps regions were compared with the cps region of NCTC11168 using BLASTn and tBLASTx programs (http://www.hgmp.mrc.ac.uk/) followed by the analysis using MSPcrunch (http://bioweb.Pasteur.fr/seqanal/interfaces/mspcrunch.html) and ACT programs (http://www.sanger.ac.uk).
Isolation and purification of CPS - The CPS was isolated from dried cell mass (approx. 1 g) by the hot water/phenol method { Westphal, 1965 } . The aqueous phase was dialyzed against water and lyophilized. The dried sample was then dissolved in water to a 1 % solution (w/v) and subjected to ultracentrifugation to yield a gel-like pellet containing LOS and supernatant containing the CPS.
Analytical methods - Sugars were determined by examining their alditol acetate derivatives by GLC-MS. Samples were hydrolyzed for 4 h using 4 M
trifluoroacetic acid at 100°C. The sample was reduced in NaBD4 overnight in H20 and acetylated with acetic anhydride at 100°C for 2 h using residual sodium acetate as the catalyst. The GLC-MS was equipped with a 30 M DB-17 capillary column (180°C
to 260°C at 3.5°C/min) and MS was performed in the electron impact mode on a Varian Saturn II mass spectrometer.
1o HR-MAS NMR allows the screening of small amounts of bacterial cells directly without having to purify surface carbohydrates HR-MAS experiments were performed on a Varian Inova 600 MHz spectrometer using a gradient 4 mm indirect detection high-resolution magic angle spinning nano-NMR probe (Varian) with a broadband decoupling coil as previously described { St Michael, 2002; Young, 2002 } .
Proton spectra of cells were acquired with the Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence {90-(z-180-z)n acquisition} to remove broad lines arising from lipids and solid-like material. The total duration of the CPMG pulse (n2~) was 10 ms with i set to (1/spin rate).
High-resolution NMR experiments on the partially purified CPS were 2o acquired using a Varian Inova 500 MHz spectrometer equipped with a Z-gradient 3 mm triple resonance (1H, 13C, 3iP) probe. The experiments were performed at 40°C
with suppression of the water resonance. The methyl resonance of acetone was used as an internal reference at 8H 2.225 ppm and ~ 31.07 ppm. Standard sequences from Varian, COSY, TOCY, NOESY, HMQC, and 31P HMQC were used.
Adherence and invasion assays - The procedure was performed as described previously by Bacon et al. (2001) with the following modifications: there were approximately 2X105 CaCo-2 epithelial cells per well infected with approximately 3X10' bacteria (multiplicity of infection = 150 bacteria per epithelial cell).
Motility assays - The OD6~ of a suspension of bacterial cells in Mueller Hinton broth was adjusted to 1.0 and 5p,L of the culture was inoculated into the centre of duplicate 0.4% agar plates. The plates were incubated at 37°C under microaerophilic conditions and the diameter was measured after 52 hours.

Serum sensitivity assays - Five ~,L of a bacterial suspension, adjusted to an OD6~ = 0.1, is added to duplicate wells containing 900~t.L, of Mueller-Hinton (MH) broth with either 100~,I. of active serum or additional MH broth. After a 1-hour incubation under microaerophilic conditions at 37°C with shaking at 100rpm, strains from each well are diluted to 10-Z and 10-3 and plated on dry MH agar plates.
The plates are incubated under microaerophilic conditions for 2 days and colony counts are performed.
1o RESULTS
Examination of CPS from whole cells by HR-MAS NMR - The capsular polysaccharide structure of the genome sequenced strain, NCTC11168 (HS:2) was described. The proton spectrum obtained from HR-MAS of suspended NCTC11168 bacterial cells closely resembled the spectrum of the purified capsular polysaccharide and clearly demonstrated the N acetyl, O-methyl, and anomeric resonances (Fig.
1 a, b). The HR-MAS NMR spectrum was obtained in a few minutes directly from 40 ul of whole cells. Hence, this method permitted quick screening of campylobacter CPS
directly from one plate of growth 0101° cells), but was sensitive enough to detect a 1/100 dilution of the suspension containing 8x107 cells (Fig. lc).
2o This method also permitted serotype comparisons between strains. The capsular polysaccharide structure from the HS:2 serostrain had not been determined.
In fact, it was previously believed that this strain did not produce high molecular weight glycans. Whole cell NMR spectra of the HS:2 serostrain and NCTC11168 are comparable (Fig. la,d). These results provide further evidence that capsular polysaccharides are the main serodeterminant in the heat-stabile typing scheme and demonstrate that HR-MAS NMR can be used to confirm serotype.
While simple HR-MAS spectra of bacterial cells can allow one to monitor glycan resonances, assignment of resonances to specific residues may require further information. In the present study, selective TOCSY and NOESY experiments were 3o employed to identify sugar residues or assign unknown resonances. For example, in the spectra of several campylobacter strains, sharp multiplets were often observed between 2.6 and 2.9 ppm (Fig. ld). A series of selective TOCSY experiments identified all the spins for this compound and they were determined to correspond to those of free aspartic acid. Addition of aspartic acid to the cells resulted in increased peak intensity between 2.6 and 2.9 ppm.
Also, selective TOCSY or NOESY could be performed on various C. jejuni serostrains to identify other sugar resonances in accordance with those reported in the literature. In the case of C. jejuni HS:41, previous studies have reported that the purified CPS was composed of a mixture of polysaccharides with the major and minor components differentiated by the presence of either 6-deoxy-altrofuranosyl or D-fucofuranosyl residues. The HR-MAS spectrum of HS:41 cells exhibited extensive spectral overlap so that signals for the signature 6-deoxy-sugars could not be assigned unambiguously. However, selective TOCSY experiments on HS:41 cells starting with selective irradiation of 'H resonances near 1.2 ppm established scalar coupling connectivities between the dominant CH3 resonance at 1.27 ppm with other sugar ring protons whose chemical shifts (5.23 ppm, 4.25 ppm, 3.9 ppm, 3.7 ppm) agreed with those of the 6-deoxy-D-altrofuranosyl moiety reported for the purified CPS
(5.185 ppm, 4.19 ppm, 4.34 ppm, 3.71 ppm, 3.89 ppm, and CH3 1.27 ppm). These results suggests that the dominant form of the HS:41 CPS contains the 6-deoxy-altrofuranosyl moiety.
HR-MAS NMR analysis of C. jejuni NCTCI1168 CPS phase variants -10 single colonies of NCTC11168 were selected and restreaked to one plate each.
The growth from a single plate was examined directly by HR-MAS NMR and also digested with proteinase K followed by deoxycholate-PAGE silver-staining or immunoblotting. Three different phenotypes were observed by these methods.
Growth from the first colony (variant 1 ) showed similar silver-staining patterns relative to the wild type population from which it was isolated from but showed increased levels of reactivity with HS:2 sera (Fig. 2a, b). Comparison of the HR-MAS NMR spectra (Fig. 2) with those of the wild type NMR spectra of purified CPS
demonstrated that variant 1 predominantly exhibited a resonance at 3.2 ppm consistent with an N-ethanolamine modification on the glucuronic acid (GIcA) (Fig.
2d) in contrast to the major wild type form which exhibited GlcA modified with 3o aminoglycerol (Fig. 2c). Variant 2 showed extremely reduced levels of silver-staining and immunoblotting although the HR-MAS spectra clearly indicated that similar amounts of polysaccharides were present in both variant and wild type samples (Fig.
2e). The HR-MAS spectrum of variant 2 revealed new resonances at 3.75 ppm (Fig.

2e) indicative of a novel modification, which had not been previously observed. In addition, the anomeric chemical shift for residue C moved down.field closer to the one for residue B. Variant 3 showed increased silver-staining but similar levels of immunoreactivity. This variant lacks the 6-O-Me group on the heptose confirmed by the loss of the resonance at 3.55 ppm (Fig. 2f).
The structural determination of the purified polysaccharide from C. jejuni NCTC1I168 variant 2 was done substantially as described in St. Michael (2002), above. Its backbone CPS structure was found to be the same as determined previously (Fig. 1), but with the addition of a modified phosphate group at C-3 of the to GalfNAc residue C (Fig. 3). The proton spectrum of the purified CPS from variant 2 is shown in Fig.3a. The sample also contained about 30% of the major wild-type CPS
whose structure is shown in Fig. 1. Comparison of the HMQC spectra of variant with the one from the wild-type sample, showed similarity in chemical shifts for residues A, B and D (Fig. 3e and Table I). Proton chemical shifts for residue C for variant 2 were identified using a selective TOCSY experiment (Fig. 3b). The 5D-and 3D-4C NOEs were also observed (Fig. 3c), as before for the wild-type CPS.
The proton spectrum for variant 2 (Fig. 3a) contained a signal for the methyl group linked to phosphate via an ester bond: 8H 3.75, b~ 54.8 ppm, which had a JP,H of 11.5 Hz. In the 31P-1H HMQC spectrum (Fig. 3d) this methyl group showed 2o correlation to the 31P signal at 13.6 ppm, which also gave a correlation to H-3 of the GalfNAc residue C. A JP,H_3c value of 8 Hz was obtained from simulation of the undecoupled 3~P spectrum. In the 31P-1H HMQC-TOCSY spectrum, correlations from the phosphorus signal at 13.6 ppm to H-2, H-3, H-4, and H-5 of the GalfNAc residue were observed. These data indicated that the methylphosphate group was linked at O-3 of the GalfNAc residue C. However, the low field chemical shift of 3IP
resonating at 13.6 ppm was inconsistent with the presence of a phosphodiester group.
Different conditions of mild acid hydrolysis of the polysaccharide were tested, in an effort to cleave furanoside bonds without complete destruction of the phosphor-containing substituent. Hydrolysis with 1% trifluoroacetic acid for 20 min. at 100° C
completely depolymerized the polysaccharide. At the same time the 31P signal at 13.6 ppm disappeared and a group of 31F signals with one major component arose at ~2 ppm. All of them correlated with methyl group signals, with the major methyl signal observed at 3.88 (1H)/56.7 (13C) ppm. Milder hydrolysis conditions (1% TFA at 60°

or 2% AcOH at 100°, lh) led to incomplete conversion of the phosphate group, but did not depolymerize the polysaccharide completely. However, the chemical behavior and 31P chemical shift of the methylphosphate group were consistent with the presence of the amide of methylated phosphoric acid R-OP=O(NHZ)OMe.
Phosphoramides can be hydrolyzed in dilute acids with the replacement of the NHZ
group with the OH group, in the conditions where alkyl esters of phosphoric acid are stable. Phosphoramides usually have 31P signals between 10 and 20 ppm (41-47), which agrees with the position of the 31P signal within the analyzed structure.
CE-MS analysis of C. jejuni NCTC11168 CPS phase variant 2 - In order to to confirm the structure of the capsular glycan derived from NMR studies, the purified CPS sample was also analyzed by using CE-MS and CE-MS/MS techniques. All the CE-MS and CE-MS/MS experiments were acquired using high orifice voltage. With this experimental setup, the polysaccharide breaks up into shorter oligosaccharide units due to the front-end collision induced dissociation. In this study, a orifice voltage of 200 V was applied and the extracted mass spectrum is shown in Fig.
4a.
Compared to the spectrum obtained with a low orifice voltage (60 V), the typical polymer peak disappeared and strong peaks that correspond to oligosaccharide or monosaccharide units appear. The ion [M + 1H]1+ = 884 corresponded to the mass of the one repeat unit minus H20. The ion m/z 791 arose from the repeat unit of the wild-type polysaccharide lacking unit E (30% of the sample) or the loss of unit E.
The ions m/z 1181 and 1472 were assigned to one repeat unit plus CE, and to one repeat unit plus BD and A, respectively.
To further investigate the composition of the CPS repeat unit, the MS/MS
experiments were conducted with the precursor ions at m/z 297, 382, 678, 884, and 1472. The MS/MS of ion m/z 297 clearly indicated the composition of C and E, whereas the MS/MS of ion m/z 382 displayed the composition of A and B.
Although the presence of the sugar residues D and A were not directly detected (Fig.
4a), the existence of these two residues in the oligosaccharide repeat unit from the tandem mass spectrum of ion m/z 884 could easily be determined. As shown Fig. 9b, there was a lost of 206 Da, which corresponded to the mass of residue D, resulting in the fragment ion m/z 678. Similarly, the fragment ions m/z 588, 456 and 429 were generated from the losses of CE (296 Da), AC (428 Da), and BD (455 Da), respectively. In Fig. 4b, the fragment ion at m/z 186 was assigned to the anhydrate C

(203 Da) and the ions at m/z 168 and 126 were generated from the consecutive neutral losses of H20 ( 18 Da) and acetyl group (42 Da), respectively. The reason for the co-existence of fragment ions that correspond to losses of D, CE, ACE, and BD
could be explained by the nature of generation of ions m/z 884 which is due to different breakage points along the polymer chain (A[CE)[BD), [CE)[BD]A, [BD)A[CE)).
Hence, all the MS and MS/MS data was consistent with the structure for the CPS
of variant 2 shown in Fig.3.
Correlation of cps genes from strains of serotypes HS:1, HS:2, HS:19, HS:23, HS:36 and HS:41 with respective CPS structures - The strategy used for sequencing l0 the variable cps loci from the different strains is described elsewhere herein and shown in Fig. 5. The overall summary of the cps sequencing results is presented in Table II. A schematic of all the cps loci compared in this study is shown in Fig. 6 with the genes involved in phosphoramide biosynthesis shown in bold (see below).
Some of the gene products are involved in the biosynthesis of activated sugars.
Such activated sugars contain energy-rich nucleotide-phosphate bonds and serve as substrates for glycosyltransferases involved in the biosynthesis of polysaccharides. In addition, nucleotide sugars may be modified by enzymes such as epimerases, dehydratases and reductases before transfer of the final product. Additional modifying enzymes can add groups such as O-methyl, phosphate, ethanolamine- and 2o aminoglycerol- to further increase the complexity of the structures.
Indeed, genes encoding these enzymes can be found in various C. jejuni cps regions. The predicted function of cps genes from strain NCTC11168 (HS:2) based on the published genome sequence { Paxkhill, 2000 } and the recently published CPS structure { St Michael, 2002} are presented in Table III. The CPS structures of NCTC11168 and the other strains used in this study are shown in. Fig.7.
There are three notable features encoded by the cps locus of NCTC 11168:
homologues of the GDP-D-glycero-D-mannoheptose pathway (HddC, GmhA2 and HddA), the presence of a UDP-glucose dehydrogenase homologue, Udg, responsible for the formation of UDP-glucuronic acid, and a UDP-pyranose mutase homologue, 3o Glf, catalysing the reversible conversion of pyranoses to furanoses.
NMR analysis of the HS:19 serostrain used in this study confirmed that the CPS structure was consistent with the published disaccharide repeat (Fig.7, results not shown). The cps region of the HS:19 serostrain did not contain homologues of the heptose pathway but did have the udg homologue (Table IV) correlating well with the presence of ~i-D-glucuronic acid which is also amidated with 2-amino-2-deoxyglycerol. NMR analysis also detected two acid-labile functional groups that were not reported previously. Both the phosphoramide modification recently described for NCTC11168 and an unknown labile group were observed during the analysis.
In contrast to NCTC11168 (HS:2) and the HS:19 serostrain, the CPS locus of G1 (HS:1) does not encode a homologue of UDP-glucose 6-dehydrogenase (Table V) and thus the strain should not have the ability to synthesise glucuronic acid.
However, to this strain contains a potential tagD homologue encoding a glycerol-3-phosphate cytidylyltransferase necessary for the formation of CDP-glycerol (Table V, G1.11).
G1 also encodes a TagF homologue, which transfers glycerol-phosphate residues from CDP-glycerol. Therefore, the repeating unit of this CPS may contain glycerophosphate residues. Indeed, the HS:1 serostrain was reported to contain glycerol-1-phosphate residues alternating with galactose in the repeating unit (Fig. 7).
The NMR spectra of G1 (Fig. 8) revealed that the structure of this CPS is consistent with the HS:1 structure. An additional anomeric resonance in the HR-MAS
spectrum of G1 was not present in the partially purified CPS sample suggesting that this resonance probably came from the medium used. Extensive NMR analysis by COSY, 2o TOCSY, NOESY and HMQC indicated the presence of only one anomeric resonance consistent with the presence of one sugar in the repeating unit. 31P NMR
experiments indicated the presence of a phosphate diester linkage, also consistent with the reported structure. The common anomeric resonances corresponding to the N linked Pgl glycan were also observed during HR-MAS analysis for both the HS:1 serostrain and G1, although they are prominent only for the HS:1 serostrain in Fig. 8. The phosphoramide modification was also observed during analysis of both strains (Fig.
8). In the 31P HMQC spectra, a strong correlation was observed between the POMe resonance at 3.8 ppm and the phosphoramide resonance at 14 ppm, indicative of a -OP=O(NH2)OMe modification.
The CPS loci of the HS:23 and HS:36 serostrains and of strain 81-176 (which reacts with both HS:23 and HS:36 antisera) all have exactly the same gene content (Figure 6 and Table II). The CPSs of HS:23 and HS:36 were found to contain repeating units of a-D-galactose, (3-D-GIcNAc- and D-glycero-D-altro-heptose or deoxy variants with and without methyl groups (Fig. 9). However, it was reported that the D-glycero-D-altro-heptose variant was not detected in the HS:23 serostrain.
Analysis of the gene products, encoded by the cps regions of serostrains HS:23 and HS:36 and of strain 81-176 (Table VI), demonstrate a potential for deoxyheptose biosynthesis due to the presence of genes hddC, gmhA2, hddA and dmhA (Fig. 6).
The latter gene homologue is suggested to be involved in conversion of heptose to deoxyheptose in Yersinia pseudotuberculosis.
HR-MAS spectra of cells and NMR spectra of the partially purified CPS from strain 81-176 demonstrated similar sugar resonances with the HS:23 and HS:36 to serostrains (Fig. 9). In all the spectra, the characteristic OMe signal at 3.5 ppm and NAc resonance at 2.05 ppm were observed. The anomeric region of the HS:23 serostrain was the simplest with two anomeric resonances in the HR-MAS
spectrum (Fig. 9a). In the iH NMR spectra of the CPS, a third anomeric resonance was observed (Fig. 9b) which was obscured in the HR-MAS spectra by the large saturated HOD peak. The anomeric region (4.7 to 5.5 ppm) for the other samples was more complex with the spectrum of strain 81-176 being the most complex.
2D-NMR experiments were done to further characterize the sugar resonances (Fig. 9). The HMQC and TOCSY spectra for the HS:23 serostrain (Fig. 9a) were the simplest with proton anomeric resonances at 5.06 ppm, 4.97 ppm and 4.77 ppm, corresponding to the Gal, Hep and GIcNAc anomeric resonances, respectively. In the HMQC spectrum, the C-6 crosspeaks of the 6-deoxy-heptose were observed at 34.8 ppm ('3C) and 2.06 and 1.71 ppm (1H).
For the HS:36 serostrain, three anomeric resonances were also observed as detected by TOCSY and HMQC experiments on the CPS (Fig. 9b). The 1H resonance at 4.92 ppm was confirmed to be a non-anomeric resonance using ~iMQC. While the anomeric carbon resonances had similar chemical shifts, the proton anomeric resonance of the heptose residue was different, probably due to different structural motifs on the heptose residue. In the TOCSY spectrum, the anomeric resonances at 4.76 ppm and 5.06 ppm exhibited connectivities that were similar to those observed for the HS:23 serostrain, indicating the presence of similar sugars in both serostrains.
In the HMQC spectrum, resonances characteristic of a 6-deoxy-heptose could not be observed, indicating that for this serostrain this modification was not predominant.

The HMQC and TOCSY spectra for strain 81-176 (Fig. 9c) showed correlation patterns similar to those observed for the HS:23 serostrain for the Gal, Hep and GIcNAc anomeric resonances, again indicating similar sugar structures to those of HS:23 and HS:36. This observation is in agreement with predictions derived from the 81-176 gene analysis. However, structural analysis of strain 81-176 also demonstrated the presence of additional resonances indicating the presence of a more complex repeating unit or the presence of another polysaccharide structure. Comparison of the NOESY spectra established that serostrains HS:23 and HS:36 and strain 81-176 exhibited similar NOE patterns for the Gal, Hep and GIcNAc residues, a result that is to consistent with the conclusions arrived at from the analysis of the TOCSY
experiments. The phosphoramide modification observed for NCTC11168 was also observed for serostrain HS:36 and strain 81-176 but not for serostrain HS:23.
The major and minor components of CPS isolated from the HS:41 serostrain were described to contain ~i-L-arabinose , 6-deoxy-(3-D-altroheptose , 6-deoxy-(3-L
altrose and ~3-D-fucose all in the furanose form (Fig. 7). NMR analysis demonstrated that the CPS of the sequenced strain used in this study is consistent with the published structure. Interestingly, sequencing results from this strain (Table VII) show three UDP-pyranose mutase glf gene homologues which are involved in pyranose to furanose ring conversions which is consistent with having three of the CPS
sugars in 2o the furanose form (note that arabinose is a pentose and therefore is naturally in the furanose configuration). The presence of genes: hddC, gmhA2, hddA and dmhA
(Fig.6) is consistent with the presence of deoxyheptose in the CPS (Fig.7).
Additional sugar dehydratases will be required for the biosynthesis of fucose and deoxyaltrose and putative homologues are observed in Table VII. Two tandem copies of the fcl gene were also found in this strain. According to the CPS structure it appears that one copy is involved in heptose biosynthesis and the other in fucose production or alternatively, they are duplicate copies that are capable of converting both substrates.
Comparative analyses of the cps regions - The derived nucleotide sequences of various biosynthetic cps regions were compared with the complete genome sequence of strain NCTC11168 using both BLASTn and tBLASTx programs. Some features are outlined below.

The cps region of serostrain HS:19 contains genes which are almost identical to genes cj1415-cj1420 of strain NCTC 11168. This region (Fig.6) is followed by gene HS19.07 with similarity in the 5' region to both gene cj1421 and cj1422.
However, no similarity between the 3' region of this gene and either the cj1421 or cj1422 genes of NCTC11168 could be found. Genes cj1423-cj1433 are not present in serostrain HS:19 and there is limited similarity to genes cj1434-cj143S, cj1437-cj1438 and cj1440-cj1442 (Fig. 6 and Table IV).
The biosynthetic region of strain G1 is the smallest (15 kb) and contains only 11 genes. Organisation of the genes from cj1415 to cj1421 in this strain is similar to that of serostrain HS:19 and strain NCTC11168. However, the remaining genes have no counterparts in the corresponding regions of these strains (Fig.6, Table V).
Genes cj141S-cj1420 are also conserved in the HS:23 serostxain, the HS:36 serostrain and strain 81-176 (Fig. 6, Table VI). However, in this case there are also a number of other conserved genes outside this region. Genes cj1423 (hddC), cj1424 (gmhA2), cj142S (hddA) and cj1427 are conserved and present in the same place as in NCTC11168, but there is almost a precise deletion of gene cj1426 (Fig. 6).
Also, a new gene (dmhA) is present between genes cj1427 and fcl. Genes cj1429 and cj1430 appear to be present (Fig. 6), but genes cj1431 and cj1440 appear replaced with two genes (81176.16 and 81176.17 as well as the corresponding ORFs in the HS:23 and HS:36 serostrains) encoding glycosyltransferases. Overall, despite gene reshuffling, the cps regions of the HS:23 and HS:36 serostrains and strain 81-176 are more similar to that of NCTC11168 than to serostrain HS:19 and strain G1.
The cps region of serostrain HS:41 is interesting in that it lacks the cj1415 cj1420 genes conserved in the other strains. However, three heptose-related genes in the middle of the cps locus of serostrain HS:41 (hddC, gmhA2 and hddA) are almost identical to those in NCTC11168, although gmhA2 and hddA are separated via insertion of gene HS41.09 encoding a putative sugar transferase with low similarity to cj1300 (Fig. 6, Table VII). The mosaic patterns of similarity and divergence indicate that these cps regions have a diverse recent ancestry, suggesting that recombination between different cps clusters has occurred.
The serostrains HS:23 and HS:36 and strain 81-176 (HS:23/36) all appeared to have the same gene content. Pair-wise alignments of the CPS biosynthetic regions (24.6 kb) of these strains showed that the HS:23 and HS:36 serostrains share 97.6%

DNA sequence identity between them while strain 81-176 shares 97.6% and 98.9%
identity with the serostrains HS:23 and HS:36, respectively. As expected from the high DNA sequence identity in this region, there is also high protein sequence identity when the individual ORFs are compared (see Table VI). All pair-wise comparisons showed above 93% protein sequence identity except for ORF HS23.08 (hddC) which shared 87.9% and 86.6% identity with the corresponding ORFs in the HS:36 serostrain and strain 81-176, respectively.
Variation in the contingency genes - The potentially phase variable cps genes of C. jejuni strains of various serotypes were investigated. The biosynthetic cps locus of C. jejuni NCTC11168 was found to contain six genes with homopolymeric G
tracts potentially prone to phase variation. It was examined whether the "ON" and "OFF"
states of these genes, if present in other strains, can also be detected. The results of this analysis, shown in Table VIII, indicate that most of the genes tested are predominantly in the "ON" state although many are demonstrated to vary. This suggests that closer examination of these genes variable modifications (for example, methyl, ethanolamine, aminoglycerol and phosphoramide) will reveal that CPS
structures can be further modulated.
Such modulation may explain the presence of variant structures in the HS:41 serostrain and in serostrains HS:23 and HS:36 compared to strain 81-I76 (HS:23/36).
The latter three strains were examined in more detail because they share the same gene content, yet produce capsules with slight differences in CPS structure (Fig. 7).
Since these three strains share >95% gene identity in their cps biosynthetic regions, phase-variable genes could be responsible for the differential expression of deoxyheptose and phosphoramide observed in this study, ie HS:23 (Gal, GIcNAc, Hep, deox he , HS:36 (Gal, GIcNAc, Hep, phosphoramide) and 81-176 (Gal, GIcNAc, Hep, deoxyhep, phosphoramide). There are six contingency genes in the cps cluster of the HS:23 and HS:36 serostrains but only five in 81-176 since the dmhA
homologue (ORF#12) is not phase-variable (Table IX). DmhA has been shown to be involved in deoxyheptose synthesis in Yersinia and interestingly, in this study, the dmhA homologue is functional in 81-176 and HS:23, but variable in HS:36. This may correspond to the detection of deoxyheptose in 81-I76 and HS:23 and the difficulty in detecting this heptose variant in HS:36. In the HS:23 serostrain, two "OFF"
genes (HS:23.07 and HS:23.20) show high sequence similarity with the putative glycosyltransferase (cj1422c) from NCTC 11168 and may play a role in adding the missing phosphoramide. However, function of these contingency genes must be proven experimentally.
In this study, CE-MS/MS and HR-MAS NMR have been used successfully to examine glycan structures from 108-101° bacterial cells. These methods can now be applied to investigate expression of glycans under different laboratory growth conditions and directly from the natural environments in which the pathogen is found.
Examination of mutants will allow the assignment of genes involved in the biosynthetic pathways of these glycans and their modifications and help to determine l0 the importance of structural phase variability in survival and pathogenesis. Due to the sensitivity and mildness of these methods, minor glycan structures that were not previously identified in the literature can also been detected.
Recently, HR-MAS NMR has been used to detect the polysaccharides of LPS
and CPS on intact bacteria. The method has also been used to detect nanomole amounts of purified LPS, purified O-linked and N linked glycopeptides, and LOS
ganglioside mimics. In this study, HR-MAS NMR was used to further examine CPS
directly from campylobacter cells. The CPS resonances could be readily identified and were in agreement with published spectra from purified CPS. In NCTC11168, the spectra clearly demonstrate the CPS anomeric protons from individual sugars and 2o modifications, allowing simple screening of potential NCTC11168 capsular mutants to determine what residues are affected. Since capsular polysaccharides are the major serodeterminant of the heat-stabile typing scheme, HR-MAS NMR also provides a quick method of determining whether strains belong to the same Penner serogroup.
HR-MAS NMR also allowed us to examine structural capsule variants from the diverse NCTC11168 population. Phase variability of campylobacter capsule structures was noted as early as 1991 by Mills et al. when several strains showed serotyping differences after in vitro laboratory passage. The authors then observed differences in antibody response with typing sera after multiple in vivo samplings of the same strain. There are an abundance of variable bacterial sugar modifications mentioned in the literature, some of which have been recently summarized. A
novel modification for C. jejuni NCTC11168 variant 2 was observed with -OP=O(NHZ)OMe on the 3-position of GaI,fNAc. The phosphoramide has not been described previously in nature and, it shows structural similarity to synthetic organophosphate insecticides.
It is generally accepted that a single microorganism can give rise to a diverse population with very different virulence properties. However, sensitive methods fox the structural analysis of bacterial populations have been limiting. As disclosed herein, CE-MS/MS can be used to examine the structure and variability in C.
jejuni LOS. HR-MAS NMR has been used to investigate CPS structure, confirm serotype, demonstrate population variability, study the effect of mutagenesis, and detect N
linked glycoprotein sugars. Campylobacter has a large repertoire of variable surface 1o glycans in addition to a conserved N linked glycan. These studies have implications in vaccine development, provide possibilities for the induction of GBS
following campylobacter enteritis, describe methods that can be adapted for the analysis of glycans from other important bacterial pathogens, expand the new field of metabolomics, and can provide more insight into the importance of bacterial LOS, capsules, and protein glycosylation allowing scientists to expand the discipline of glycomics beyond the gene complement and glycan structure.
The sequences of capsule biosynthetic loci from six C. jejuni strains were compared using a PCR amplification procedure based on the presence of highly conserved genes in this region. CPS structure prediction based on the analysis of these sequences showed a good correlation with NMR and sugar analysis and with published data. The presence of additional genes in the cps regions suggests a potential for the biosynthesis of CPSs with modified structures. There is extensive duplication of glycosyltransferase genes in these loci resulting in approximately double the number of transferases predicted by the structure.
Striking similarity between the cps regions of both the HS:23 and HS:36 serostrains and that of strain 81-176, which is of mixed HS:23/HS:36 serotype (Fig.6), suggests a common origin. Some variation in the respective CPS structures may be attributed to the presence of phase variable genes. Phase-variable expression of methyl, ethanolamine, aminoglycerol and phosphoramide groups on the CPS of strain 3o NCTC11168 has been observed.
Sequence data was used in further comparison of the cps regions with that of the C. jejuni NCTC11168 strain. Both highly conserved and variable genes were found. The biosynthetic genes that are proximal to the transport- and assembly-related kps genes were usually more conserved. The most conserved genes were the five to six genes near the kpsC gene. Interestingly, the study of Streptococcus pneumoniae cps loci also revealed a non-random variation of CPS-related genes, with the highest difference for those closest to the central region. The current data suggest that recombination events leading to variant forms of CPS of C. jejuni usually occur in the middle of this region with the exception of the heptose biosynthetic genes (Fig. 10). In addition, most biosynthetic cps genes of serostrains HS:41 and strain NCTC11168, except for three genes in the middle, were found to have very low levels of similarity.
This finding suggests that the cps regions of these strains are the most distantly related.
In addition to the mechanism of variation attributed to horizontal gene transfer, extensive intragenomic variation in the cps regions has been observed. Some genes, e.g. cj1421 and cj1422 in NCTC1I168 share long regions of identity, which may have resulted from gene duplication. In other strains only one copy of these genes is present. Other genes may have also arisen from deletions resulting in formation of hybrid genes. Such deletions/duplications may also play an important role in structural variation of CPSs. One interesting feature related to the mechanism of genetic variation was the finding of mosaic structure of some genes and their respective products. The N-terminal region of the Cj 1440 homologue from serostrain 2o HS:19 (H19.11) revealed similarity to many C. jejuni glycosyltransferases, with the first 169 as residues almost identical to the N-terminal residues of Cj 1440 protein of NCTC 11168. However, the C-terminus of HS 19. I I revealed no similarity to the corresponding region of Cj 1440, and resembled instead that of the Cj 1438 glycosyltransferase. The finding supports the possibility of intra-cistron recombinations between the genes performing a similar function (e.g. encoding glycosyltransferases), which may result in altered substrate specificity and may contribute to antigenic variation of the CPS. This is similar to the observation in the LOS biosynthesis locus of the HS:10 serostrain which contains a (3-1,4-N-acetylgalactosaminyltransferase (CgtA) and a (3-1,3-galactosyltransferase (CgtB) that 3o have diverged mostly in their C-termini when compared with the corresponding glycosyltransferases in the HS:19 serostrain {Gilbert, 2002}. Furthermore, a number of genes in the cps region have homopolymeric tracts that may also contribute to variation.

The elucidation of the high conservation of some genes in the biosynthetic cps region along with the variation of others, serves as a basis for a PCR based typing procedure, which can provide a number of advantages over a classical Penner typing scheme. The limited number of antisera available for serotyping (usually a panel of 66 antisera) used in the standard Penner typing protocol results in up to 20% of strains being untypeable. PCR amplification of the cps loci e~ allows for differentiation of these strains based on their potential of CPS production. For example, PCR
analysis allowed detection of CPS-related genes in the untypeable strain X, known to produce a CPS. An advantage of a PCR-based typing scheme based on the sequences derived from the cps regions is that it is based on the presence of the genes, rather than on their expression, which may be affected by a number of factors, including growth conditions. In addition, slight variation in the method of antigen preparation and conditions of passive hemagglutination may affect the results of typing using the classical Penner typing protocol. For example, the results of passive hemagglutination depend on the origin of erythrocytes. Therefore, a PCR-based approach based on genetic difference in the cps regions can produce a more reliable and comprehensive typing scheme.
Multi-strain comparison of C. jejuni CPS loci has revealed a high conservation in genes involved in heptose biosynthesis and those flanking the kps regions, particularly near kpsC. The findings suggest that CPS clusters are exchanged between C. jejuni and other bacteria and may in part be responsible for the structural variation observed. Other putative mechanisms of structural variation revealed here include gene duplication, deletion, recombination and contingency gene variation.
Furthermore, there still remain a remarkably large number of genes with as yet unknown function that may be involved in the biosynthesis of CPSs with modified structures. Analysis of the polysaccharides using NMR has provided novel CPS
structural information, including the demonstration that the recently identified phosphoramide modification is common to many C. jejuna CPSs, and where known, the predicted CPS structure showed good correlation with published structural data.
3o This "belt-and-braces" sequencing approach has provided an opportunity to test hypotheses formed regarding the structure of the capsular polysaccharides synthesized by the respective loci. In addition, these comparative studies form the basis for further work in the elucidation of heptose biosynthesis in C. jejuna. This study demonstrates the extensive variability of the CPS structural determinant in C. jejuni and underpins the genetic basis fox Penner serotyping. The commonality of CPS-related heptose biosynthetic pathways among bacteria and the presence of a mobile genetic element responsible for heptose biosynthesis in various strains of C. jejuni has also been described herein. Heptose and related biosynthetic pathways are useful targets, as heptose is not ordinarily found in eukaryotes. There is provided herein a method to modulate heptose biosynthesis in C jejuni comprising modulating the expression of the mobile genetic element identified and/or the activity of the amino acid sequence encoded thereby.
1o Also provided is the use of the mobile genetic element, and functional portions thexeof, in modulating heptose metabolism in bacteria, including bacteria strains which ordinarily metabolize heptose using enzymes substantially identical to those encoded by the mobile genetic element and strains which normally do not. Also provided is use of the mobile genetic element for one or more functional portions thereof in "rescuing" heptose metabolism in a cell or organism in which ordinary heptose metabolism has been compromised either deliberately (e.g. by an exogenous agent) or by alteration of the cell or organism or a predecessor. Also provided is a construct comprising the mobile genetic element and/or one or more functional portions thereof and a promoter element.
C. jejuni strains from different disease presentations and geographical locations were surveyed for the phosphoramide (Table Xa). Examination of the closely related Campylobacter coli demonstrated that the phosphoramidate t~ris modification is absent from this species (Table Xb) and also absent in other species sampled with the exception of one C. fetus isolate from a human with bacterial septicemia. Multiple colonies from selected mutants were analysed to demonstrate their potential role in phosphoramide biosynthesis in NCTC11168. Mutagenesis of cj1416c, cj1417c, cj1418c and cj1421 c resulted in loss of phosphoramide while inactivation of the second copy of cj1421 (ie. cj1422c) had no effect in (Table Xc). However, based on the comparative sequencing results described above, 3o cj1422c homologues are found in some phosphoramide producing strains without cj1421c and appear to be responsible for the addition of phosphoramide in these strains (for example the variable phosphoramide observed in the HS:23, HS:36 and HS:23/36 group). Interestingly, mutagenesis of cj1435c and cj1437c resulted in loss of capsule and yet the phosphoramide signal was still detected. These results suggested that this modification can either be added to additional structures and/or that we are detecting biosynthetic intermediates in the phosphoramide pathway.
The phosphoramidate is being added to one additional sugar by 1422 beyond those previously described herein.
Thus, additional surface structures useful in the detection and control of Campylobacter are identified by screening of cells for the presence of the phosphoramidate and identification of the sugar of attachment.
Also provided is the modified CPS structure of NCTC 11168, and the use of it, and antigenic portions of it, in the identification, and modulation of C.
jejuni.
A summary of the phosphoramide survey results is shown in Table XI
demonstrating that the phosphoramide is common to 70% of C. jejuni isolates and 0%
of C. coli isolates examined. Many strains were tested for the presence of genes cj1416c, cj1421c and cj1422c by PCR. The results summarizing the PCR probing and sequencing (which also includes cj1417c and cj1418c) are summarized in Table XIII
and the primers used for probing are listed in Table XIV.
Phosphoramide analysis demonstrated that C. jejuna, RM1221 is unable to produce phosphoramide and this is consistent with this strain lacking the genes involved in its biosynthesis (Fig. 11). Comparison of the G jejuna NCTC11168 capsule locus with other sequenced epsilon proteobacteria demonstrates that these strains also lack these genes and are likely unable to synthesize the phosphoramide (Fig. 11 ). Thus, this structure appears to be unique to C, jejuna with rare exceptions in other Campylobacter species.
It is interesting to note that the phosphoramide is added to capsules of strains with different serotypes and thus to different structures. A more thorough analysis of the capsule structures of two serostrains known to produce phosphoramide, HS:1 and HS:19 was performed. As mentioned above, the HS:l CPS structure consists of galactose and glycerol-phosphate. However, an unusual beta-fructofuranose that has not previously been described (see Table II) was detected. The beta-fructo-furanose 3o identified herein is useful in identifying certain bacteria. Conjugates with the beta-fructo-furanose are useful in vaccine production. This beta-fructo-furanose can make glycans more immunogenic. It is to this unusual sugar that the phosphoramide is being attached (Fig. 12). More convincing results were obtained with HS:19 (Fig. 13).

The phosphoramide is attached at the 4-position of GIcNAc in contrast to NCTC11168 where the phosphoramide is attached at the 3-position of GalfNAc. As mentioned above, the phosphoramide is detected in select mutants that lack the CPS.
This suggests that the phosphoramide is added to alternate structures or that intermediate forms can be detected. Occasionally additional phosphoramide signals are observed during phosphate scans of capsulated C. jejuni isolates (Fig.
14). Thus, in addition to the possibilities mentioned, phosphoramides may be attached to a varying CPS backbone which could lead to additional signals.
In order to determine whether the expression of phosphoramide on C. jejuni l0 CPS has any biological relevance, tissue culture and serum sensitivity assays were performed. Preliminary adherence and invasion assays comparing wildtype to the phosphoramide mutants demonstrated that loss of phosphoramide caused decreased adherence to CaCo-2 cells while invasion appeared unaffected (Fig. 15). To ensure that the differences in the tissue culture assays were not due to differences in motility of the mutants, motility assays were done comparing the wildtype strains with their respective mutants and demonstrated that all mutants had similar levels of motility compared to the parent (Fig. 16). Preliminary serum sensitivity assays comparing wildtype to the 1416-1 mutant indicated that expression of the phosphoramide increases G jejuni sensitivity to pooled human serum (Fig. 17). These results suggest 2o that it would be beneficial for C. jejuni to suppress phosphoramide expression while traveling to its optimal location and then expressing the phosphoramide in order to efficiently bind to host cells.
The presence of the identified phosphoramide across most serotypes of C.
jejuni makes it a useful target for attack on these cells, as well as for the identification of these cells.
Molecules having a good binding affinity for this phosphoramide ("binders") can be employed, either alone or as conjugates or on the surface of liposomes or other suitable cargo carriers or matricies to bind to C. jejuni cells. Where such molecules are functionally associated with a toxin or similar substance, they may be used to reduce C. jejuni viability or proliferation on a surface or in a solution, fluid, or semifluid of concern. Similarly, when such molecules are associated with a marker, such as an enzyme able to catalyze a colourometric reaction, or a fluorescent or radioactive marker, they can be used to identify and/or localize C. jejuni.
One example of suitable binders for this purpose are antibodies having specificity for the phosphoramide. Such antibodies may be single domain antibodies. In some cases only fragments of antibodies having the desired specificity will be employed. Such fragments may be expressed as part of a fusion protein with a "cargo"
polypeptide of interest.
Another example of suitable binders are bacteriophages or portions thereof having a good affinity for the phosphoramide. In some instances the phage particles or portions will also be capable of lysing the C. jejuni cells. Parts of interest can include any functional part. In some instances phagetail sheaths and/or tail spike proteins may to be employed.
Other binder molecules can be identified by screening of materials for specific binding to the phosphoramide. Thus, in one embodiment the invention provides a use of the phosphoramide in identifying compounds, or materials useful in identifying or reducing the viability of C. jejuni.
In some instances binders which recognize the phosphoramide regardless of its sugar of attachment will be desired.
In some instances binders which recognize the phosphoramide in association with one or more particular sugars of attachment will be desired.
Binders which specifically recognize the phosphoramide structure unique to 2o Campylobacter and no other phosphate compounds produced in nature are considered to be_particularly useful binders.
In an embodiment of the invention there is provided a method of modulating the adhesion of C. jejuni cells to a surface, comprising modulating the concentration of binders in the surrounding fluid. It will be understood that the surface may include a non-living material, cells, and/or cell-derived materials.

Table I
Chemical shiftsa (ppm) for the G jejuni NCTC11168 variant 2 CPS
Unit 1 2 3 4 5 6 7 8 A ~ 106.2 81.2 70.7 84.0 63.0 $H 5.35 4.21 4.30 4.13 3.89, 3.70 B ~ 99.0 73.1 73.8 76.4 72.7 171.3' 53.9 61.3b $H 5.14 3.94 4.10 3.93 4.38 8.32' 4.03 3.72, 3.676 C ~ 106.4 62.5 79.6 82.2 78.3 61.9 174.9' 22.9 $H 5.13 4.27 4.88 4.48 3.97 3.82, 3.77 8.31' 2.05 D $~ 98.1 72.2 73.8 70.2 72.3 79.5 63.1 60.4 8n 5.61 3.53 3.72 3.56 4.08 3.80 3.86 3.55 E $~ 54.8 SH 3.75 a Measured at MHz ( H) in DZO
at (~
0.2 ppm error for 8o and 0.02 ppm for 8H).

Internal t at 8H 2.225 ppm and 8~
acetone 31.07 ppm.

resonance se b (CH2)2 of glycerol (8B
and 9B).

' C=ONH.8~ from HMBC
at 25C. 8H from rH spectrum in 90% H20 10%D20 at 25C.

Table. II. Biosynthetic cps regions of various strains of C. jejuni.
Strain SerotypeSize Number ContingencySugar-phosphatePutative Number of of genesgenes nucleotidyl-glycosyl- different sugar transferasestransferasesresidues found*

NCTC11168 HS:2 3418028 6 2 8 4 NCTC 12517HS:19 1672713 2 1 4 2"

Gl HS:l 1518011 2 2 2 2#

8I-176 HS:23/362462521 5 2 7 6 CCUG 10954HS:23 2462721 6 2 7 6 ATCC 43456HS:36 2462521 6 2 7 6 176.83 HS:41 3411830 5 2 8 4 TAccording to structural analysis Additional uncharacterised labile substituent is present N

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Table XIV The sequences of primers used for the identification and characterization of Cj1416c, Cj1421 c and Cj1422c. The chromosomal location represents the location of the primer on the forward strand in the genome of NCTC 11168.
Gene Primer Name Sequence Chromosomal Location C' 1416cC' 1416cR1 ATGAATGCAATTATCTTAGCAGCAG 1348851 - 1348876 Cj 1416cCj 1416cF758TTTTCAATTTGCACCTCTAAATCAC 1349585 - 1349610 Cj1421c C'1421cR7 AACCCAAATTCAGCCATAGAAAGAG 1356023 - 1356047 C'1421c C'1421cF1839CTAAATATCACCATCCAACTCCTTG 1354215 - 1354239 Cj1421c 1421 s ec TTGCCTAATACTTTGGAAGG 1354907 - 1354926 C' 1421 1421 s ec AAAAACCCACTGAACTATCC 1354521 - 1354540 c B 1 Cj 1421c1421-R AAAGGAAAAACCCATGCTCA 1356066 - 1356047 C'1421c 1421-F TGGGTATTTAAGTTGGGGAAA 1355853 - 1355873 C'1421c 1421LR GACCCATCATGGTATGGCAATC 1355121 - 1355142 Cj1421/221421-22specB4AACCTGTATTGATTTTGCTTAGAGATCC 1356997 - 1357024 C' 1421/221421/22s TCCATAAAAACGATTGCGGC 1354880 - 1354899 ecF2 Cj 1422cCj 1422cR7 AACCCAAATTCAGCCATAGAAAGAG 1357956 - 1357980 C' 1422cC' 1422cF1869AGAAAATGATAAAGATGGGTTTTGA 1356117 - 1356141 Cj 1422c1422 spec GGATTTTTTTTGCATACAAGTGGAG 1357226 - 1357250 C'1422c 1422 s ec GAGCTTTGATACTTTCAAGAGATGG 1356806 - 1356830 Cj 1422c1422-R TTGGTGTGCCTGAGGATTATT 1358059 - 1358079 C' 1422c1422-F TGGGTATTTAAGTTGGGGAAA 1357785 - 1357805 C' 1422c1422LR GAGAGATTTTTAAAAGAAGTTGGTG 1357191 - 1357215 C' 1422c1422LF CTGGGGAAGAAAAATTCAGTAT'TTTTG 1356639 - 1356665 Table XV
Part A

cj1421c:

S MLNPNSAIERVKNHLAYKLGQTVIEHRHNGGGYIALFKKLYKIKKQHKKEQ

KIYQQIIQV

FPQLKYPSLETCSDYNEALRCKFHLSYMIGEVLIKAYQNWYKGGGFKLKNN

IKKANKEFQ

IFREILKEFKELNGETLKAIQDNKQLFLKEFPRIKNILKTHQDYQPILDNI

FHNFNYFIK

NFDLIEEWLLSDDFKEKYKKENHPYPSLLDPKKLNDENEKINYHNIPAELA

WKMNLPLPP

NYEFMWFFSHGAGAFTLGQFFYHLFKINILDYFCGGDGDIRYYKFYNKLLE

LKDKRNIIT

INDIDPSWYGNQHKRDKLFSSFQKITPILFQIRDPIELIKHAYGRKWGNNL

AKTKEFDLS

YQFNDIITEVEVYNYNLPNTLEGQRPQSFLWKSLIECFDKFNDCFYLDISK

IRGEETIHT

LNYLSNKFNLKQIKINDKEFVTKSYFKGNLYFLLPLTLYLNKEDLNTNIPN

KKINKNNSL

IININFFQNDNNLFNLYSELSILDMDSSVGFYIDKQDYNKLKNDSIFYKQV

IDYLRNFAY

ELKNRIQIEEDLMLKVEDVLRHLYNNKNARVSAKNILDEELVYIKQHRPDI

VASWKYYQE

FEKMCKELDGDI

Part B
cj 1422c:
MLNPNSAIERVKNHLAYKLGQTVIEHRHNGGGYIALFKKLYKIKKQHKKEQ

KIYQQIIQV

FPQLKYPSLETCSDYNEALRCKFHLSYMIGEVLIKAYQNWYKGGGFKLKNN

IKKANKEFQ

IFREILKEFKELNGETLKAIQDNKQLFLKEFPRIKNILKTHQDYQPILDNI

FHNFNYFIK

NFDLIEEWLLSDDFKEKYKKENHPYPSLLDPKKLNDENEKINYHNIPAELA

WKMNLPLPP

NYEFVGFFLHTSGEKAMERFLKEVGVVLIGAFGYEDGKRYISIFNFLISEA

CACNDLKFA

IGILDVNCQKYDKFCFLLQNKPVLILLRDPIDSLKSFINVRHQKNGFNEIL

KIDINNTDF

DKINDRIVYVHESNGCFNPDTNQKFPSLESIKALSDTNHWMLMYNIRRNKT

IEFFRFNKI

IKVNNKVKI

FVSNRFSVKNIQIMENCIDITDKFKEIFHENLIIFCSKDHFDSLINNQTLY

NVVLEYINK

FLISLKKRINVEKNKEVKVDDVLDYFKKNISVAKSYKDILDEELVYIKQHR

PDIVASWKY

YQEFERMCKELDENNQNPSLSFSNQ

Part B: IDENTIFICATION OF NOVEL O-METHYL PHOSPHORAMIDATE
STRUCTURES AND USES THEREOF
There is disclosed herein O-methyl phosphoramidate structures that are attached to various capsular polysaccharide sugars in different linkages that can be used in the identification and targeting of C. jejuni.
Campylobacter jejuna is the major bacterial cause of gastrointestinal disease in developed countries and infection can lead to the development of the neuropathy known as Guillain-Barre syndrome.
Genome sequencing of C. jejuni NCTC11168 demonstrated that the strain contained four gene clusters necessary for carbohydrate biosynthesis including the capsular biosynthesis locus, containing a Kps transport system similar to that found in other encapsulated organisms. This gene cluster also encodes enzymes involved in the biosynthesis and transfer of a branched tetrasaccharide repeat to the outer membrane surface of C. jejuni. Recently, the CPS structure of NCTC11168 was determined to contain 6-O-methyl-D-glycero-a-L-gluco-heptose, j3-D-glucuronic acid modified with 2-amino-2-deoxyglycerol, (3-D-GalfNAc and (3-D-ribose.
Early structural studies of C. jejuni CPS showed that these molecules are highly heterogeneous from one strain to another. Microarray hybridisation analysis also demonstrated some differences in the CPS-related genes between strains of various serotypes. In addition, the biosynthetic region of the cps loci is prone to phase variation due to the presence of multiple genes with homopolymeric tracts. It was subsequently shown that CPS structures from a single isolate (NCTC11168) can vary in structure. However, the genetic mechanisms underlying the structural heterogeneity and antigenic variation remained unknown.
Since capsular polysaccharides are the outermost structure on the bacterial cell they play an important role in the interaction between the pathogen, host, and environment. In C, jejuni 81-176, the capsule is involved in INT407 cell invasion, virulence in ferrets, serum resistance and maintenance of bacterial cell surface charge.
In C. jejuni NCTC11168, the capsule is necessary for colonization of poultry, the primary source of C. jejuni infection. The C. jejuni, CPS structures are also highly variable, a fact reflected by the increasing number of serostrains described for this species of Campylobacter which is currently greater than 60, each with a unique CPS
structure [Karlyshev et al. 2004]. Moreover, for each serogroup it is possible to have phase-variable changes such as expression of the methyl, ethanolamine and aminoglycerol modifications reported for C. jejuni NCTC 11168 CPS [Szymanski et a1.2003].
In a recent study, the CPS biosynthetic regions of C. jejuni strains of serotype:
HS:41, HS:23/26, HS:36, HS:23, HS:19 and HS:1 were sequenced [Karlyshev et al.
2004]. Comparison of the determined cps sequences of the HS:19, HS:41 and HS:l serostrains with the sequenced NCTC11168 strain provided evidence for multiple mechanisms of CPS structural variation in C. jejuni including exchange of capsular genes and entire clusters by horizontal transfer, gene duplication, deletion, fusion and contingency gene variation [Karlyshev et al. 2004]. In contrast to the NCTC11168 and HS:19 serostrain, the biosynthetic region of strain HS:1 was the smallest and was shown to contain eleven genes. Unlike these other strains, the cps locus of HS:l does not encode homologues of UDP-glucose 6-dehydrogenase or the heptose pathway.
Based on these observations, it was inferred that HS:1 likely does not have the ability to make glucuronic acid or heptose [Karlyshev et al. 2004]. Of importance, the cps locus of HS:1 was shown to contain a potential tagD homologue encoding a glycerol-3-phosphate cytidylyltransferase necessary for the biosynthesis of CDP-glycerol.
Moreover, it was shown that HS:l encodes a TagF homologue responsible for transferring glycerol-phosphate residues from CDP-glycerol. From the genetic analyses of HS:1, it was concluded that the repeating unit of its CPS may contain glycerol-phosphate residues [Karlyshev et al. 2004].
Disclosed herein are the results of an investigation of the structure of CPS
for C.
jejuni serostrains HS:l and HS:19. Initially, CPS was extracted from bacterial cells using a traditional hot water/phenol method [Westphal & Jann 1965]. However, due to the high degree of structural heterogeneity observed in CPS samples isolated using this method, a novel, more sensitive enzymatic CPS extraction method designed to preserve the labile side chains of HS:1 CPS was developed. High resolution NMR
at 600 MHz with an ultra-sensitive cryogenically-cooled probe was then used to elucidate the structure of hot water/phenol and enzyme-purified CPS.
Concurrently, high resolution-magic angle spinning NMR (HR-MAS) at 500 MHz was used to examine the molecular structure of CPS on the surface of bacterial cells.
Finally, CE-MS operating at high orifice voltage was used to analyze the structure of purified HS:1 CPS and corroborate NMR findings. In this study, we present the advantages of this sensitive enzyme CPS isolation method and HR-MAS for examining bacterial CPS, s report the structures of C. jejuni HS:1 and HS:19 serostrain CPS.
The elucidation of the complete structures for C. jejuni capsular polysaccharides demonstrated the commonality of the O-methyl phosphoramidate and the exact location and linkage for attachment. This information identified structures that are useful in the identification and targeting of C. jejuni thereby providing means l0 for diagnosing and limiting this problematic pathogen.
There is disclosed herein O-methyl phosphoramidate structures found on the surface of a majority of Campylobacter jejuni isolates and uses thereof.
C. jejuni produces a capsular polysaccharide (CPS) that is the major antigenic 15 component of the classical Penner serotyping system. High resolution magic angle spinning (HR-MAS) NMR was used to examine capsular polysaccharides directly from campylobacter cells and showed profiles similar to those observed for purified polysaccharides analysed by solution NMR. This method also exhibited the potential for campylobacter serotyping, mutant verification, and preliminary sugar analysis.
2o HR-MAS NMR examination of growth from individual colonies of C. jejuni NCTC11168 indicated that the capsular glycan modifications are also phase variable.
These variants show different staining patterns on deoxycholate-PAGE and reactivity with immune sera. One of the identified modifications, that showed both reduced reactivity with silver staining and rabbit sera, was a novel -OP=O(NHZ)OMe (O-25 methyl phosphoramidate). This modification was attached to the 3-position of the CPS
Galf NAc.
Biosynthetic cps regions were sequenced, ranging in size from 15 to 34 kb, from C. jejuni strains of HS:1, HS:19, HS:23, HS:36, HS:23/36 and HS:41 serotypes and compared with the sequenced strain, NCTC11168 (HS:2). Extensive structural 30 studies, including HR-MAS NMR, demonstrated polysaccharide heterogeneity in campylobacter CPS and demonstrated the presence of additional CPS
modifications and the commonality of the recently described O-methyl phosphoramidate.

Development of a novel HRMAS filtering method has allowed investigation of multiple isolates of C. jejuni from various clinical presentations and geographical locations and revealed that the O-methyl phosphoramidate is common to approximately 70% of all strains examined (see Part A). This modification appears generally specific to C. jejuni and was not observed in the closely related Campylobacter coli. Structural analysis of the HS:1 and HS:19 strains demonstrated that the O-methyl phosphoramidate can be attached to different sugars in different linkages. Multiple O-methyl phosphoramidate signals are observed during HRMAS
analysis indicating that the modification is attached to vaxying capsule backbones, l0 attached to alternate structures and/or being detected as biosynthetic intermediates.
In this study, the structure of C. jejuni serostrains HS:1 and HS:19 CPS were investigated. CPS was extracted using a conventional hot water/phenol method and a more sensitive enzymatic isolation method developed to preserve the labile side chains of C. jejuni HS:1 CPS. NMR at 600 MHz equipped with an ultra-sensitive, cryogenically-cooled probe and CE-MS operating at high orifice voltage were used to determine the structure of CPS isolated from bacterial cells. The molecular structure of cell-bound CPS was then examined in vivo using HR-MAS NMR on the surface of whole HS:l bacterial cells. Analysis of enzyme-purified and cell-bound CPS
revealed the structure of C. jejuni HS:1 CPS to be [-4)-a-D-Gal-(1-2)-(R)-Gro-(1-P]n with labile a-D-Gal-3-2-(3-fructofuranose and a-D-Gal-2-2-~3-fructofuranose branches containing a common O-methyl phosphoramidate modification (OMePN) on position 3 of (3-fructofuranose (where n is greater than or equal to 1). In contrast, the structure of hot water/phenol-purified CPS was highly heterogeneous due to the loss of this labile keto sugar side chain and phosphoramidate during extraction.
Collectively, the results of this study highlighted the advantages of this sensitive enzymatic isolation method and HR-MAS NMR for determining the structure of bacterial CPS and demonstrated that the backbone structure of HS:1 CPS very closely reflects its sequenced cps loci.
Preliminary examination of select C. jejuni mutants in human cell culture assays has demonstrated that the O-methyl phosphoramidate is required for efficient adherence, but is not necessary for cell invasion. Furthermore, the expression of this modification renders the bacteria more sensitive to human sera (see Part A) demonstrating the biological relevance of this modification.
BRIEF DESCRIPTION OF THE FIGURES OF PART B
Figure 18. Determined structures for the defructosylated repeating unit (CPS-1) and complete CPS structure (CPS-2) of the C. jejuni HS:1 serostrain. For CPS-2, the repeating unit is [-4)-a-D-Galp-(1-2)-(R)-Gro-(1-P-]" with MeOPN-3-~-D-fructofuranose branches at 1o C-2 and C-3 of Gal. Structural heterogeneity is due to variable phosphoramidate groups on non-stoichiometric fructose branches. Residue A is oc-D-Galp, oc-D-galactopyranose, residue B is GroP, glycerol-phosphate, residue C is ~i-D-Fruf, (3-D-fructofuranose;
and MeOPN is O-methyl phosphoramidate, CH30P(O)(NH2)(OR).
Figure 19. NMR analysis of purified and cell-bound 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 'H NMR spectrum (10 °C) of cell-bound CPS. N linked glycan anomeric resonances are indicated with astexisks. (e) 1D-NOESY spectrum (400 ms) of Gal 2o 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 =10 Hz) for cell-bound CPS. (i) HR-MAS 31P
HSQC
spectrum (23 °C, 512 transients, 64 increments, IJp,H =10 Hz) for cell-bound CPS. For the selective 1D experiments, excited resonances are underlined.

Figure 20. 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-(60 ms). (e) 31P HSQC with 1JP,H =10 Hz, 64 transients and 240 increments. (f) with IJc,H 140 Hz, 8 transients and 256 increments. For the selective 1D
experiments, excited resonances are underlined. Ff and Fp represent the fructofuranose and fructopyranose monosaccharides, respectively.
1o Figure 21. 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) HSQC with 1JP,H = 20 Hz, 8 transients and 32 increments. (h) 13C HSQC with IJc,H = 150 ~5 Hz, 80 transients and 256 increments. For the selective 1D experiments, excited resonances are underlined Residue C represents Fru represents with MeOPN present and residue *C, Fru with no MeOPN.
Figure 22. Mass spectrometry analysis of C. jejuni HS:1 CPS. (a) CE-ESI-MS
analysis of an 2o 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 25 100-800.

Figure 23. Is a proposed structure for G jejuni HS:19 CPS containing a labile sorbofuranose side chain and an O-methyl phosphoramidate modification located on H-4 of GlcNAc.
Figure 24. Is the NMR analysis of a sample of hot water/phenol purified C.
jejuni HS:19 CPS. 1H NMR analysis of hot water/phenol purified CPS (A). 31P HMQC
analysis showing the attachment of the O-methyl phosphoramidate modification on H-4 of GIcNAc (B). 31P HMQC-TOCSY analysis of HS:19 CPS showing attachment of the O-methyl phosphoramidate modification on H-4 of GIcNAc and the TOCSY
correlation between H-4 and H-3!H-5 of GIcNAc (C). 13C HMQC analysis showing proton/carbon correlations for Glc, GIcNAc, the O-methyl phosphoramidate modification and sorbose for purified CPS (D).
EXAMPLES
While the invention is discussed with respect to particular examples and embodiments, it will be readily understood that it is not so limited, but in fact includes all variants and alternative embodiments thereof. While possible mechanisms and/or modes of action may be discussed, it will be understood that the invention is not so limited.

Solvents and reagents Unless otherwise stated, all solvents and reagents were purchased from Sigma Biochemicals and Reagents (Oakville Ont. Canada .
Media and growth conditions C. jejuni serostrains HS:1 and HS:19 were routinely maintained on Mueller Hinton agar (Difco, Kansas City M0, USA) plates under microaerophilic conditions (10% COZ, 5% OZ, 85% NZ) at 37°C. For the purposes of CPS extraction, 6 L of each strain was grown in four 1.5 L shaker flasks, inoculated with four plates of each, in 1o brain heart infusion broth (BHI) (Difco, Kansas City MO) under microaerophilic conditions at 37°C for 24 h while being agitated (100 rpm). Bacterial cells were then harvested from BHI broth by centrifugation (8K rpm for 20 min), the supernatant was discarded, and the bacterial pellet was placed in 70% ethanol. The ethanol solution was then roto-yapped at 37°C to remove ethanol, flash frozen in an acetone/dry ice bath, lyophilized and stored at -20°C until extraction.
Hot water/phenol isolation of CPS
Bacterial CPS was first extracted using the hot water/phenol extraction method according to Westphal and Jann [1965]. Briefly, lyophilized bacterial cells harvested from 6 L of BHI broth (6 g wet pellet mass) were placed in a stainless steel bender cup with 150 ml of 90% phenol, heated to 96°C with a water bath and blended vigorously for 15 min. Blended bacterial cells were allowed to cool for 30 min before being placed in cellulose dialysis tubing (molecular weight cutoff 12K Da, Sigma Oakville Ont.) and dialyzed against running water for 72 hr to remove phenol. Dialyzed, blended cells were then roto-yapped at 37°C to reduce the volume of water to approximately 100 ml, which was then ultracentrifuged (45K rpm, 15°C) for 2 hr. The pellet was discarded and the supernatant, which contained CPS, was flash frozen in an acetone/dry ice bath and lyophilized.
The powdery bacterial extract was re-suspended in 5 ml of HZO and CPS was 3o purified with a Sephadex ° superfine G-50 column (Sigma, Oakville Ont.) equipped with a Waters differential refractometer (model 8403, Waters, Mississauga, Ont.) using H20:pyridine:HOAc (250:1:2.5) as mobile phase (5 ml/min). 1H NMR at 400 MHz (Varian, Palo Alto CA) was then used to screen fractions and those found to contain CPS were combined, flash frozen in an acetone/dry ice bath and lyophilized.
Lyophilized bacterial CPS was then re-suspended in 2 ml of H20 and purified using a Gilson liquid chromatograph (model 306 and 302 pumps, 811 dynamic mixer, 802B
maometric module, Gilson, Middleton WI) with a Gilson UV detector at 220 nm (model UV/Vis-151 detector, Gilson, Middleton WI) equipped with a tandem QHP
HiTrapTM ion exchange column (Amersham Biosciences, Piscataway NJ). An H20/H20-NaCI (1M) gradient was used as mobile phase (3 ml/min: 0 min 100% H20, 24 min 71% H20, 35 min 48% H20). Fractions containing CPS were combined, flash frozen in an acetone/dry ice bath and lyophilized. Purified bacterial CPS was then de salted using a SephadeR superfine G-15 desalting column (Sigma, Oakville Ont.) using H20:pyridine:HOAc (250:1:2.5, 5 mUmin) as mobile phase. 1H NMR at 400 MHz (Varian, Palo Alto CA) was then used to screen the fractions, those found to contain CPS were combined, flash frozen in an acetone/dry ice bath, lyophilized and stored at -20°C.
Enzymatic isolation of CPS
Because of the structural heterogeneity observed in hot water/phenol-purified CPS, a more sensitive method of CPS was required. Accordingly, a novel method was developed and is based on the methodologies of Darveau & Hancock [1983], Huebner et al. [1999] and Hsieh at al. [2003]. Briefly, bacterial cells harvested from

6 L of BHI broth (6 g wet pellet mass) were suspended in 30 ml of phosphate buffered saline (PBS) (pH 7.4). Thirty milligrams of lysozyme (approx. 1 mg lysozyme/ml, Sigma, Oakville Ont.) and 2000 units of mutanolysin (approx. 10 000 U/ml, Sigma, Oakville Ont.) were added to the bacterial cell suspension, which was then incubated at 37°C
and agitated (100 rpm) for 24 hr. The cell suspension was then passed twice through an emulsiflex and to this was added 4 mg of DNAse I and 4 mg of RNAse (50 wg/ml DNAse I and 100 ~,g/ml RNAse, Sigma, Oakville Ont.) prior to being incubated for 4 hr at 37°C while being agitated at 100 rpm. Following digestion with nucleases, 6 mg of pronase and 6 mg of protease was added (100 g,g/ml pronase and protease, Sigma, Oakville Ont.) to the cell suspension which was then incubated at 37°C
overnight while being agitated (100 rpm). The digested cell suspension was then dialyzed against running water for 72 hr (cellulose dialysis tubing, molecular weight cutoff 12K Da, Sigma Oakville Ont.). Following dialysis, the cell extract was roto-yapped at 37°C to reduce the volume of water to 100 ml prior to being ultracentrifuged for 2 hr (45 K
rpm, 15°C). The pellet was discarded and the supernatant, containing crude CPS
extract, was lyophilized. Pure CPS was then obtained from the crude extract using the same procedure described above.
Sugar composition analysis The sugar composition of C. jejuni HS:1 and HS:19 CPS was determined using l0 the alditol acetate method according to Sawardeker et ad. [1965]. Briefly, 1 mg of pure CPS was hydrolyzed by adding 0.5 ml of 3M trifluoroacetic acid and heating at 100°C
for 2 hr. Hydrolyzed CPS was then dried under a nitrogen stream at room temperature prior to being reduced through the addition of 300 p,l H20 and 5 mg of NaBH4.
The reduction reaction was allowed to proceed for 1 hr at room temperature before being 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 X 1 ml), with a drying step using a nitrogen stream at room temperature between each addition of MeOH. Acetic anhydride (0.5 ml) was then added to the reduced sugars, which were then heated at 85°C for 30 min prior to being dried at room temperature under a nitrogen stream. Alditol acetate derived CPS sugars were suspended in 1.5 ml of CH2C12 and analyzed using an Agilent 6850 series GC
system, equipped with an Agilent 19091L-433E 50% phenyl siloxane capillary column (30 m X 250 ~tm X 0.25 pxn) (170°C to 250°C, 2.8°C ~miri 1) (Agilent Technologies, Palo Alto CA). Authentic pure standards for common keto- and aldo-sugars were purchased (Sigma, Oakville Ont.) and their alditol acetate derivatives were prepared using the same protocol outlined above. The sugar 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.
Determination of absolute configuration Absolute configuration (D or L) of HS:l and HS:19 CPS sugars were assigned by characterization of their butyl glycosides in GC according to Loetein et al.
[1978].

Approximately 1 mg of pure CPS was placed in 300 p,l of R- and S-butanol (Sigma, Oakville Ont.) with 30 pl of acetyl chloride. The mixture was then heated at 85°C for 3 hr prior to being dried under a nitrogen stream at room temperature. After the addition of acetic anhydride (500 ~,l) and pyridine (500 pl), the solution was heated at 85°C for 3 hr before being dried a second time under a nitrogen stream at room temperature. R-and S-butyl glycosides were then placed in 1.5 ml of CH2Clz and were analyzed using an Agilent 6850 series GC system, equipped with an Agilent 19091L-433E 50%
phenyl siloxane capillary column (30 m X 250 N,m X 0.25 um) (170°C to 250°C, 2.8°C ~miri 1) (Agilent Technologies, Palo Alto CA). Authentic pure standards for l0 common D- and L-aldo sugars were purchased (Sigma, Oakville Ont.) and their R- and S-butyl glycosides were prepared using the same method. The absolute configuration of HS:1 CPS sugars were then unambiguously determined by comparing the retention times of R- and S-butyl CPS sugars with those of the pure standards of known configuration.
High resolution NMR spectroscopy Two milligrams of pure, lyophilized CPS was suspended in 150 ~.1 of 98% DZO
buffered with NH4HC03 (54 mM, pD 8.6), placed into a 3 mm NMR tube (Wilmad, Buena NJ) and analyzed by NMR. At the same time, 2 mg of pure, lyophilized CPS
was suspended in 150 p.l of 98% D20 with no buffer (pH 2.2), and was allowed to degrade over a period of two weeks in order to remove labile side chains and analyze the unsubstituted CPS backbone. For both buffered and unbuffered samples, proton, i3C HSQC, HMBC and selective one-dimensional TOCSY, NOESY and NOESY-TOCSY NMR experiments were performed at 600 MHz (rH) using a Varian 5 mm, Z-gradient triple resonance cryogenically-cooled probe (Varian, Palo Alto CA).
The methyl resonance of acetone was used as an internal reference at 8H 2.225 ppm and 8~
31.07 ppm. 31P HMQC experiments were acquired using a Varian Inova 500 MHz spectrometer equipped with a Varian Z-gradient 3 mm triple resonance (1H, 13C, 3iP) probe (Varian, Palo Alto CA). External 85% phosphoric acid was used a reference (8P
0 ppm). Experiments were performed at 25°C with suppression of the deuterated H20 (HDO) resonance at 4.78 ppm. Standard homo- and heteronuclear correlated two-dimensional pulse sequences from Varian for COSY, HSQC, HMBC, and 31P HMQC
~o experiments were used for general assignments. Selective one-dimensional TOCSY
experiments with a Z-filter, and one-dimensional NOESY experiments were performed fox complete residue assignment. Mixing times of 50-80 ms were used for one-dimensional TOCSY experiments, and 800 ms for the one-dimensional NOESY
experiments [Uhrin & Brisson 2000].
HR-MAS NMR spectroscopy For HR-MAS analysis, cells were prepared as according to Szymanski et al.
(2003]. Briefly, G jejuni HS:1 or HS:19 overnight growth from one agar plate ~ (Mueller Hinton, Difco, Kansas City MO) was harvested and placed in 1 ml of 10 mM
potassium-buffered saline (pH 7) prepared with 98% D20 containing 10% sodium azide (w/v) for 1 hr at room temperature to kill cells. Cells were then pelleted by centrifugation (9800 rpm for 2 min) and washed once with 10 mM potassium-buffered saline in D20. The pellet was then resuspended in 20 ~,l of D20, and 10 p,l of 1 % TSP
was added as internal standard (0 ppm). Approximately 40 p,l of cell suspension was then loaded into a 40 ~.l nano NMR tube (Varian, Palo Alto CA) using a long tipped pipette cut diagonally approximately 1 cm from the end.
HR-MAS experiments were performed using a Varian Inova 500 MHz spectrometer equipped with a Varian 4 mm indirect detection gradient nano-NMR
probe with a broadband decoupling coil (Varian, Palo Alto CA) as previously described [St. Michael et al. 2002, Young et al. 2002, Szymanski et al. 2003].
Spectra from 40 ~.1 samples were spun at 3 kHz and recorded in ambient temperature (21 °C).
Experiments were performed with suppression of the HOD signal at 4.8 ppm. 1H
NMR
spectra of bacterial cells were acquired using the Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence (90-(i-180-~)n acquisition) (Meiboom & Gill 1958) to remove broad lines arising from lipids and solid-like materials. The total duration of the CPMG pulse (n*2 i) was 10 ms with ~ set to (1/MAS spin rate). The proton spectrum of the cell-bound CPS on bacterial cells was obtained using 256 transients (50 min). The proton decoupled 31P HMQC experiment was acquired using 512 transients and 64 increments (33 hr), and the NOESY experiment was acquired using 16 transients and 256 increments (3 hr).
7t Mass spectrometry analysis (CE-ESI-MS and CE-ESI-MS/MS) Mass spectroscopy (CE-ESI-MS and CE-ESI-MS/MS) analyses were performed as previously described [St. Michael et al. 2002). Briefly, a crystal Model 310 capillary electrophoresis (CE) instrument (AYI Unicam, Boston MA) was coupled to an API 3000 mass spectrometer (Perkin-Elmer/Sciex, Woodbridge Ont.) via a microion-spray interface. A sheath solution (isopropanol/methanol, 2:1) was delivered at a flow rate of 1 pl-min r to a low dead volume tee (250 ~.m internal diameter, Chromatographic Specialties, Brockville Ont.). All aqueous solutions were filtered through a 0.45 p.m filter (Millipore, Bedford MA) before use. An electrospray stainless steel needle (27 gauge) was butted against the low dead volume tee and enabled the delivery of the sheath solution to the end of the capillary column.
Separations were achieved on approximately 90 cm of bare fused-silica capillary (192 ~m outside diameter x 50 pm i.d., Polymicro Technologies, Phoenix AZ) using 10 rnM
ammonium acetate/ammonium hydroxide in deionized water, pH 9.0, containing 5%
methanol. A voltage of 20 kV was typically applied at the injection. The outlet of the capillary. was tapered to c. 15 N,m internal diameter using a laser puller (Sutter Instruments, Novato CA). Mass spectra were acquired with dwell times of 3.0 ms per step of 1 m/z 1 unit in full-mass scan mode. MS/MS data were acquired with dwell times of 1.0 ms per step of 1 m/z 1 unit. Fragment ions formed by collision activation of selected precursor ions with nitrogen in the RF-only quadrupole collision cell, were mass-analyzed by scanning the third quadrupole.
RESULTS
I) Examination of CPS from whole cells by HR-MAS NMR
The capsular polysaccharide structure of the genome sequenced strain, (HS:2) was described. The proton spectrum obtained from HR-MAS of suspended NCTC11168 bacterial cells closely resembled the spectrum of the purified capsular polysaccharide and clearly demonstrated the N acetyl, O-methyl, and anomeric resonances. The HR-MAS NMR spectrum was obtained in a few minutes directly 3o from 40 ul of whole cells. Hence, this method permitted quick screening of campylobacter CPS directly from one plate of growth 0101° cells), but was sensitive enough to detect a 1/100 dilution of the suspension containing 8x10 cells.

II) Campylobacter jejuni HS:1 CPS (please see updated results section in Part D
Section 1) III) Campylobacter jejuni HS:19 CPS
Based on the results of high resolution NMR experiments, CE-ESI/MS and CE
ESIMS/MS, the molecular structure for G jejuni HS:19 CPS is proposed to be [-4)-(3 D-GlcA6(NGro)-(1-3)-(3-D-GIcNAc-(1-]n containing a labile sorbofuranose side branch on position 3 of GIcA(NGro), and a rare O-methyl phosphoramidate modification on position 4 of GIcNAc (Fig. 23). To determine the structure of HS:19 CPS, intact CPS
to structure was examined.
High resolution 1H NMR analysis of hot water/phenol purified HS:19 CPS
revealed two anomeric signals at 4.65 and 4.62 ppm corresponding to GIcA and GIcNAc, respectively (Fig. 24A) (Table XVII). 1D-31P HMQC analysis of HS:19 CPS
showed strong correlation between the methyl group of the OMePN modification and H-4 of GIcNAc and provided direct evidence indicating that the OMePN is attached to position 4 of GIcNAc for HS:19 CPS (Fig. 24B). 3IP HMQC-TOCSY analysis of HS:19 CPS confirmed the attachment of the OMePN on H-4 of GIcNAc, and also revealed chemical shift data for GIcNAc as TOCSY correlations were observed for H-4, and H-3 / H-5 of GIcNAc (Fig. 24C). Finally, a ~ 3C HMQC experiments was used to 2o obtain carbon chemical shift data for the Glc, GIcNAc/OMePN and fructose sugars comprising HS:19 CPS (Fig. 24D).
The results of CE-ESI/MS and CE-ESIMS/MS analysis corroborated NMR
findings, and confirmed the proposed structure of hot water/phenol purified C.
jejuni HS:19 CPS (Fig. 23) (Table XVIII). CE-ESI/MS analysis (positive ionization mode, +200V orifice voltage) operating at high orifice voltage revealed a heterogeneous mixture of ions (Table XVIII). The ion m/z 412.3, observed during CE-ESI/MS
analysis, confirmed that the labile sorbofuranose side chain is located on the GIcA(NGro) sugar (Table XVIII). Of particular importance, the ions m/z 279.3 and m/z 296.8 provided unequivocal evidence showing that the OMePN modification is located on the GIcNAc sugar for C. jejuni HS:19 CPS (Table XVIII). Finally, the ion mJz 708.3 observed during CE-ESI/MS analysis and representing one complete repeat of the CPS structure, supported NMR data and corroborated the proposed CPS
structure for HS:19 CPS (Fig. 23) (Table XVIII).

In an embodiment of the invention there is provided [-4)-a-D-Galp-(1-2)-(R)-Gro-(1-Pjn with labile oc-D-Gal-2-2-~3-fructofuranose and a-D-Gal-3-2-(3-fructofuranose branches containing a O-methyl phosphoramidate modification (OMePN) on position 3 of (3-fructofuranose.
In an embodiment of the invention there is provided ~3-fructofuranose having OMePN attached on position 3.
In an embodiment of the invention there is provided a keto sugar having OMePN attached thereto and use of this sugar in the preparation of or as all or a component of a vaccine and/or antigenic target and/or in the identification of molecules or compounds having binding affinity for a C. jejuni CPS.
In an embodiment of the invention there is provided [-4)-~i-D-GIcA6(NGro)-(1-3)-(3-D-GlcNAc-(1-jn containing and an O-methyl phosphoramidate modification on position 4 of GIcNAc. In some cases it also includes a labile sorbofuranose side branch on position 2 or 3 of GIcA(NGro), In an embodiment of the invention there is provided sorbofuranose having GIcA(NGro) attached thereto.
In an embodiment of the invention there is provided sorbofuranose having GIcA
attached thereto and use of this sugar in the preparation of or as all or a component of vaccine andJor antigenic target and/or in the identification of molecules or compounds having binding affinity for a C. jejuni CPS.
In an embodiment of the invention there is provided an O-methyl phosphoramidate and a pharmaceutical carrier and the use of this composition as a vaccine.

In an embodiment of the invention there is provided a method of increasing potential immune response or otherwise modulating to C. jejuni comprising administering to a mammal, bird, fish, reptile or insect an O-methyl phosphoramidate linked to a sugar moiety.
In an embodiment of the invention there is provided use of an O-methyl phosphoramidate in identifying molecules or compounds having binding affinity for a C jejuniCPS.

Part B: Table XV. Proton and carbon chemical shifts 8 (ppm) for C. jejuni HS:1 CPS

Atom Type 8H 8c Type 8H 8c A1 CH 5.20 98.9 CH 5.40 98.8 A2 CH 3.88 70.4 CH 4.28 68.2 A3 CH 3.98 69.9 CH 4.40 68.5 A4 CH 4.53 75.6 CH 4.69 77.1 AS CH 4.17 71.6 CH 4.16 71.8 A6 CHZ 3.74 61.6 CHZ 3.75 61.6 B 1B 1' CHZ 4.04/4.11 65.3 CHZ 4.11 /4.1564.1 B2 CH 3.97 78.1 CH 4.02 77.2 B3 CHZ 3.76 62.1 CHZ 3.76 62.1 C1 CHZ 3.68/3.7763.9 C2 C - 104.1 C3 CH 4.84 79.5 C4 CH 4.52 73.2 CS CH 3.84 81.2 C6 CHZ 3.77/3.8662.5 D CH3 3.80 54.8 I and CPS-2 structures.
1H and'3C chemical shifts 8 (ppm) were referenced to an internal acetone standard ('H 2.225 ppm and i3C 31.07 ppm). 31P chemical shifts for CPS-1 were 0.69 ppm and 14.72 ppm for the phosphate backbone and O-methyl phosphoramidate, respectively. The 3'P chemical shift for CPS-2 was 1.43 ppm. 31P chemical shifts were referenced to an external 85% phosphoric acid standard (0 ppm).

Table XVI. CE/MS/MS analysis of C. jejuni HS:O1 purified CPS (-200V orifice voltage). Analysis performed in negative ionization mode. For real masses, add one mass unit to the observed and calculated masses.
Mass (Da) Structure Observed Calculated Difference 110.3 110.1 0.2 Gro + H20 153.1 153.1 0.0 GroP - H20 171.3 171.1 0.2 GroP

205.0 205.1 0.1 Hex + P - (H20)3 223.3 223.1 0.2 Hex + P - (H20)2 240.3 241.1 0.8 Hex + P - (H20) 254. 8 254.2 0.6 Hex + OMePN - HZO

259.0 259.1 0.1 Hex + P

297.3 297.2 0.1 Hex + GroP - (H20)2 315.3 315.2 0.1 Hex + GroP - HZO

333.5 333.2 0.3 Hex + GroP

377.5 377.2 0.3 Hex + GroP + P - (H20)z 385.3 385.2 0.1 (Hex)Z + P - (HZO)z 395.3 395.2 0.1 Hex + GroP + P - H20 398.3 398.2 0.1 (Hex)z + OMePN - (H20)2 407.5 407.3 0.2 Hex + GroP + Gro 416.8 416.3 0.5 (Hex)2 + OMePN - H20 453.3 453.3 0.0 (Hex)Z + OMePN + H20 459.3 459.5 0.2 (Hex)Z + GroP - (H20)2 469.3 469.3 0.0 Hex + (GroP)Z - H20 477.3 477.3 0.0 (Hex)Z + GroP - H20 487.3 487.3 0.0 Hex + (GroP)2 490.8 490.4 0.4 (Hex)2 + Gro + OMePN - HZO

495.5 495.4 0.1 (Hex)Z + GroP

509.6 509.4 0.2 (Hex)2 + Gro + OMePN

539.3 539.3 0.0 (Hex)2 + GroP + P - (Hz0)2 551.5 551.4 0.1 (Hex)Z + GroP + Gro - Hz0 557.5 557.3 0.2 (Hex)2 + GroP + P - HZO

570.3 570.4 0.1 (Hex)Z + GroP + OMePN - H20 613.5 613.4 0.1 (Hex)2 + (GroP)2 - (H20)2 621.5 621.5 0.0 (Hex)Z + GroP - (H20)2 631.3 631.4 0.1 77 (Hex)Z + (GroP)2 - Hz0 639.3 639.5 0.2 (Hex)3 + GroP - H20 667.5 667.4 0. I (Hex)2 + (GroP)2 + HZO

701.5 701.4 0.1 (Hex)3 + GroP + P - (HZO)2 723.3 723.5 0.2 (Hex)Z + (GroP)Z + Gro 732.3 732.5 0.2 (Hex)3 + GroP + OMePN - H20 775.5 775.5 0.0 (Hex)3 + (GroP)2 - (HZO)2 793.5 793.5 0.0 (Hex)3 + (GroP)z - HZO

793.3 793.5 0.2 (Hex)2 + GroP + P + OMePN - (HZO)z 829.5 829.6 0.1 (Hex)3 + (GroP)Z + HZO

855.3 855.5 0.2 (Hex)3 + (GroP)2 + P - (HZO)2 886.8 886.6 0.2 (Hex)3 + (GroP)Z + OMePN - H20 894.5 894.6 0.1 (Hex)4 + GroF + OMePN - H20 929.8 929.6 0.2 (Hex)3 + (GroP)3 - (HZO)2 947.5 947.6 0.1 (Hex)3 + (GroP)3 - Hz0 987.5 987.7 0.2 (Hex)4 + GroP + (OMePN)Z - H20 1048.5 1048.7 0.2 (Hex)4 + (GroP)2 + OMePN - H20 1109.5 1109.7 0.2 (Hex)4 + (GroP)3 - HZO

Table XVII. Proton and carbon chemical shifts b (ppm) for hot water/phenol purified C. jejuni HS:19 CPS
Atom Type $H 8c A1 CH 4.65 101.0 A2 CH 3.71 73.8 A3 CH 3.71 73.8 A4 CH 3.90 79.0 AS CH 3.94 75.1 A7 CH 4.07 53.9 A8 CHZ 3.75/3.65 61.5 Bl CH 4.62 100.7 B2 CH 3.97 56.1 B3 CH 4.24 75.8 B4 CH 4.26 74.2 BS CH 3.61 75.5 B6 CH2 3.93/3.74 61.3 B7 C - 175.0 B8 CH3 2.11 23.5 C CH3 3.76 54.8 Dl CHZ 3.73/3.64 61.5 D2 C - 104.3 D3 CH 4.17 79.2 D4 CH 4.41 76.1 DS CH 4.39 79.2 D6 CHz 3.79/3.69 62.9 'H and'3C chemical shifts b (ppm) were referenced to an internal acetone standard ('H 2.225 ppm and 13C
31.07 ppm). 31P chemical shifts for CPS-1 were 15.0 for the O-methyl phosphoramidate and was referenced to an external 85% phosphoric acid standard (0 ppm).

Table XVIII. CE/MS/MS analysis of C. jejuni HS:19 purified CPS (+200V orifice voltage). Analysis performed in positive ionization mode. For real masses, subtract one mass unit to the observed and calculated masses.
Mass (Da) Structure Observed CalculatedDifference 204.3 204.2 0.1 HexNAc - HZO

213.8 213.2 0.6 HexNGro - (H20)3 231.8 231.2 0.6 HexNGro - (HZO)2 249.8 250.2 0.4 HexNGro - HZO

279.3 279.2 0.1 HexNAcOMePN - (H20)z 296.8 297.2 0.4 HexNAcOMePN - HZO

412.3 412.4 0.1 HexNGro + Hex - HZO

435.5 435.9 0.4 HexNGro + HexNAc - (H20)Z

453.3 453.4 0.1 HexNGro + HexNAc - Hz0 528.3 528.4 0.1 HexNGro + HexNAcOMePN - (HZO)Z

546.3 546.4 0.1 HexNGro + HexNAcOMePN - H20 615.3 615.6 0.3 HexNGro + HexNAc + Hex - H20 656.5 656.6 0.1 HexNGro + (HexNAc)2 - H20 691.3 690.8 0.5 HexNGro + HexNAcOMePN + Hex - (H20)2 708.3 708.6 0.3 HexNGro + HexNAcOMePN + Hex - H20 749.8 749.6 0.2 HexNGro + HexNAcOMePN + HexNAc - HZO

795.3 795.7 0.4 (HexNGro)Z + HexNAcOMePN - HZO

842.3 842.7 0.4 HexNGro+ (HexNAcOMePN)Z - H20 865.3 864.8 0.5 (HexNGro)2 + HexNAc + Hex - Hz0 887.5 887.8 0.3 (HexNGro)Z + (HexNAc)2- (HZO)Z

905.8 905.8 0.0 (HexNGro)2 + (HexNAc)2- HZO

957.8 957.8 0.0 (HexNGro)Z + HexNAcOMePN + Hex - HZO

999.3 998.8 0.5 (HexNGro)2 + HexNAcOMePN+ HexNAc - HZO

1091.8 1091.9 0.1 (HexNGro)2 + (HexNAcOMePN)2 - H20 Part C: BIOSYNTHESIS OF O-METHYL PHOSPHORAMIDATE AND USES
THEREOF
There is disclosed herein genes involved in the biosynthesis of the novel cell surface structures collectively known as O-methyl phosphoramidates and uses thereof in the identification and targeting of C. jejuni.
Campylobacter jejuni is the major bacterial cause of gastrointestinal disease in developed countries and infection can lead to the development of the neuropathy known as Guillain-Barre syndrome.
Genome sequencing of C. jejuni NCTC 11168 demonstrated that the strain contained four gene clusters necessary for carbohydrate biosynthesis including the capsular biosynthesis locus, containing a Kps transport system similar to that found in other encapsulated organisms. This gene cluster also encodes enzymes involved in the biosynthesis and transfer of a branched tetrasaccharide repeat to the outer membrane surface of C. jejuni. Recently, the .CPS structure of NCTC11168 was determined to contain 6-O-methyl-D-glycero-a-L-gLuco-heptose, ~i-D-glucuronic acid modified with 2-amino-2-deoxyglycerol, (3-D-GalfNAc and (3-D-ribose.
Early structural studies of C. jejuni CPS showed that these molecules are highly heterogeneous from one strain to another. Microarray hybridisation analysis also demonstrated some differences in the CPS-related genes between strains of various serotypes. In addition, the biosynthetic region of the cps loci is prone to phase variation due to the presence of multiple genes with homopolymeric tracts. It was subsequently shown that CPS structures from a single isolate (NCTC 11168) can vary in structure. However, the genetic mechanisms underlying the structural heterogeneity and antigenic variation remained unknown.
Since capsular polysaccharides are the outermost structure on the bacterial cell they play an important role in the interaction between the pathogen, host, and environment. In C. jejuni 81-176, the capsule is involved in INT407 cell invasion, virulence in ferrets, serum resistance and maintenance of bacterial cell surface charge.
In C. jejuna NCTC11168, the capsule is necessary for colonization of poultry, the primary source of C. jejuni infection.
gl There is disclosed herein genes involved in the biosynthesis of O-methyl phosphoramidate structures found on the surface of a majority of Campylobacter jejuni isolates and uses thereof.
C. jejuni produces a capsular polysaccharide (CPS) that is the major antigenic component of the classical Penner serotyping system. High resolution magic angle spinning (HR-MAS) NMR was used to examine capsular polysaccharides directly from campylobacter cells and showed profiles similar to those observed for purified polysaccharides analysed by solution NMR. This method also exhibited the potential to for campylobacter serotyping, mutant verification, and preliminary sugar analysis.
HR-MAS NMR examination of growth from individual colonies of C. jejuni NCTC 11168 indicated that the capsular glycan modifications are also phase variable.
These variants show different staining patterns on deoxycholate-PAGE and reactivity with immune sera. One of the identified modifications, that showed both reduced reactivity with silver staining and rabbit sera, was a novel -OP=O(NHZ)OMe (O-methyl phosphoramidate). This modification was attached to the 3-position of the CPS GalfNAc.
Biosynthetic cps regions were sequenced, ranging in size from 15 to 34 kb, from C. jejuni strains of HS:l, HS:19, HS:23, HS:36, HS:23/36 and HS:41 serotypes 2o and compared with the sequenced strain, NCTC11168 (HS:2). Extensive structural studies, including HR-MAS NMR, demonstrated polysaccharide heterogeneity in campylobacter CPS and demonstrated the presence of additional CPS
modifications and the commonality of the recently described O-methyl phosphoramidate.
Development of a novel HRMAS filtering method has allowed investigation of multiple isolates of G jejuni from various clinical presentations and geographical locations and revealed that the O-methyl phosphoramidate is common to approximately 70% of all strains examined (see Part A). This modification appears specific to C. jejuni and was not observed in the closely related Campylobacter coli.
Structural analysis of the HS:l and HS:19 strains demonstrated that the O-methyl phosphoramidate can be attached to different sugars in different linkages.
Multiple O-methyl phosphoramidate signals axe observed during HRMAS analysis indicating that the modification is attached to varying capsule backbones, attached to alternative structures and/or is detected as biosynthetic intermediates.
Sequential inactivation of the cps biosynthetic genes in C. jejuni NCTC11168 followed by O-methyl phosphoramidate filter analysis allowed identification of s multiple genes encoding enzymes involved in the biosynthesis of O-methyl phosphoramidate: cj1416c, cj1417c, cj1418c, cj1421 c and the duplicated gene, cj1422c (Table XIX and XX). This work demonstrated that cj1422c encodes an enzyme also capable of transferring the O-methyl phosphoramidate (Figure 25 and 19). However, this structure is added at an alternative site on the CPS
backbone (Figure 27), the C~ position on the D-glycero-a-L-glcsco-heptopyranose (see Part E).
A summary of the mutants examined is presented in Tables XIX and XX. All or most of these genes are missing in other Campylobacter species and genome sequenced strains belonging to the epsilon proteobacteria confirming their inability to synthesize O-methyl phosphoramidate (see Figures 6 and 11 of Part A). Preliminary examination I5 of the C. jejuni mutants in human cell culture assays has demonstrated that the O-methyl phosphoramidate is required for efficient adherence, but is not necessary for cell invasion. Furthermore, the expression of this modification renders the bacteria more sensitive to human sera (see Part A).
2o BRIEF DESCRIPTION OF THE FIGURES AND TABLES
FIGURE 25 Is a whole cell 1H HR-MAS spectra of samples of wildtype and cj1421/1422 mutants of Campylobacter jejuna strain UK11168-H . Spectra show the anomeric proton region and capsular polysaccharide peaks are labeled A-D:
2s (A) ~3-D-Ribf, (B) a-D-GlcpA6(NGro), (C) ~3-D-GalfNAc and (D) 6-O-Me-D-glycero-a-L-glcHepp FIGURE 26 Is a whole cell 1H 3jP filtered 1H-HRMAS spectra of samples of wildtype and cj1421/1422 mutants of Campylobacter jejuni strain UK11168-H .
3o The experiment selects for only phosphoramidate residues. The wildtype exhibits two resonances indicating that cj1421 and cj1422 are being expressed. In the case of the double mutant and the strain with cj1421 off, the signal may be due to either a metabolite or to non-capsular polysaccharide phosphoramidates.
Figure 27 Is a whole cell 1H 31P -filtered 1H-HRMAS spectra of cj1421/1422 mutants of Campylobacter jejuni strain UK11168-H. The 1H-31P filter is a modification of that used in Figure 19 that allows 31P-coupled protons of the capsule carbohydrate residues to be detected, indicated by arrows. The small signal at 4.88 ppm in the spectrum of the mutant. Cj1422-3 (cj1421 on l cj1422 mutant) corresponds to the H3 proton of residue C (Gal, f NAc moiety ) in Figure 1. In the case of the Cj1421-1 mutant ( cj1421 mutant / cj1422 on) the carbohydrate proton signal ( arrow ) indicates that the phosphoramidate residue is attached other than at the O-3 position of the Gal, f NAc moiety.
TABLE XIX Is a summary of mutants examined involving genes which are believed to be involved in O-methyl phosphoramidate biosynthesis. The table demonstrates that cj1416c, cj1417c and cj1418c are involved in early steps of O-methyl phosphoramidate biosynthesis since they are not dependent on the on/off status of the transferases. Mutation of any of these 3 genes causes loss of POMe detection. In contrast, cj1421 c and cj1422c can each add O-methyl phosphoramidate to the CPS and thus must be both knocked out in order to see a loss of O-methyl phosphoramidate.
TABLE XX Is a summary of NMR spectra data representing selected permutations of cj1421 c and cj1422c expression. These results demonstrate that both genes encode enzymes capable of adding O-methyl phosphoramidate to CPS, but that there are slight differences to the overall CPS NMR frequencies due to the addition of O-methyl phosphoramidate to different locations within the polymer. In addition, the O-methyl phosphoramidate signal intensity demonstrates that even in mutants that lack CPS O-methyl phosphoramidate, we are able to detect a weak signal due to the accumulation of metabolite intermediates.

EXAMPLES
While the invention is discussed with respect to particular examples and embodiments, it will be readily understood that it is not so limited, but in fact includes all variants and alternative embodiments thereof. While possible mechanisms andlor modes of action may be discussed, it will be understood that the invention is not so limited.
The inclusion of a reference is not an admission or suggestion that it is relevant to the patentability of anything disclosed herein.
Bacterial strains and growth conditions - Campylobacter jejuni NCTC 11168 l0 (HS:2) was isolated from a case of human enteritis and later sequenced by Parkhill et al.. All campylobacter strains were routinely grown on Mueller Hinton agar (Difco) under microaerophilic conditions at 37°C. C. jejuni NCTC 11168 mutants were grown on Mueller Hinton agar with 30 ~ g/mL kanamycin.
Preparation of cells for HR-MAS NMR - C. jejuni overnight growth from one agar plate 0101° cells) was harvested and suspended in 1 mL of 10 mM
potassium buffered saline (pH 7) made in D20 containing 10% sodium azide (w/v). The suspension was incubated for 1 h at room temperature to kill the bacteria. The cells were pelleted by centrifugation (7 500 X g for 2 min) and washed once with 10 mM
potassium buffered saline in D20. The pellet was resuspended by adding 20 ~L
of 2o D20 containing TSP and then 40 ~L of the suspension was inserted into the rotor for analysis.
HR-MAS NMR spectroscopy - HR-MAS experiments were performed using a Varian Inova 500 MHz spectrometer equipped with a Varian nano-NMR probe as described in Szymanski et al. (2003). Spectra from 40 ~L samples were spun at KHz and recorded at ambient temperature (21°C). The experiments were performed with suppression of the HOD signal at 4.8 ppm. Proton spectra of bacterial cells were acquired with the Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence [90-(i-180-i)n acquisition] to remove broad lines arising from lipids and solid-like material. The total duration of the CPMG pulse (n*22) was 10 ms with ~ set to (1/MAS spin rate).
One-dimensional selective TOCSY experiments with various spin-lock times from 30-150 ms and selective NOESY with mixing times from 100-400 ms were performed substantially as described in Uhrin and Brisson (2000) in NMR in Microbiology, p.165-210 Horizon Science Press, UK, and in Brisson et.al. (2002) in NMR
spectroscopy of glycoconjugates p.59-93, Wiley-BCH, Weinheim. For use under MAS conditions, the TOCSY sequences were modified so that the DIPSI-2 mixing sequence was replaced with the adiabatic WURST-2 pulses. Selective experiments were described as EXP[selected spins, selective excitation bandwidth, mixing time]
where EXP is TOCSY or NOESY. Typically, proton spectra of bacterial cells could be obtained using 256 to 1024 transients (15 min to 1 hour). ). 1D IH-3'P
correlated spectra were acquired using the first increment of the lH-31P CPMG-correlated experiment ( B. Luy and J.P. Marino, J. Amer. Chem. Soc.2001, 123,11306-11307) and with 1H-31P scalar coupling constant of 10 Hz.. The 31P transmitter was set on resonance for the O-methyl phosphoramidate group. Typically 256 to 1024 transients were collected.
RESULTS
Examination of CPS from whole cells by HR-MAS NMR - The capsular polysaccharide structure of the genome sequenced strain, NCTC11168 (HS:2) was described. The proton spectrum obtained from HR-MAS of suspended NCTC 11168 bacterial cells closely resembled the spectrum of the purified capsular polysaccharide and clearly demonstrated the N acetyl, O-methyl, and anomeric resonances. The HR-2o MAS NMR spectrum was obtained in a few minutes directly from 40 ul of whole cells. Hence, this method permitted quick screening of campylobacter CPS
directly from one plate of growth 0101° cells), but was sensitive enough to detect a 1/100 dilution of the suspension containing 8xI07 cells.
HR-MAS NMR analysis of C. jejuni NCTC11168 mutants - The HR-MAS
spectrum of variant 2 (see attached document Part A) revealed new resonances at 3.75 ppm indicative of a novel modification, which had not been previously observed. In addition, the anomeric chemical shift for residue C moved downfield closer to the one for residue B.
The structural determination of the purified polysaccharide from C. jejuni 3o NCTC11168 variant 2 was done essentially as described in St. Michael (2002). Its backbone CPS structure was found to be the same as determined previously, but with the addition of a modified phosphate group at C-3 of the GalfNAc residue C.
The sample also contained about 30% of the major wild-type CPS. Comparison of the HMQC spectra of variant 2 with the one from the wild-type sample, showed similarity in chemical shifts for residues A, B and D. Proton chemical shifts for residue C for variant 2 were identified using a selective TOCSY experiment. The 5D-4C and 3D-4C NOES were also observed as before for the wild-type CPS (see Part A for full structure eludication by NMR and mass spectrometry).
Correlation of cps genes from strains of serotypes HS:1, HS:2, HS:19, HS:23, HS:36 and HS: 41 with respective CPS structures - The strategy used for sequencing the variable cps loci from the different strains is described in Part A. The overall summary of the cps sequencing results and a schematic of all the cps loci compared is shown in Part A with the genes involved in O-methyl phosphoramidate biosynthesis shown in bold (see below). Some of the gene products are involved in the biosynthesis of activated sugars. Such activated sugars contain energy-rich nucleotide-phosphate bonds and serve as substrates for glycosyltransferases involved in the biosynthesis of polysaccharides. In addition, nucleotide sugars may be modified by enzymes such as epimerases, dehydratases and reductases before transfer of the final product. Additional modifying enzymes can add groups such as O-methyl, phosphate, ethanolamine- and aminoglycerol- to further increase the complexity of the structures. Indeed, genes encoding these enzymes can be found in various C.
jejuni cps regions.
Variation in the contingency genes - The potentially phase variable cps genes of C. jejuni strains of various serotypes were investigated. The biosynthetic cps locus of C. jejuni NCTC11168 was found to contain six genes with homopolymeric G
tracts potentially prone to phase variation (See for examples Tables VIII and IX in Part A) A comparison of the anomeric region of the 1H HR-MAS whole cell spectra of the wildtype and the cj1421/1422 mutants of C. jejuni strain UK

indicates that while the core structure of the capsule remains the same the genes modify the structure in different ways. When cj1421 and cj1422 are "knocked-out" or are both unexpressed, the same capsule structure is produced as evidenced by identical anomeric 1H chemical shifts for residues A-D ( top two spectra , Figure 25).

When cj1421 is expressed and cj1422 is "knocked-out", the anomeric proton resonance of GalfNAc (residue C, second spectrum Figure 26) shifts downfield near that of residue B, a result of the O-methyl phosphoramidate substitution at O-3. The 1H spectrum of the mutant with cj1421 "knocked-out" and cj1422 expressed ( middle spectrum Figure 25) exhibits an anomeric resonance of residue C that is shifted upheld from that of the other mutants suggesting that the core capsule structure is modified by gene cj1422 in a way that differs from that arising from gene cj1421.
Figure 19 shows the corresponding 1H HR-MAS spectra of the same cells as to Figure 18 but acquired to detect only those protons having scalar coupling to the phosphorous atom of the O-methyl phosphoramidate ( which we refer to here as the O-methyl phosphoramidate filter analysis,lH-3'P filter). The wildtype strain exhibits two methyl resonances (Figure 26, bottom) indicating that two distinct O-methyl phosphoramidate residues are present in approximately 1:1 ratio. Silencing gene cj1422 results in one O-methyl phosphoramidate resonance being detected in the ~1P filtered experiment ( Figure 26, second from bottom) having the same chemical shift as one of the resonances detected for the widtype strain ( Figure 26, bottom).
Silencing gene cj1422 also results in one O-methyl phosphoramidate resonance being detected in the 1H-31P filtered experiment ( Figure 26, middle from bottom) having the same chemical shift as the second resonance detected for the widtype strain ( Figure 26, bottom). The results indicate that the two genes are responsible for generating O-methyl phosphoramidate at two distinct sites on the capsular polysaccharide.
Silencing both cj1421 and cj1422 results in both strong 1H-31P filtered signals being absent (Figure 26, top two spectra) and only weak residual signals being detected. It is believed that the latter signals may arise from either metabolites or O-methyl phosphoramidate derivatives of other cellular molecule(s).
To confirm further that genes cj1421 and cj1422 are responsible for modification of capsule at different sites, the 1H-31P filter was modified to reduce the potential effects of transverse relaxation and as a result increase the signal intensity for carbohydrate protons that are scalar coupled to the phosphorous atom of the O-methyl phosphoramidate. The modified 1H-~1P filter experiment was conducted with 8s the two mutants in which genes cj1421 or cj1422 only were silent. In addition to the strong O-methyl phosphoramidate 1H signal the modified experiment allowed the much weak capsule carbohydrate resonance to be detected. In the case of the mutant with cj1422 silent and cj1421 expressed" a weak 1H resonance at 4.88p was detected Figure 27, bottom) which is attributable to the H-3 proton of the GaI,fNAc ( residue C) at the site of attachment of the O-methyl phosphoramidate residue. This data is consistent, with the previously published data for the capsular polysaccharide of C.
jejuni NCTC11168. The modified 1H-3lP filtered spectrum of the mutant with cj1421 silent and cj1422 expressed exhibited a new resonance at ~4.35p confirming that the site at which the O-methyl phosphoramidate moiety is attached to the core capsule structure differs with that arising from gene cj1421.
In this study, HR-MAS NMR have been used successfully to examine glycan structures from NCTC 11168 and variants in cj1421 c and cj1422c. A novel modification for C. jejuni NCTC11168 variant 2 was previously observed with -OP=O(NH2)OMe on the 3-position of Gal, fNAc. Herein we demonstrate that this structure is located at an alternative site on the CPS of NCTC11168 These structures could not be resolved in the previous study due to their low abundance in the wild type population. The O-methyl phosphoramidate has not been described previously and, it shows structural similarity to synthetic organophosphate insecticides.
Current studies have identified the relevance of the O-methyl phosphoramidate, identified the genes necessary for biosynthesis, and examined the commonality of this structure (see Part A). There is provided herein further data on the importance of these genes in O-methyl phosphoramidate biosynthesis and demonstrate that one of the proposed transferases, Cj 1422c, is capable of adding the moiety elsewhere on the CPS.
It is generally accepted that a single microorganism can give rise to a diverse population with very different virulence properties. However, in the past, sensitive methods for the structural analysis of bacterial populations have been limiting. As disclosed herein, HR-MAS NMR has been used to investigate CPS structure, demonstrate population variability and study the effect of mutagenesis.
Campylobacter has a large repertoire of variable surface glycans in addition to a conserved N linked glycan. These studies have implications in therapeutic development against novel targets on CPS, describe analytical methods that can be adapted for the analysis of small amounts of glycans from other important bacterial pathogens (e.g. HRMAS and/or various filtering methods can be used to detect small quantities of sugars from any bacteria, such as Neisseria in which HRMAS
NMR was applied to demonstrate that CPS O-acetyl groups are expressed in non-stoichiometric ratios in vivo) expand the new field of metabolomics, and can provide more insight into the importance of bacterial LOS, capsules, and protein glycosylation allowing scientists to expand the discipline of glycomics beyond the gene complement and glycan structure. These studies were possible by characterizing the hypermotile version of the genome sequenced NCTC11168 strain which expressed cj1421c and cj1422c in the majority of the population (see Table VIII in Part A).
In an embodiment of the invention there is provided use of a nucleic acid sequence having significant sequence identity to at least one sequence found in Table XIII and/ or IX and/or nucleic acid sequences complementary thereto (with or without the homopolymeric G tract region or its complementary region), in identifying genes and gene products useful in the recognition of C. jejuni, diagnosis of related conditions, and targeting of binders to C. jejuni. In some instances the level of sequence identity will be at least 70%, 75% 80%, 85 %, 90 %, 95%, 98%, 99% or %.
In an embodiment of the invention there is provided an isolated polypeptide sequence encoded by a C. jejuni gene comprising nucleic acid sequence containing the appropriate homopolymeric G tract region. Also provided is use of such a polypeptide sequence in generating and/or identifying binders to C.
jejuni. In some instances the level of sequence identity between a sequence of interest and a sequence found in Table VIII or IX may be at least 70%, 75% 80%, 85 %, 90 %, 95%, 98%, 99% or 100 %.
In an embodiment of the invention there is provided use of a homopolymeric G tract region to modulate transcription of a gene.

In an embodiment of the invention there is provided a method of modulating C. jejuni adherence and/or sensitivity to sera comprising modulating O-methyl-phosphoraxnidate synthesis.
In an embodiment of the invention there is provided use of a transferase substantially similar in amino acid sequence to the transferase encoded by Cj1421c and/or Cj 1422c in causing linkage of O-methyl-phosphoramidate to CPS.
In an embodiment of the invention there is provided a method of producing antigenic material useful in inducing an immune response to a C, jejuni strain, said method comprising: a) obtaining the functional protein product of: Cj 1416c, Cj I417c, to Cj 1418c and either Cj 1421c, or Cj 1422c;
b) exposing capsular polysaccharide of a type found in the C. jejuni strain to the protein products obtained in step (a) in the presence of biosynthetic intermediates necessary for O-methyl phosphoramidate productions. Biosynthetic intermediates may be provided within the context of a defined reaction mixture or an undefined or poorly defined mixture such as that obtainable form crude biological preparations.
In an embodiment of the invention there is provided a method of producing material useful in identifying molecules and/or compounds having a binding affinity for CPS of at least one C. jejuni strain, said method comprising: a) obtaining the functional protein product of: Cj 1416c, Cj 1417c, Cj 1418c and either Cj 1421c, or Cj1422c;
b) exposing capsular polysaccharide of a type found in the C, jejuni strain to the protein products obtained in step (a) in the presence of biosynthetic intermediates necessary for O-methyl phosphoramidate productions. Biosynthetic intermediates may be provided within the context of a defined reaction mixture or an undefined or poorly defined mixture such as that obtainable form crude biological preparations.

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Part D - Section 1 Campylobacter jejuni HS:1 CPS
Overview 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 ~3-D-fructofuranoses substituted at C-3 with CH30P(O)(NH2)(OR) groups. Structural heterogeneity was due to non-stoichiometric 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 I5 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 non-stoichiometric 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 biosynthetic region of the C. jejuni HS:1 strain is small and contains only eleven genes (Fig. la). Of importance, the cps locus of the HS:1 strain contains a tagD homologue encoding a glycerol-3-phosphate cytidylyltransferase necessary for the biosynthesis of CDP-glycerol. Moreover, the HS:1 strain encodes a tagF homologue responsible for transferring glycerol-phosphate residues from CDP-glycerol. These genetic findings for the cps loci of this HS:1 strain corroborate the structures identified for the C. jejuni HS:1 serostrain HMW
LPS (CPS), where the repeat unit was [-4)-a-D-Gal-(1-2)-Gro-(3-P-]" [17;18].
However important discrepancies were observed 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 [I4]. For instance, the presence of at least two acid-labile groups was detected and provided evidence showing that one of these was likely an MeOPN

CH30P(O)(NH2)(OR) modification similar to the one identified on the CPS
structure of the genome-sequenced strain of C. jejuni, NCTC 11168.
The chemical structure of CPS for the HS:1 serostrain of C. jejuni is described herein.
Initially, CPS was isolated from bacterial cells using a traditional hot water/phenol method;
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:l CPS. Accordingly, the methods of Darveau & Hancock, Huebner et al. and Hsieh et al. 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 was used to analyze the structure of purified HS:1 CPS, corroborate NMR findings and characterize the extent of heterogeneity for HS:l CPS.
Results - Part D - Section 1 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. 28b). The complete CPS structure for HS:1 is complex due to the presence of a non-stoichiometric fructose branch at C-3 of galactose, and variable MeOPN
groups at C-3 of both fructose branches at C-2 and C-3 of galactose (Fig.
28b). 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 IH NMR spectroscopy Using the hot water/phenol extraction method, 7.3 mg of pure CPS was obtained from 6 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:l CPS in non-buffered D20 (pD 2.2), the auto-hydrolyzed defructosylated repeating unit was obtained (CPS-1), as well as other hydrolysis fragments. The'H NMR
spectrum of this auto-hydrolyzed CPS sample showed sharp spectral lines and one major anomeric signal for Gal H-1 (Fig. 29a). Signals originating from the methyl group of the MeOPN
modification, normally present at 3.78 ppm, were absent. 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 29b). 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. 29c).
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.
29d) 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.
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. Furthermore, an intense increase in adsorption at 340 nm following treatment with a hexokinase-phosphoglucoisomerase-glucose-6-dehydrogenase-NADP fructose assay kit (Sigma, Oakville, Canada) indicated that fructose also had the D configuration.
The chirality of naturally occurring glycerols can be determined chemically, enzymatically or can be deduced from the biosynthetic pathway responsible for their production. When CDP-glycerol is used as a precursor to incorporate glycerol in the growing repeating chain of teichoic acids, the resulting glycerol-1-phosphate unit has the D, or R
configuration. Alternatively, when glycerophosphate is biosynthetically derived from phosphatidylglycerol, the resulting product is L- or S-glycerol-1-phosphate.
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, 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 [-4)-a-D-Galp-(1-2)-(R)-Gro-(1-P]n repeating unit (CPS-1) as well as other hydrolysis products (Fig. 30). The 1D-TOCSY of Gal H-1 revealed J-correlated peaks for Gal H-2, H-3 and H-4 (Fig. 30a). The 1D-NOESY of Gal H-revealed NOES for Gal H-3 and H-5 (Fig. 30b), and the 1D-TOCSY of Gal H-5 identified the Gal H-6 resonances (Fig. 30c). A 1D-NOESY-TOCSY experiment with selective excitation of Gal H-1/Gro H-2 was used to identify the glycerol resonances (Fig. 30d).
The Gal H-1/Gro C-2 HMBC correlation confirmed the Gal-(1-2)-Gro linkage. The 31P HSQC
spectrum (Fig. 30e) 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. 30g) and HMBC spectrum were used to assign the ~3C resonances (Table XXI) and signals consistent with those reported for fructofuranose and fructopyranose monosaccharides were observed.
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 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 since 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 labeled A2a, A2b, A3a, A3b, A4a and A4b, respectively. (Fig. 31a). 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 Fzu H-4 and Fru H-6/6' (Fig. 31b). 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.
3Ic). 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. 31d).
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 G jejuni HS:1.
The 1D-NOESY of Fru H-3 revealed enhancements for Fru H-1/1', H-4 and H-5 (Fig. 31e) while the 1D-TOCSY of Fru H-4 showed correlations to Fru H-3 and H-5 (Fig.
31f).
The 31P HSQC experiment revealed rnonophosphate diester linkages between Gal C-4a, C-4b and Gro C-1 with different chemical shifts at ~ 0.40 ppm and 0.49 ppm, respectively (Fig. 31g). A proton-phosphorus correlation at 8P 14.67 ppm 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 ~3-D-fructofuranoside residues (Fig. 31g). The phosphorus chemical shift of the MeOPN was consistent with those reported for phosphoramidates in the literature.
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 31H, Table XXII). The small upheld shift changes caused by fructosylation of Gal at C-2 and C-3 of 2.1 ppm and 1.2 ppm, respectively, were consistent with those reported for the CPSs of Escherichia coli strains 04:K52:H- and 013:K11:H11 (Table XXI and XXII). The 13C HSQC spectrum also showed minor contaminating signals similar to those reported for C. jejuni HS:1 LPS
(CPS) as well as for peptides and nucleic acids. Furthermore, signals belonging to non-substituted (3-fructofuranoside indicated that fructose branches were variably substituted with MeOPN
groups.
Mass spectrometry analysis CE-ESI-MS analysis corroborated the structure proposed fox HS:1 CPS and clearly established that two branches are present in the repeating unit with various degrees of heterogeneity (Fig. 32 and Table XXIII). 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. 32a). In particular, ions observed at m/z 315.0, 407.1 and 631.2, corresponding to the masses of Hex + GroP, Hex + GroP +
Gro + H20 and (Hex)2 + (GroP)Z, 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-Gale-(1-2)-(R)-Gro-(1-P-]n.
Due to the high-molecular-weight of HS:1 CPS, a high negative orifice voltage (-400 V) was used to promote in-source collision-induced dissociation for an intact enzyme purified sample of HS:l CPS (CPS-2) to facilitate its analysis by CE-ESI-MS (Fig. 32b).
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)Z, (Hex)4 + GroP + MeOPN, (Hex)3 +
GroP +
(MeOPN)Z + 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 mlz 732.5, corresponding to one full repeat of HS:1 CPS, showed an ion at mlz 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. 32c).
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 ppm was detected in their corresponding 1H spectra (Fig. 29c and 29d). Because loss of the fructose branch at Gal C-2 would have caused an upheld shift of Gal H-1 similar to that observed for the hot water/phenol purified CPS sample or the auto-hydrolyzed CPS sample (Fig. 29a and 29b), 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 non-stoichiometric branching at C-3 of Gal. The larger carbon chemical shift difference observed for Gal C-3a and b (0.9 ppm) compared to the one for Gal C-2a and b (0.2 ppm) 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.
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. 29). The 1D NOESY of Gal H-1 for an enzyme purified CPS sample showed NOEs for Gal H-2, Gro H-1/1', Gro H-2 and Gro H-3/3' (Fig. 29e). Likewise, in the HR-MAS NOESY trace of Gal H-1 (Fig. 29f) for HS:l cells, the same NOE pattern was observed. The 1D HR-MAS NOESY for the Gal H-4a and H-4b resonances (Fig. 29g) 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. 31).
The 31P HSQC HR-MAS spectrum of whole HS:l cells showed proton-phosphorus correlations for Gal H-4a and b with at 8P 0.33 ppm a.nd ~ 0.49 ppm, respectively (Fig. 29h).
The correlation between MeOPN at ~ 14.67 ppm with H-3 of fructofuranose was also observed. Hence, the NOEs and 3IP 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 presentlpresent). 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.
33. Molecular dynamics simulations showed that regardless of the substitution pattern of Gal, the average inter-proton 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 inter-residue NOE observed between Gal H-4a and fructose H-4 for an intact enzyme purified CPS sample (Fig. 31b) could have originated from either fructose branch since inter-proton distances were comparable for both branches and ranged from 3-5 A. The results of molecular dynamics simulations also suggested that NOEs observed at 3.86 ppm in the NOESY spectra of Gal H-4a and Gal H-4b (Fig. 31b and c) likely arose from Fru H-6/6' since the minimum inter-proton distance for these protons was approximately 2 A. In contrast, inter-proton 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 inter-residue NOES.
Discussion - Part D - Section 1 The CPS structure for the representative HS:1 serostrain of C. jejuni was investigated to complement data recently reported for CPS biosynthesis in strain Gl (HS:1), and to determine the structure of labile CPS constituents not detected by previous studies examining HMW LPS (CPS) fox the HS:1 serostrain. Together, different analytical methods showed that the HS:1-type CPS of C. jejuni is complex and has a teichoic acid-like [-4)-a-D-Galp-(1-2)-(R)-Gro-(1-P-]" repeating unit with a (3-D-fructofuranose branch at C-2 of Gal, a non-stoichiometric 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 and a more sensitive enzymatic approach, it was 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 ppm when the fructose branch at Gal C-2 was present and; another at 5.20 ppm 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, ZO 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 ppm. 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:l cells. For this study, use of this gentle enzymatic method coupled with HR-MAS NMR proved important in determining the structure and location of the fructose branches and MeOPN groups since both are labile structures that are easily hydrolyzed by high temperature and moderately acidic conditions.
Because CPS is considered to be an important virulence factor for C. jejuni, sensitive analytical techniques that facilitate the study of its fragile CPS structures are fundamental in increasing 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 013:K11:H11.
NMR and mass spectrometry analyses of an auto-hydrolyzed defructosylated sample of enzyme purified HS:1 CPS showed that it resembled teichoic acid, and consisted of a [-4)-a-D-Gale-(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. Moreover, these findings supported those reported for HMW LPS
(CPS) isolated from this strain of C. jejuni by McDonald, who showed it to consist of a [-4)-oc-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 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 Gal-Gro linkage since it refers to the original work by McDonald [17]. Also, in the present work, since 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.
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 G jejuni NCTC 11168. Phosphoramidate structures are quite rare in nature and are not believed to have 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. 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 non-specifically with accessible amino acids of proteins. 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. 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. Although a two component system regulating growth and colonization in response to environmental temperature was reported for C. jejuni, the relationship between the biological roles reported for phosphoramidates in other bacteria and the MeOPN CPS modification in C.
jejuni has not been clarified in publications to date.
In an embodiment of the invention there is provided use of the phosphoramide disclosed herein, and its variants, as well as MeOPN - substituted and unsubstituted fructose branches in modulating signal transduction in a cell.
In conclusion, in this study we determined the complete structure of the CPS
for the C. jejuni HS:l serostrain was described as was the importance of using mild isolation methods and non-invasive analytical techniques for examining CPS in this bacterium due to the presence of highly labile constituents that are easily overlooked using conventional methods.
As a result of using HR-MAS NMR to examine CPS directly on the surface of bacterial cells it was shown that the HS:1-type CPS of C. jejuni consists of a (-4)-a-D-Gasp-(1-2)-(R)-Gro-(1-P-]" repeating unit with two labile fructofuranoside branches and variable MeOPN modifications. "n" may be any positive integer, but is preferably 4 or greater. In some instances n will be 10-100, 100-500, 500-1500, 1500-5000, 5000-10,000, 10,000-30,000, 30,000-100,000, or 100,000-1,000,000. Hence, this strain of C, jejuni can achieve a structurally variable and complex CPS from its relatively small CPS biosynthetic locus. This structural heterogeneity may be a mechanism to convey antigenic variation and protection from host defenses.
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.
Experimental Procedures - Part D - Section 1 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:I serostrain (ATCC 43429, designation MK5-57630) was routinely maintained on Mueller Hinton (MH) agar (Difco, Kansas City, USA) plates under microaerophilic conditions (10% C02, 5% OZ, 85% NZ) at 37 °C. For large scale extraction of CPS, 6 L of C. jejuni HS:1 was grown in Brain Heart Infusion (BHI) broth (Difco, Kansas City, USA) under microaerophilic conditions at 37 °C for 24 h with agitation at 100 rpm.
Bacterial cells were then harvested by centrifugation (9 kG for 20 min) and placed in 70%
ethanol. Cells were removed from the ethanol solution by centrifugation (9 kG
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. Briefly, bacterial cells harvested from 6 L of BHI broth were blended in 90%
phenol at 96 °C for 15 min, allowed to cool for 30 min and then dialyzed (MWCO 12 KDa, Sigma, Oakville, Canada) against running water for 72 h. The volume of the bacterial extract was then reduced to approximately 100 ml under vacuum (37 °C), ultracentrifuged (I40 kG, 15 °C) for 2 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 re-suspended in H20 and purified using a Sephadex~ superfine G-50 column (Sigma, Oakville, Canada) equipped with a Waters differential refractometer (model 8403, Waters, Mississauga, Canada). IH
NMR at 400 MHz (Varian, Palo Alto, 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 H20 and purified using a Gilson liquid chromatograph (model 306 and 302 pumps, 811 dynamic mixer, 802B
manometric module, Gilson, Middleton, USA) with a Gilson UV detector (220 nm) (model UV/Vis-151 detector, Gilson, Middleton, USA) equipped with a tandem QHP HiTrapTM ion exchange column (Amersham Biosciences, Piscataway, 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 Sephadex~ superfine G-15 column (Sigma, Oakville Ont.) 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.
Enzymatic isolation of CPS
An enzymatic method of isolating CPS from G jejuni HS:I cells was developed based on the methodologies of, Huebner et al. and Hsieh et al. Bacterial cells harvested from 6 L of BHI broth were suspended in PBS buffer (pH 7.4). Lysozyme was then added to a final concentration of 1 mg~rnL-I (Sigma, Oakville, Canada) prior to the addition of mutanolysin to a final concentration of 67 U~mL-1 (Sigma, Oakville, Canada). The bacterial cell suspension was then incubated for 24 h at 37 °C with agitation at 100 rpm. The mixture was then emulsiflexed twice (21000 psi) to lyse cells, and DNAse I and RNAse (130 p,g~mL-1 DNAse I
and RNAse, Sigma, Oakville, Canada) was added prior to being incubated for 4 h at 37 °C
with agitation at 100 rpm. Following digestion with nucleases, pronase and protease was added to a final concentration of 200 p,g~mL-1 (Sigma, Oakville, Canada) before being incubated at 37 °C overnight with agitation at 100 zpm. The crude CPS
extract was then dialyzed against running water for 72 h (MWCO 12 kDa, Sigma, Oakville, Canada), ultracentrifuged for 2 h (140 kG, 15 °C) and the supernatant, containing crude CPS, was lyophilized to dryness. CPS was then purified using essentially the same chromatographic protocol described above.

Sugar composition analysis of enzyme purified CPS
The composition of an enzyme purified sample of C. jejuni HS:l CPS was determined using the alditol acetate method adapted from Sawardeker et al. A 1 mg sample of CPS was hydrolyzed by adding 0.5 mL of 3 M trifluoroacetic acid and heating at 100 °C fox 2 h.
Hydrolyzed CPS was then dried under a nitrogen stream at room temperature prior to reduction with 5 mg of NaBH4 in 300 ~,L of HZO. The reaction was allowed to proceed for 1 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 x 1 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 min prior to being dried at room temperature under a nitrogen stream. Alditol acetate derived CPS sugars were then suspended in 1.5 mL of CHZC12 and analyzed using an Agilent 6850 series GC system, equipped with an Agilent 19091L-433E 50% phenyl siloxane capillary column (30 rn X 250 ~n X 0.25 uxn) (170 °C to 250 °C, 2.8 °C-miri 1) (Agilent Technologies, Palo Alto, USA). Alditol acetate derivatives of authentic standards for common keto and aldo sugars (Sigma, Oakville, Canada) 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.
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. Approximately 300 ~,L of R-butanol and 30 ~L of acetyl chloride (Sigma, Oakville, Canada) was added to 1 mg of enzyme purified CPS. The mixture was then heated at 85 °C for 3 h prior to being dried under a nitrogen stream at room temperature. Following the addition of 500 p.L of acetic anhydride and pyridine, the mixture was heated at 85 °C for 3 h before being dried a second time. The R-butyl glycoside of galactose was then suspended in 1.5 mL of CHZC12 and analyzed using an Agilent 6850 series GC system, equipped with an Agilent 19091L-433E 50% phenyl siloxane capillary column (30 m X 250 ~m X 0.25 p,m) (170 °C to 250 °C, 2.8 °C-miri 1) (Agilent Technologies, Palo Alto, USA). 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 standaxd prepared using the same method (Sigma, Oakville, Canada).

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, Oakville, Canada) and a 1 mg sample of hydrolyzed enzyme purified HS:l CPS according to Rodriguez et al. As recommended by the manufacturer, the CPS sample was first treated overnight with (3-D-glucose oxidase (100 p,g~mL-i, 37 °C, Sigma, Oakville, Canada) to eliminate traces of D-glucose.
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-IO galactose standard to the R-butyl glycosides of an enzyme purified HS:1 CPS
sample, galactose was shown to have the D configuration. Furthermore, an intense increase in adsorption at 340 nm following treatment with a hexokinase-phosphoglucoisomerase-glucose-6-dehydrogenase-NADP fructose assay kit (Sigma, Oakville, Canada) indicated that fructose also had the D configuration. Both galactose and fructose were found to have the D-configuration and such a configuration is preferred. However, it will be understood that in some applications an enantiomer could be used to develop diagnostics or therapeutics.
HR-MAS NMR spectroscopy of cell-bound CPS
For HR-MAS analysis, G jejuni HS:1 cells were prepared as according to Szymanski et al. Overnight growth from one MH agar plate was harvested and placed in 1 mL of IO mM
potassium-buffered 98% D20 (pD 7.0) (Cambridge Isotopes Laboratories Inc, Andover, USA) containing 10% sodium azide (w/v) for 1 h at room temperature to kill cells. Cells were then pelleted by centrifugation (8900 G for 2 min), and washed once with 10 mM
potassium buffered D20. Approximately 10 ~,L of 1 % (w/v) TSP was then added as an internal standard (0 ppm) to the cell suspension prior to being loaded into a 40 ~,L nano NMR
tube (Varian, Palo Alto, USA) using a long tipped pipette cut diagonally approximately 1 cm from the end.
HR-MAS experiments were performed using a Varian Inova 500 MHz spectrometer equipped with a Varian 4 mm indirect detection gradient nano-NMR probe with a broadband decoupling coil (Varian, Palo Alto, USA) as previously described. Spectra from 40 ~L cell samples were spun at 3 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. 1H
NMR spectra of bacterial cells were acquired using the Carr-Purcell-Meiboom-GiII (CPMG) pulse sequence (90-(z-180-i)n-acquisition) to remove broad signals originating from lipids and solid-like materials, and the total duration of the CPMG pulse (n*2 i) was 10 ms with i 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 ZD-NOESY spectrum for cell-bound CPS was acquired using 16 transients1256 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).
High resolution NMR spectroscopy To obtain the hydrolyzed defructosylated repeating unit of HS:l CPS (CPS-1), a 3 mg sample of enzyme purified CPS was suspended in 150 pL of non-buffered 99% DZO
(pD 2.2) (Cambridge Isotopes Laboratories Inc, Andover, USA) and placed in a 3 mm NMR
tube (Wilmad, Buena, 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 1 h. For analysis of hot water/phenol purified CPS and enzyme purified CPS samples (CPS-2), a 3 mg sample of each was suspended in 150 p.L of NHaHC03 buffered 99% D20 (54 mM, pD 8.6), placed in 3 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 (IH) using a Varian mm, Z-gradient triple resonance cryogenically cooled probe (Varian, Palo Alto, USA). The methyl resonance of acetone was used as an internal reference (8H 2.225 ppm and 8~ 31.07 ppm). The 31P HSQC experiments were performed using a Varian Inova 500 MHz spectrometer equipped with a Varian Z-gradient 3 mm triple resonance (1H, 13C, 3iP) probe.
The 1D 31P spectra were acquired using a Varian Mercury 200 MHz (1H) spectrometer and a Nalorac 5 mm four nuclei probe. For all 31P experiments, spectra were referenced to an external 85% phosphoric acid standard (8P 0 ppm). NMR experiments were typically performed at 25 °C with suppression of the deuterated HOD resonance at 4.78 ppm. 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.
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, USA) coupled to a mass spectrometer (Applied Biosystems/Sciex, Concord, Canada) via a Turbo "V"
CE-MS
probe. A sheath solution (isopropanol:methanol, 2:1) was delivered at a flow rate of 1 p,L~miri 1. Separations were achieved on approximately 90 cm of bare fused-silica capillary (360 pm outside diameter x 50 ~m i.d., PolymicroTechnologies, Phoenix, USA) and 15 mM
ammonium acetate/ammonium hydroxide in deionized water (pH 9.0) containing 5%
MeOH
as mobile phase. 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 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 amuls.
Molecular dynamics simulations Molecular dynamics modeling was used to verify NOEs observed for C. jejuni HS:I
CPS during NMR analyses. CPS molecular models were constructed using the Biopolymer module of the Insight II Software package (Accelrys Inc, San Diego, USA), and then subjected to a 3000-step 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, non-bond 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, San Diego, USA) and data generated during the first 100 ps was discarded to allow the systems to reach equilibrium. A Verlet algorithm with a 2 fs timestep, extensible systematic forcefield, group-based nonbond method with a cutoff distance of 9.5 A and a distance-dependent dielectric value of 4 was then used for the simulations with trajectory frames being saved every 0.25 ps. The molecular model of an energy minimized structure was drawn using VMD.

Table XXI. NMR proton and carbon chemical shifts 8 (ppm) for an auto-hydrolyzed enzyme purified sample of C. jejuni HS:1 CPS (CPS-1) and corresponding hydrolysis products.

Atom Type 8H
A1 CH 5.20 98.9 A2 CH 3.87 70.4 A3 CH 3.98 69.9 A4 CH 4.54 75.5 AS CH 4.17 71.5 A6/A6' CH2 3.74/3.74 61.6 Bvgl~ CHZ 4.11/4.05 65.2 B2 CH 3.97 77.9 B3/B3' CHZ 3.76/3.76 62.1 Frufl/1' CH2 3.56/3.64 63.4 Fru, f Z C - 105.1 Fruf3 CH 4.10 76.1 Fruf4 CH 4.10 75.2 FrufS CH 3.82 81.4 Fruf6/6' CHZ 3.67/3.79 63.2 Frupl/1' CHZ 3.55/3.70 64.5 Frup2 C - 98.8 Frup3 CH 3.79 68.3 Frup4 CH 3.88 69.3 FrupS CH 4.02 69.4 Frup6/6' CHZ 3.7014.02 64.1 The 31P chemical shift for the monophosphate diester linkage was 8P 0.49 ppm.

Table XXII. NMR proton and carbon chemical shifts 8 (ppm) for an intact enzyme purified sample of C. jejuni HS:1 CPS (CPS-2).

Atom Type 8H S~
A1 CH 5.40 98.8 A2a CH 4.29 68.5 A2b CH 4.28 68.3 A3a CH 4.33 69.4 A3b CH 4.40 68.7 A4a CH 4.74 77.2 A4b CH 4.69 77.3 AS CH 4.16 72.0 A6/A6' CHZ 3.76/3.76 61.6 B1B1' CHZ 4.15/4.11 64.5 B2 CH 4.02 77.1 B3B3' CHZ 3.84/3.76 61.6 Cl/Cl' CH2 3.78/3.63 62.4 C2 C - 104.1 C3 CH 4.84 79.7 C4 CH 4.52 73.2 C5 CH 3.85 81.2 C6/C6' CH2 3.86/3.77 62.5 MeOPN CH3 3.81 54.9 *Cl/Cl' CH2 3.78/3.63 62.4 *C2 C - 104.1 *C3 CH 4.12 77.0 *C4 CH 4.12 76.6 *CS CH 3.75 81.5 *C6/C6' CHZ 3.86/3.77 62.5 *Chemical shift data 8 (ppm) for unsubstituted (3-D-fructofuranoside (MeOPN is absent). The 31P chemical shifts for the monophosphate diester linkages of Gal H-4a and b were 8P 0.40 ppm and 0.49 ppm, respectively. The ~1P chemical shift for the MeOPN groups was 14.67 ppm, and a scalar coupling 3JP,H of 11.1 Hz was observed.

Table XXIII. Negative ion CE-ESI-MS data (-400 V orifice voltage), calculated masses and proposed fragments for auto-hydrolyzed (CPS-1) and intact (CPS-2) samples of HS:1 CPS.
Observed Calculated D'ference 153.1 153.1 0.0 GroP

171.3 171.1 0.2 GroP + HZO

223.3 223.1 0.2 Hex + P - (H20)2 254.8 254.2 0.6 Hex + MeOPN

259.0 259.1 0.1 Hex + P + H20 297.3 297.2 0.1 Hex + GroP - HZO

315.3 315.2 0.1 Hex + GroP

333.5 333.2 0.3 Hex + GroP + H20 377.5 377.2 0.3 Hex + GroP + P - H20 385.3 385.2 0.1 (Hex)2 + P - H20 395.3 395.2 0.1 Hex + GroP + P

398.3 398.2 0.1 (Hex)2 + MeOPN - HZO

407.5 407.3 0.2 Hex + GroP + Gro + H20 416.8 416.3 0.5 (Hex)2 + MeOPN

453.3 453.3 0.0 (Hex)2 + MeOPN + (H20)Z

459.3 459.5 0.2 (Hex)2 + GroP - H20 469.3 469.3 0.0 Hex + (GroP)2 - HZO

477.3 477.3 0.0 (Hex)2 + GroP

487.3 487.3 0.0 Hex + (GroP)2 + H20 490.8 490.4 0.4 (Hex)2 + Gro + MeOPN

495.5 495.4 0.1 (Hex)2 + GroP + H20 509.6 509.4 0.2 (Hex)2 + Gro + MeOPN

539.3 539.3 0.0 (Hex)2 + GroP + P - H20 551.5 551.4 0.1 (Hex)2 + GroP + Gro 557.5 557.3 0.2 (Hex)2 + GroP + P

570.3 570.4 0.1 (Hex)2 + GroP + MeOPN

578.8 578.4 0.4 (Hex)3 + MeOPN

613.5 613.4 0.1 (Hex)2 + (GroP)2 - H20 621.5 621.5 0.0 (Hex)2 + GroP - H20 631.3 631.4 0.1 (Hex)2 + (GroP)2 639.3 639.5 0.2 (Hex)3 + GroP

652.3 652.5 0.2 (Hex)3 + Gro + MeOPN

658.2 658.4 0.2 (Hex)3 + MeOPN + P

Molecular mass (m/z) Structure Observed Calculated Difference 667.5 667.4 0.1 (Hex)z + (GroP)z + (HZO)2 671.8 671.5 0.3 (Hex)3 + (MeOPN)z 701.5 701.4 0.1 (Hex)3 + GroP + P - Hz,O

723.3 723.5 0.2 (Hex)z + (GroP)z + Gro +

732.3 732.5 0.2 (Hex)3 + GroP + MeOPN

751.4 751.4 0.0 (Hex)3 + (MeOPN)z + P

775.5 775.5 0.0 (Hex)3 + (GroP)z - Hz0 793.5 793.5 0.0 (Hex)3 + (GroP)z 793.3 793.5 0.2 (Hex)z + GroP + MeOPN+ P

801.6 801.6 0.0 (Hex)4 + GroP

829.5 829.6 0.1 (Hex)3 + (GroP)2 + (H20)2 855.3 855.5 0.2 (Hex)3 + (GroP)z + P - H20 886.8 886.6 0.2 (Hex)3 + (GroP)z + MeOPN

894.5 894.6 0.1 (Hex) + GroP + MeOPN

905.5 905.5 0.0 (Hex)3 + GroP + (MeOPN)z + P

929.8 929.6 0.2 (Hex)3 +
(GroP)3 -Hz.O

947.5 947.6 0.1 (Hex)3 +
(GroP)3 987.5 987.7 0.2 (Hex)4 + GroP + (MeOPN)z 1048.5 1048.7 0.2 (Hex)4 + (GroP)z + MeOPN

1109.5 1109.7 0.2 (Hex)4 + (GroP)3 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 CH30P(O)(NHZ), 93.2; P
(phosphate), 80.0; H20, 18Ø For these gas-phase (IS-CID) degradation products, no Hz0 molecule is added to the residues unless specifically indicated.

Figure legends Fig. 28. 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, Gl [14]. The phase variable genes in Gl which could be involved in the structural heterogeneity described in this report are indicated by white arrows. b) For CPS-2, the repeating unit is [-4)-oc-D-Galp-(1-2)-(R)-Gro-(1-P-]n with MeOPN-3-(1-D-fructofuranose branches at C-2 and C-3 of Gal. Structural heterogeneity is due to variable phosphoramidate groups on non-stoichiometric fructose branches. Residue A is a-D-Galp, oc-D-galactopyranose, residue B is GroP, glycerol-phosphate, residue C is ~i-D-Fruf, ~i-D-fructofuranose; and MeOPN is O-methyl phosphoramidate, CH30P(O)(NH2)(OR).
Fig. 29. NMR analysis of purified and cell-bound 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. (fj 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, IJP,H =10 Hz) for cell-bound CPS. (i) HR-MAS 31P
HSQC
spectrum (23 °C, 512 transients, 64 increments, 1JP,H =10 Hz) for cell-bound CPS. For the selective 1D experiments, excited resonances are underlined.
Fig. 30. 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-(60 ms). (e) 31P HSQC with 1JP,H =10 Hz, 64 transients and 240 increments. (f) with IJc,H 140 Hz, 8 transients and 256 increments. For the selective 1D
experiments, excited resonances are underlined. Ff and Fp represent the fructofuranose and fructopyranose monosaccharides, respectively.

Fig. 31. 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) HSQC with 'JP,H = 20 Hz, 8 transients and 32 increments. (h) 13C HSQC with IJc,H = 150 Hz, 80 transients and 256 increments. For the selective 1D experiments, excited resonances are underlined. Residue C represents Fru represents with MeOPN present and residue *C, Fru with no MeOPN.
Fig. 32. 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:l 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.
Fig. 33. Molecular model for the G jejuni HS:1 CPS. The [-4)-a-D-Galp-(1-2)-(R)-Gro-(1-P-]n repeating unit with both MeOPN-substituted ~i-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.

References The inclusion of a reference is not an admission or suggestion that it is relevant to the patentability of anything disclosed herein.
[1] Karlyshev,A.V., Champion,O.L., Churcher,C., Brisson,J.R., Jarrell,H.C., Gilbert,M., Brochu,D., St Michael,F., Li,J., Wakarchuk,W.W., Goodhead,L, Sanders,M., Stevens,K., White,B., Parkhill,J., Wren,B.W., & Szymanski,C.M.
(2005) Analysis of Campylobacter jejuni capsular loci reveals multiple mechanisms for the generation of structural diversity and the ability to form complex heptoses. Mol. Microbiol. 55, 90-103.
[2] Szymanski,C.M., St Michael,F., Jarrell,H.C., Li,J., Gilbert,M., Larocque,S., Vinogradov,E., & Brisson,J.R. (2003) Detection of conserved N-linked glycans and phase-variable lipooligosaccharides and capsules from campylobacter cells by mass spectrometry and high resolution magic angle spinning NMR spectroscopy. J. Biol. Chem. 278, 24509-24520.
[3] McDonald,A.G. (1993) Lipopolysaccharides from Campylobacter, Ph.D.
Thesis. York University, North York, Canada.
[4] Moran,A.P., Penner,J.L., & Aspinall,G.O. (2000) Campylobacter Lipopolysaccharides. In Campylobacter (Nachamkin,I. & Blaser,M.J., eds), pp. 241-257. American Society for Microbiology, Washington, D.C.
[5] Westphal,0. & Jann,K. (1965) Bacterial lipopolysaccharide. Extraction with phenol-water and further applications of the procedure. Methods Carbohydr.
Chem. 5, 88-91.
[6] Darveau,R.P. & Hancock,R.E. (1983) Procedure for isolation of bacterial lipopolysaccharides from both smooth and rough Pseudomonas aeruginosa and Salmonella typhimurium strains. J. Bacteriol. 155, 831-838.

[7] Huebner,J., Wang,Y., Krueger,W.A., Madoff,L.C., Martirosian,G., Boisot,S., Goldmann,D.A., Kasper,D.L., Tzianabos,A.O., & Pier,G.B. (1999) Isolation and chemical characterization of a capsular polysaccharide antigen shared by clinical isolates of Enterococcus faecalis and vancomycin-resistant Enterococcus faecium. Infect. Immun. 67, 1213-1219.

[8] Hsieh,Y.C., Liang,S.M., Tsai,W.L., Chen,Y.H., Liu,T.Y., & Liang,C.M.
(2003) Study of capsular polysaccharide from Vibrio parahaemodyticus. Infect.
Immun. 71, 3329-3336.

[9] Li,J., Wang,Z., & Altman,E. (2005) In-source fragmentation and analysis of polysaccharides by capillary electrophoresis/mass spectrometry. Rapid Commun. Mass Spectrom. 19, 1305-1314.

[10] Rodriguez,M.L., Jann,B., & Jann,K. (1990) Structure and serological properties of the capsular K11 antigen of Escherichia coli 013:K11:H11.
Carbohydr. Res. 196, 101-109.

[11] Sawardeker,J.S., Sloneker,J.H., & Jeanes,A. (1965) Quantitative determination of monosaccharides as their alditol acetates by gas liquid chromatography.
Anal. Chem. 37, 1602-1604.

[12] Loentein,K., Lindberg,B., & Lonngren,J. (1978) Assignment of absolute configuration of sugars by G.L.C. of their acetylated glycosides formed from chiral alcohols. Carbohydr. Res. 62, 359-362.

[13] F.St.Michael, C.M.Szymanski, J.Li, K.H.Chan, N.H.Khieu, S.Larocque, W.W.Wakarchuk, J.R.Brisson, and M.A.Monteiro. Eur. J. Biochem. 269, 5119 (2002).

[14] N.M.Young, J.R.Brisson, J.Kelly, D.C.Watson, L.Tessier, P.H.Lanthier, H.C.Jarrell, N.Cadotte, F.St Michael, E.Aberg, and C.M.Szymanski. J. Biol.
Chem. 277, 42530 (2002).
References from Part "E"

[15] Darveau,R.P. & Hancock,R.E. (1983) Procedure for isolation of bacterial lipopolysaccharides from both smooth and rough Pseudomonas aeruginosa and Salmonella typhimurium strains. J. Bacteriol. 155, 831-838.

[16] Huebner,J., Wang,Y., Krueger,W.A., Madoff,L.C., Martirosian,G., Boisot,S., Goldmann,D.A., Kasper,D.L., Tzianabos,A.O., & Pier,G.B. (1999) Isolation and chemical characterization of a capsular polysaccharide antigen shared by clinical isolates of Enterococcus faecalis and vancomycin-resistant Enterococcus faecium. Infect. Immun. 67, 1213-1219.

[17] Hsieh,Y.C., Liang,S.M., Tsai,W.L., Chen,Y.H., Liu,T.Y., & Liang,C.M.
(2003) Study of capsular polysaccharide from Vibrio parahaemolyticus. Infect.
Immun. 71, 3329-3336.

[18] Karlyshev,A.V., Champion,O.L., Churcher,C., Brisson,J.R., Jarrell,H.C., Gilbert,M., Brochu,D., St Michael,F., Li,J., Wakarchuk,W.W., Goodhead,L, Sanders,M., Stevens,K., White,B., Parkhill,J., Wren,B.W., & Szymanski,C.M.
(2005) Analysis of Campylobacter jejuni capsular loci reveals multiple mechanisms for the generation of structural diversity and the ability to form complex heptoses. Mol. Microbiol. 55, 90-103.

[19] Li,J., Wang,Z., & Altman,E. (2005) In-source fragmentation and analysis of polysaccharides by capillary electrophoresislmass spectrometry. Rapid Commun. Mass Spectrom. 19, 1305-1314.

Part D - Section 2 Because Campylobacter jejuni is the leading cause of bacterial food-borne gastroenteritis throughout the world, there is intense effort to determine the mechanisms of infectivity associated with this bacterium. Capsular polysaccharide (CPS) is an important virulence factor in C. jejuni and a recent study that examined the genome-sequenced NCTC11168-26 strain identified several phase-variable CPS modifications including an unusual O-methyl phosphoramidate (OMePN) group on C-3 of a GalfNAc residue.
There is disclosed herein the examination of the OMePN group using homo- and heteronuclear HR-MAS NMR experiments of whole bacterial cells grown on lsNH4C1-enriched media.

HSQC NMR experiments showed that the level 1sN labeling within the OMePN
reached 80%, and a large 1sN_31P scalar coupling provided direct evidence that confirmed the structure of the OMePN as CH30P(O)(NHZ)(OR). Because 1sN was detected within the major outer membrane protein as well as NAc and NGro groups of CPS and N-linked protein glycan, ammonium was concluded to be an important building block used in the synthesis of amino acids and glycan structures in C. jejuhi. HR-MAS NMR
studies of IsN-labeled cells revealed an unanticipated level of complexity as multiple OMePN
signals were observed within the 31P HSQC spectra for the NCTC11168-26 and 11168-H
strains.
While some signals originated from the OMePN at C-3 of GaljNAc, others were attributed to a novel OMePN located on D-glycero-a-L-gluco-heptopyranose. Together, these HR-MAS NMR findings shed light on nitrogen metabolism in C. jejuni, confirm the chemical structure of the OMePN and demonstrate that it occurs on both furanose and pyranose capsular polysaccharide sugars for this bacterium.
There is disclosed herein a method of labeling the OMePN with 1sN by growing C.
jejuni on lsNH4Cl-enriched media. The amount of 1sN incorporation and the structure of the OMePN were determined directly on the surface of bacterial cells using homo-and heteronuclear HR-MAS NMR experiments. Since nitrogen metabolism in campylobacters is uncharacterized, CE-ESI/MS and NanoLC-MS/MS were used to determine the extent of isN labeling within select proteins and glycan structures.

Experimental - Part D - Section 2 Solvents and reagents Unless otherwise stated, all solvents and reagents were purchased from Sigma Biochemicals and Reagents (Oakville, Canada).
Bacterial strains, media and growth conditions C. jejuni 11168-H (HS:2) and C. jejuni 11168-26 (HS:2) are identical strains of the bacterium that were isolated from different geographical locations. Both isolates were routinely maintained on Mueller Hinton (MH) (Difco, Kansas City, USA) plates, while the C. jejuni kpsM mutant (St. Michael, 2002) was grown on MH plates supplemented with 30 qg mL-1 kanamycin under microaerophilic conditions (10% COZ, 5% OZ and 85% N2) at 37°C. 14N- and 15N-labeled MH plates were prepared by combining filter sterilized (0.22 p.m, Millipore, Billerica, USA) aqueous solutions of varying concentrations of (Sigma, Oakville, Canada) or 15NH4C1 (Cambridge Isotopes Laboratories Inc, Andover, USA) with autoclaved MH agar to a final volume of 20 mL prior to pouring the plates. To obtain 14N- and 15N-labeled bacterial cells, strains were grown for a total of 48 h under microaerophilic conditions on 14N- or 15N-labeled plates, with transfer onto fresh plates after 24 h.
HR-MAS NMR spectroscopy For HR-MAS analysis, bacterial cells were prepared as described in Part D, Section 1 and in Szymanski, (J.B.C, 2003). For HR-MAS analysis of 15N labeling within the N
linked protein glycan (9, 20), bacterial cells were harvested and killed as previously described, however; as a final step they were washed twice in a 90% H20 KH2P04 buffer (10% D20, pH 5.5) before being loaded into a 40 ~.L nano NMR tube (Varian, Palo Alto, USA). HR-MAS experiments were performed using a Varian Inova 500 MHz spectrometer equipped with a Varian 4 mm indirect detection gradient nano-NMR probe with a broadband decoupling coil (Varian, Palo Alto, USA). Spectra from 40 ~L cell samples were spun at 3 kHz and recorded at ambient temperature (23°C) and all experiments were performed with suppression of the HOD signal by presaturation as previously described in Szymanski (J.B.C., 2003). 1H NMR spectra of bacterial cells were acquired using the Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence (90-(i-180-i)n acquisition) to remove broad signals originating from lipids and solid-like materials (21), and the total duration of the CPMG pulse (n*2 i) was 10 ms with i set to (1/MAS spin rate). 1H NMR spectra for cell-bound CPS on bacterial cells were typically obtained using 256 transients (11 min). 1D and 2D 31P HSQC were acquired with the standard Varian HSQC pulse sequence with 1D
spectra representing the first increment of the standard HSQC experiment.
Unless otherwise specified, 1D 31P HSQC spectra were obtained with 600 scans and 1JP,H =10 Hz (20 min) while 2D 31P HSQC spectra were obtained using 128 scans, 64 increments and IJP,H =10 Hz (8 h).
Mass spectrometric analysis of CPS
For mass spectrometric analysis of 14N- and 15N-labeled CPS, C. jejuni NCTC11168-26 was grown on four MH plates containing 100 mM of ~4NH4C1 or for 48 h as described above. Cells were harvested and blended in 90% phenol at 96°C for min to extract CPS, allowed to cool for 30 min and then dialyzed (MWCO 12 kDa, Sigma, Oakville, Canada) against running water for 72 h to eliminate traces of phenol. Cells were then ultracentrifuged (140,000 X g, 15°C) for 2 h and the supernatant, which 15 contained crude CPS, was lyophilized to dryness. CE-ESI/MS analysis was performed using a Crystal Model 310 Capillary Electrophoresis instrument (ATI Unicam, Boston, USA) coupled to an API 3000 mass spectrometer (Perkin-Elmer/Sciex, Wellesley, USA) via a microIon spray interface. A sheath solution (isopropanol:methanol, 2:1) was delivered at a flow rate of 1 p,L~miri 1. Separations were achieved on approximately 90 cm of bare fused-silica capillary (360 ~m outside diameter x 50 pm i.d., Polymicro Technologies, Phoenix, USA) and 15 mM ammonium acetate/ammonium hydroxide in deionized water (pH 9.0) containing 5% MeOH as mobile phase. 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 1 unit in full-mass scan mode.
Fragment ions formed by collision activation of selected precursor ions with nitrogen in the RF-only quadrupole collision cell were mass analyzed by scanning the third quadrupole.
All samples were analyzed in positive ion mode using an orifice voltage of 50 V.
Mass spectrometric analysis of proteins For mass spectrometric analysis of 14N- and 15N-labeling in proteins, C.
jejuni NCTC11168-26 was grown on two MH plates containing 100 mM of 14NH4C1 or for 48 h as described above. Cells were harvested and proteins were extracted for 20 min with 500 ~.L of cold glycine (0.2 M, pH 9.2). Extracts were then centrifuged (8,900 X g for 2 min) to pellet cells and the supernatant was retained. A 2 X SDS PAGE sample buffer (10 mL 1.5 M Tris pH 6.8, 6 mL 20% SDS, 30 mL glycerol, 15 mL ~i-mercaptoethanol, 1.8 mg bromophenol blue, 40 mL H20) was then added to the extracts (bufferaupernatant, 3:1 v/v ratio) prior to heating at 100°C for 15 min. To purify proteins, 20 p,L
of crude protein extract was loaded onto a 10% acrylamide gel (30% acrylamide:Bis solution, 37:1, BioRad, Mississauga, Canada) and separation of proteins was achieved over 45 min (200 V, 0.04 A). Protein bands were visualized by staining with colloidal coomassie blue for 45 min (Biosafe Coomassie, 6250 stain, BioRad, Mississauga, Canada) and pure protein bands were manually excised from the gel. Proteins were then destained with a 1:1 ratio of 30 mM
potassium ferricyanide and 100 mM sodium thiosulphate, digested with Promega modified trypsin (Madison, USA) overnight in 50 mM NH4HC03 and peptides were extracted with 5% HOAc/50% aqueous MeOH as previously described in Young (J.B.C., 2002).
Peptide fragments were then analyzed by NanoLC-MS/MS using a Q-TOF2 mass spectrometer (Micromass, Waters, Milford, USA) in automated mode as previously reported in Young (J.B.C., 2002).
Results Growth of C. jejuni cells on lSNHdCI plates To establish unambiguously the structure of the OMePN residue, is was sought to demonstrate a 3'P-1sN scalar coupling in the 31P HSQC spectra of CPS
biosynthetically labeled with lsN. Since the biosynthetic pathways responsible for OMePN
production in C.
jejuni are unknown, ammonium chloride utilization by this bacterium was examined. In order to label the OMePN with lsN, C. jejuni NCTC11168-26 cells were grown on six different concentrations of lsNH4C1 for 48 h (0, 10, 25, 50, 75 and 100 mM) and then examined with HR-MAS NMR. Although bacteria exhibited moderately reduced growth at 75 and 100 mM NH4Cl, the 1H HR-MAS NMR spectrum for cells grown on 100 mM of lsNH4Cl had identical spectral features compared to control cells grown on unlabeled MH
plates (Fig. 35a vs. 35b). Because expression of sensitive phase-variable CPS
modifications such as ethanolamine, methyl and OMePN groups was not affected, it was concluded that growth on lsNH4C1-enriched media did not introduce obvious structural artifacts within the CPS.

HR-MAS NMR analysis of 15N labeling within the OMePN
To quantify the levels of IsN labeling within the OMePN modification, C.
jejuni NCTC11168-26 cells grown on different concentrations of lsNH4Cl were examined using 3iP HSQC HR-MAS NMR (Fig. 36). Because of the low natural abundance of lsN, 3iP-isN
scalar couplings were not observed for the OMePN residue in spectra of control cells grown on unlabeled MH plates resulting in one phosphorous signal at 13.6 ppm (Fig. 36a).
Upon the addition of 10 mM lsNH4C1 to the media however, additional phosphorous signals were observed as a result of a lJlsrr,3ir scalar coupling (Fig. 36b).
The magnitude of the 1J isrr,3iP scalar coupling observed for the OMePN at 39 Hz is consistent with values reported for iJ isN,3iP scalar couplings in the literature. Integration of these new signals revealed that the addition of 10 mM of lsNH4Cl to the media resulted in approximately 35%
1sN labeling within the OMePN. Increasing concentrations of lsNH4C1 within the media resulted in higher levels of 1sN incorporation within the OMePN. For instance, bacteria grown on 25 mM, 50 mM and 75 mM of lsNH4C1 exhibited 50%, 65% and 75% 1sN
labeling, respectively, within their OMePN modifications (Fig. 36c, d and e).
Bacteria grown on 100 rnM of lsNH4C1 exhibited the highest incorporation of 1sN within the OMePN at 80% (Fig. 36f).
HR-MAS NMR analysis of 15N labeling within the N-linked protein glycan HR-MAS NMR was used to determine if 1sN was being incorporated into the N-linked protein glycan that modifies several glycoproteins in C. jejuni. The acapsular mutant of C. jejuni, kpsM, was grown on agar enriched with 100 mM lsNH4Cl and analyzed with IH HR-MAS NMR in a 90% H20 KH2P04 buffer (10% DZO, pH 5.5) (Fig. 37). By comparing the 1sN-1H coupled proton spectrum to the decoupled spectrum, at least three signals in the 8.1-8.6 ppm region exhibited 1sN coupling (Fig. 37a and b).
Based on its chemical shift and coupling constant, an 1sN-coupled signal at 8H 8.21 ppm (lJlsrr>iH 7.5 Hz) was determined to originate from the NAc protons of GaINAc residues within the N-linked protein glycan (St. Michael Eur. J. Biochem., 2002) thereby confirming 1sN
incorporation within this glycan structure.
Mass spectrometric analysis of 15N labeling within CPS
To confirm 1sN incorporation within the OMePN and determine if the NGro and NAc CPS groups were being labeled, CPS was partially purified from NCTC11168-26 cells grown on 100 mM lsNH4C1 or 14NH4Cl and analyzed with CE-ESI/MS. Approximately mg of cells (wet pellet mass) was harvested from four lsNH4Cl enriched plates that yielded 16 mg of crude CPS. A comparable amount of cells (281 mg) and crude CPS (12 mg) were recovered from four plates enriched with 14NH4C1. Due to the presence of contaminating ions originating from proteins, nucleic acids and lipids, precursor ion scans for D-glycero-a-L-gluco-heptopyranose (m/z 205) were used to mass analyze both CPS samples (Fig. 38a and b). CE-ESI/MS analysis of CPS prepared from cells grown on 14NH4C1 labeled media showed the mass of one repeat to be m/z 884 [M + 1H]'+, and was consistent with what has been reported for C. jejuni NCTC11168-26 (Fig. 5a). An intense ion at m/z 791, representing one repeating unit minus the OMePN [M+ - 93]'+, showed that the dominant isotope of nitrogen within the OMePN for these cells was 14N. Because only one sharp signal was observed for m/z 884, it was concluded that the amount of 15N
labeling for the NGro and NAc CPS groups was negligible. In contrast, three distinct ions at m,/z 885, 886 and 887 were observed for one CPS repeat for cells grown on 'SNH4Cl media that confirmed 'SN labeling within the OMePN, NAc and NGro CPS groups (Fig. 5a vs.
b).
Furthermore, a strong ion at m/z 792.5 representing one CPS repeat minus the OMePN
[M+ - 94J'+ showed that 15N was the dominant isotope of nitrogen within the OMePN fox these cells.
Mass spectrometric analysis of 15N labeling within proteins Following staining of the acrylamide gel with colloidal coomassie blue, a strong protein band at 47 kDa was identified as the major outer membrane protein (MOMP) using in-gel tryptic digestion and NanoLC-MS/MS. To analyze 15N incorporation within this protein, the isotope profiles of the tryptic peptides from'4N and'SN labeled MOMP as well as their MS/MS fragment ions were compared. As an example, the MS/MS spectra obtained for the 14N- and 15N-labeled tryptic peptide 3s3VGADFVYGGTK363 are presented in Figure 5c and d, respectively. The y8 (m/z 886.5) and y9 (m/z 957.5) fragment ions are presented in the insets. Based on their isotope profiles we calculate that approximately 40°10 of the nitrogen atoms in the'SN-labeled ions have been substituted with'SN. This is in general agreement with the calculations performed for other peptides that were derived from proteins extracted from cells grown in the'SNH4Cl media.
HR-MAS NMR analysis of 15N-labeled OMePN in C. jejuni NCTC11168-26 and To characterize the distribution of OMePN on the surface of bacterial cells, C. jejuni NCTC11168-26 and 11168-H cells were grown on media enriched with 100 mM
14NH4Cl or 15NH4Cl and examined with HR-MAS NMR (Fig. 39 and 40). The ~'P HSQC
spectrum of 14N labeled NCTC11168-26 cells revealed five distinct signals with chemical shifts characteristic of phosphoramidates between 8P 13.2 and 13.8 ppm (Fig. 39a).
The 31P
HSQC spectrum of 15N labeled cells confirmed that these signals originated from OMePNs as they exhibited 1J15rr>3iP scalar couplings between 32-40 Hz (Fig. 39b). Two OMePN
signals were observed within the 1H HR-MAS spectrum for G jejuni 11168-H cells as overlapping doublets at 3.78 ppm and 3.76 ppm (3Jp,H 12 Hz) (Fig. 40a). The 3'P HSQC
spectrum of 1$N-labeled 11168-H cells showed these two signals at 8P 13.6 ppm and 14.1 ppm had lJisrr,3iP scalar couplings of 42 Hz and 40 Hz, respectively, and therefore confirmed that they originated from OMePNs (Fig. 40b).
That several OMePN signals were detected for C. jejuni NCTC 11168-26 and 11168-H
cells suggested either environmental heterogeneity on the cell surface resulting in multiple signals for the OMePN at C-3 of GalfNAc, or the presence of other OMePN(s) substituted elsewhere. To investigate these possibilities, a 1D HR-MAS 1H-31P HSQC
spectrum was acquired (JP,H 15 Hz) since OMePN cross peaks to carbohydrate residues are not typically observed for the 2D 31P HSQC experiment (Fig 41). In addition to the expected OMePN
methyl resonances and GalfNAc H-3 resonance at 4.94 ppm, another signal at 4.34 ppm was detected for 11168-H cells (Fig. 41 a). Inversion relative to that at 4.94 ppm of this novel resonance at 4.34 ppm was most likely the result of 1H homonuclear couplings during the 1/2J period that have magnitudes approaching the heteronuclear coupling (12 Hz), suggesting that the 31P is scalar coupled to either a geminal proton, or to a proton attached to a pyranose ring with a gluco configuration.
To establish that the resonance at 4.34 ppm was not an artifact, a 1D
difference spectrum was acquired using the pulse sequence shown in Figure 41c. During odd acquisitions, a CPMG pulse train was simultaneously applied to 1H and 31P
during the 1/2J
coherence transfer step to maintain in-phase 1H magnetization similar to the CPMG-INEPT
transfer. During even acquisitions, 31P ~t pulses were applied off-resonance (28 kHz) and subtracted. After an evolution time of 1/2J (J = 12 to 15 Hz), the heteronuclear single quantum coherence (IXSZ) is present but does not evolve into a detectable signal during acquisition when 31P is decoupled. Subtraction of the spectrum acquired without 3iP ~
pulses produces a difference spectrum in which only those protons scalar coupled to 31P are visible. This is similar to a heteronuclear spin-echo difference spectrum, however; with this experiment, the evolution time is reduced by a factor of two thereby reducing attenuation of the signal. The resulting spectrum for 11168-H cells exhibited peaks at 4.94 ppm and 4.34 ppm that were in-phase (Fig. 41b). Because both peaks were in-phase, it was concluded that the resonance at 4.34 ppm was not an artifact since the 1H CPMG-pulse train of this difference experiment selectively suppresses the evolution of homonuclear couplings (Jn,H).
It appears that the signal at 4.34 ppm originates from a novel OMePN located at the 3-O
or 4-O position of the D-glycero-a-L-gluco-heptopyranose CPS residue. For example, addition of the OMePN to the GalfNAc 3-O position in NCTC11168-26 was shown to cause a downfield shift of H-2 (0.2 ppm), H-3 (0.64 ppm) and H-4 (0.24 ppm) relative to that of the unsubstituted GalfNAc. Similarly, addition of OMePN to the 3-O
position of Fru in the CPS of the HS:1 serostrain lead to a downfield shift of H-3 (0.74 ppm) and H-4 (0.7 ppm) compared to Fru without OMePN. Inspection of the chemical shifts reported for the unsubstituted CPS of NCTC11168-26 reveals that the 3-O (3.72 ppm) and 4-O
(3.52 ppm) positions of D-glycero-a-L-gluco-heptopyranose are both candidates for the OMePN at 4.34 ppm, since this would correspond to downfield shifts of 0.61 ppm and 0.82 ppm, respectively, at these locations.
Discussion - Part D - Section 2 In section 1 use of HR-MAS NMR to show in vivo that the HS:1 serostrain of C.
jejuni has a structurally heterogeneous CPS that is partly due to variably substituted OMePN
groups was described. In section 2, we confirmed the chemical structure of the OMePN
CPS modification using HR-MAS NMR spectroscopy of cells grown on 15NH4Cl-enriched media was confirmed. Also provided are new findings on nitrogen metabolism and the biosynthetic pathway responsible for OMePN production in C. jejuni.
By monitoring the expression of phase-variable CPS modifications such as the -OCH3, ethanolamine and OMePN groups with 1H HR-MAS NMR, it was determined that the concentrations of NH4Cl used during this study did not affect the structure of the CPS.
These phase-variable CPS groups in C. jejuni are useful biomarkers since their expression is highly variable. The -0CH3 CPS modification is particularly useful for monitoring structural CPS changes with iH NMR due to the intensity and unique chemical shift of the signals originating from its three protons (Fig. 35).

HR-MAS NMR is well suited to studying the OMePN in C. jejuni since it is a highly labile structure that is readily hydrolyzed by chemical reagents used to purify CPS from bacteria. A large 1sN-31P coupling of Jlsrr,sir 39 Hz observed in the 1H-31P
HSQC for cells grown on IsNH4Cl-enriched media provided for the first time direct evidence confirming the P-N bond for the OMePN. That the amount of 1sN labeling within the OMePN
reached 80% and was directly correlated to the amount of lsNH4Cl in the media indicates that ammonium can be a primary source of nitrogen used for OMePN biosynthesis in C.
jejuni.
Because of phylogenetic similarities, it was previously thought that metabolism in C.
jejuni closely resembled Helicabacter pylori, however; the genome sequence fox C. jejuni NCTC11168-26 showed that only 55.4% of G jejuni genes have orthologues in H.
pylori.
Consequently, the factors that contribute to the metabolic versatility of C.
jejuni allowing it to adapt its metabolism to many environments within and outside its hosts are still poorly understood. That 1sN was detected within the major outer membrane protein, as well as the NAc and NGro groups of the CPS shows that C. jejuni can exploit exogenous sources of inorganic nitrogen to make amino acids and glycan structures. This result is consistent with the genome sequence of the NCTC11168-26 strain where several genes related to amino acid biosynthesis were shown to be present. That 1sN was found within the N
acetyl groups of the N-linked protein glycan supports a study that reported 60-80% 1sN
labeling of N
linked protein glycan NAc groups for animal cells grown on ~sNH4Cl-enriched media.
It is interesting that two OMePNs were unambiguously identified for the 11168-H
strain differing by ~8H 0.2 ppm; one at C-3 of Gal, f NAc (8H 3.76 ppm) and a novel OMePN
at C-3 or C-4 of D-glycero-a-z-gluco-heptopyranose (8H 3.78 ppm). Inspection of the 3iP
HSQC spectra for NCTC11168-26 cells shows that four of the five OMePN signals can be grouped into two general populations that also differ by 08H 0.2 ppm (8H 3.80 ppm and 8H
3.78 ppm) suggesting that a novel heptose-associated OMePN is present for this strain as well (Fig. 39). The diverse range of phosphorous chemical shifts exhibited by these OMePNs is possibly due to structural heterogeneity for cell-bound CPS or alternatively;
moderately different pH environments since the chemical shift of phosphoramidates vary considerably with slight changes in pH (26). That a weak OMePN signal at 8H
3.70 ppm and 8P 14.7 ppm was observed for NCTC11168-26 cells is suggestive of yet another OMePN at an unknown location. It is conceivable that OMePN could be located on glycan structures other than CPS. For example, the harsh chemical treatments presently used to purify LOS and other Glycan structures would likely hydrolyze the OMePN
resulting in it being overlooked by previous studies examining these structures in C. jejuni.
This is the first time that OMePN has been found on a pyranose ring since it was shown to be substituted on furanose sugars for the two structures reported to date for C. jejuni NCTC11168-26 (HS:2, C-3 of GaI,fNAc) and the HS:1 serostrain (C-3 of Fruf).
That OMePNs are found on both pyranose and furanose sugars in the 11168-H and NCTC11168-26 strains reveals that biosynthesis of this CPS modification is complex and suggests that the transfer of OMePN to CPS carbohydrates is possibly regulated by more than one gene. In light of the disclosure herein, one can develop reagents against OMePN to be used as diagnostics. In the case of the 11168-H strain, this study has shown that the phosphoramidate modification occurs at predominantly two sites in the CPS with approximately equal frequency. This raises the question as to whether each repeating unit in the CPS has two sites simultaneously decorated with the OMePN modification, or only one OMePN with equal frequency throughout the CPS. For the latter situation, two additional structures potentially exist since repeats with only one OMePN could be homogeneously or randomly distributed. These possibilities present largely different CPS
epitopes and have interesting implications regarding the details of CPS assembly from subunits containing the OMePN modification. While the basic CPS structure of the 11168-H strain has been established, it will be important to examine the structure of purified intact CPS with respect to the topology of phosphoramidation.
The 15N labeling method disclosed herein for C. jejuni can be used in a variety of ways, including labeling proteins, sugars and phase-variable modifications for NMR
analysis. In some instances, the amount of 15N incorporation within proteins and glycan structures can be augmented by growing G jejuni on minimal media instead of complex MH broth.

Figure captions Figure 34. CPS structure for C. jejuni NCTC11168-26 showing phase-variable modifications such as the 6-O-methyl group on D-glycero-a-L-gluco-heptopyranose, NGro on a-D-GlcpA and the OMePN on C-3 of (3-D-GalfNAc (8, 9). Abbreviations: Gro, glycerol; OMePN, O-methyl phosphoramidate.
Figure 35. HR-MAS NMR analysis investigating the effects of adding 'SNH4C1 to growth media in C. jejuni. (a) HR-MAS 'H NMR spectrum of C. jejuni NCTC11168-26 cells grown on MH plates enriched with 100 mM 'SNH4C1. (b) HR-MAS 'H NMR spectrum of C. jejuni NCTC11168-26 cells grown on regular MH media (control).
Abbreviations: Etn, ethanolamine; OMe, 6-O-methyl group of D-glycero-a-L-gluco-heptopyranose residue;
OMePN, O-methyl phosphoramidate.
Figure 36. HR-MASH-3'P HSQC NMR spectra showing'SN labeling within the OMePN
for C. jejuni NCTC11168-26 cells. All spectra are shown as one-dimensional traces of the 3'P dimension centered on the same OMePN modification. (a) Control cells grown on unlabeled MH plates. (b) Cells grown on MH plates enriched with 10 mM
of'$NH4C1. A
doublet generated by a 'SN-3'P scalar coupling is evident with a 'JN,P 39 Hz (~ 1 Hz).
(c)(d)(e) and (f) Cells grown on MH plates enriched with 25, 50, 75, and 100 mM of 'sNH4Cl, respectively.
Figure 37. HR-MAS 'H NMR spectra (90% H20/10% D20 KHZP04 buffer, pH 5.5) for acapsular cells, C. jejuni kpsM, grown on media enriched with 100 mM of 'SNH4Cl showing incorporation of 'SN within the common N linked protein glycan. (a) The 'SN-coupled HR-MASH NMR spectrum. Arrows indicate protons coupled to'SN. (b) The'SN-decoupled HR-MASH NMR spectrum.
Figure 38. Mass spectrometry analysis of 'SN incorporation within the CPS
structure and major outer membrane protein (MOMP) extracted from C. jejuni NCTC11168-26 cells. (a) CE-ESI/MS spectrum for CPS partially purified from cells grown on media enriched with 100 mM of 14NH4C1 and; (b) CE-ESUMS spectrum for partially purified CPS
prepared from cells grown on media enriched with 100 mM of lsNH4Cl (precursor ion scans mlz 250, positive ion mode, orifice voltage 50 V). (c) NanoLC-MS/MS spectrum of the doubly protonated MOMP peptide fragment ion at m/z 557.3 (3s3VGADFVYGGTK363) prepared from cells grown on MH plates enriched with 100 mM of 14NH4Cl and; (d) the MS/MS
spectrum obtained for the same peptide fragment ion prepared from cells grown on the lsNH4Cl media. The region around the y8 (mlz 886.5) and the y9 (m!z 957.5) fragment ions is shown in the insets in order to demonstrate the differences in the isotope profiles between the 14N-only and 1sN-labeled samples.
Figure 39. HR-MAS NMR analysis of the OMePN CPS modification on the surface of C.
jejuni NCTC11168-26 cells. (a) 31P HSQC NMR spectrum showing five distinct signals originating from OMePNs on the surface of cells grown on media enriched with 100 mM of 14NH4C1 and; (b) the same NMR experiment for cells grown on media enriched with 100 mM of lsNH4Cl. Nitrogen-phosphorous coupling constants (lJlsN,3lP, ~ 1 Hz) are indicated for OMePN residues.
Figure 40. HR-MAS NMR analysis of C. jejuni 11168-H cells. (a) HR-MAS 1H NMR
spectrum for cells grown on unlabeled MH plates showing two distinct OMePNs as overlapping doublets at 3.78 ppm and 3.76 ppm (3JP,H 12 Hz). Two anomeric signals were observed for residue C ((3-D-GaI,fNAc) representing two different forms of the sugar; one form where the OMePN is present at C-3 of the sugar (5.10 ppm) and another where the OMePN is absent (5.02 ppm). (b) HR-MAS 3'P HSQC NMR spectrum for cells grown on 100 mM of lsNH4C1 showing two OMePNs. Nitrogen-phosphorous coupling constants (IJISN,mP, ~ 1 Hz) are indicated for both OMePN residues. Abbreviations: A, (3-D-Ribf; B, a-D-GlcpA6(NGro); D, 6-O-Me-D-glycero-a-L-gluco-heptopyranose; OMePN, O-methyl phosphoramidate.
Figure 41. (a) 1D 1H-31P HSQC HR-MAS spectrum of the C. jejuni 11168-H strain showing proton resonances of CPS carbohydrates linked to OMePN residues. The peak at 4.94 ppm corresponds to the GalfNAc H-3 OMePN (total number of scans was 2048 and 1JH,P was 15 Hz). (b) 1H-31P correlation difference spectrum showing proton resonances of CPS carbohydrates linked to OMePN residues. The spectrum was acquired using the pulse sequence shown in (c) (total number of scans was 4096 and 1JH,P was 12 Hz).
(c) Pulse sequence used to obtain the 1H-31P correlation difference spectrum. Narrow and thick bars represent 90° and 180° RF pulses, respectively. The 90°
pulse durations were 4.5ps (1H) and 23 ps (31P). The 180° pulse durations for the CPMG pulse train were 27 ~s (yBl = 9.26 kHz) and 102 ps (yBl = 2.45 kHz) for 1H and 31P, respectively. The 31P pulses were on-resonance for the OMePN residue during odd scans, and off-resonance (28 kHz) for even scans. The receiver phase was alternated between x and -x. Pulse phases in the sequence are: [x,y,x,y,y,x,y,x,-x,-y,-x,-y,-y,-x,-y,-x]. The number of cycles (N) and the T
interval (-150 ~s) were calculated so that the duration of N consecutive XY-16 cycles corresponded to 0.5 JlH,3iP (12 Hz). Abbreviations: AQ, acquisition; OMePN, O-methyl phosphoramidate; Presat, presaturation.

Part E, Section 1 Expression of Campylobacter jejuni Phosphoramidate (OMePN) in vivo and Direct Detection using High Resolution Magic An~le Spinning (HR-MAS) NMR
1. Establishment of High Dose Model System in Specific Pathogen Free (SPF) Leghorn Chicks One day after hatching, each chick received an inoculum of 3 x 101° - 1 x 1011 colony forming units (cfu) of C. jejunj 11168H in sterile phosphate buffered saline. Due to the presence of the nutrient-filled yolk sac, chicks do not eat considerable amounts of feed for the first 48 hours after hatching. In this model, chicks were given only water for the 48 hours proceeding oral gavaging with C.
jejuni. This is to minimize the particulate matter in the chick's cecal contents that can interfere with the detection of the bacteria.
A 48 hour infection model was chosen due to the relatively short time period without feed, but long enough to ensure proper colonization in the cecum.
Originally, both 24 and 48 hour models were used, however both showed similar results and so the 48 hour model was chosen. The rate of passage through the gastrointestinal (GI) tract of leghorns is approximately 4 hours. Therefore, after 48 hours, all non-adhered bacteria remnant of the oral gavaging should be eliminated.
After the 48 hour incubation time, the chicks were sacrificed, their ceca were excised and the cecal contents collected. Serial dilutions of the cecal contents were plated onto C. jejuni selective Karmali plates in order to quantitate colonization levels. An inoculation of approximately 1011 cfu/ml of C. jejuni resulted in colonization levels that ranged from 10~-109cfu/ml after 48 hours (Table XXIV).
Bacteria present in the cecal contents were then washed with 10% sodium azide in D20 and analyzed directly by HR-MAS NMR.
2. Direct HR-MAS Analysis of Chick Cecal Contents Whole cell HR-MAS NMR of C. jejuni 11168H resulted in spectra similar to the representative spectrum shown in Figure 42b, which exhibits peaks corresponding to the different components of the capsular polysaccharide (Figure 42c) including the monosaccharides and phase variable modifications such as the rare OMePN
structure.
Figure 42c illustrates one of two populations of the CPS repeat in which the phosphoramidate was present on C3 of the GaljNAc residue. The next section in Part E of this provisional will provide evidence demonstrating that the phosphoramidate can also exist on C4 of the heptose residue. Conventional HR-MAS proton NMR
methods are unable to detect the Campylobacter jejuni capsular polysaccharide (CPS) present in the collected cecal contents (Figure 42a). However, using newly developed P-filtered heteronuclear correlation methods, the OMePN residues substituted at C3 on the GalfNAc and at the C4 position on the D-glycero-a-L-gluco-heptopyranose can be detected with confidence (Figure 43b).
A total of 19 chick cecal samples have been analyzed in the establishment of this model. Thirteen chicks representing triplicate experiments have been used for the 48 hour model where the detection rate of the OMePN moiety has been 100%
(Table XXIV, 6 chicks used for the original 24 hour model are not shown).
C. jejuni 11168H was not detected in the cecal contents using conventional HR-MAS proton NMR due to contaminating peaks, including those that overlap the methyl protons of the OMePN, and sensitivity issues in detecting the anomeric resonances. However, the OMePN CPS modification was detected using newly developed P-filtered heteronuclear NMR experiments. Based on the known CPS
structure and known chemical shift values, it appears that the GalfNac and the D-glycero-a-L-gluco-heptopyranose residues are also present and therefore C.
jejuni CPS was indirectly detected. These studies demonstrate that the phosphoramidate is indeed expressed in vivo in the chicken and that C. jejuni can be detected directly in these samples by HR-MAS NMR using the unique OMePN modification as a diagnostic marker. Thus, there is provided in an embodiment of the invention, use of an agent having affinity for the OMePN modification or otherwise able to allow its ready identification in the identification of C. jejuni in samples. For example, one could employ binders such as peptides, polypeptides and proteins (including antibodies and portions thereof) having binding affinity for the OMePH
modification to allow ready detection of C. jejuni. Binders may be conjugated to reporters (e.g.
fluorescent, enzymes catalyzing a colourometric reaction, etc.) and/or may be modified or selected so as to interact with a reporter to produce an assayable signal.

E
Chip ~Iumt~ .
' ~ ~~ : l3e~e~'k~n ~c~ml~y _ , a~

91 1.46E+09 +

154 1.18 E+07 +

157 1.48E+07 +

158 4.28E+07 +

159 1.13 E+07 +

161 1.35 E+07 +

163 1.85E+07 +

168 6.33E+09 +

170 1.57E+10 +

20 9.53E+07 +

222 2.43E+08 +

223 6.67E+08 +

224 1.38 E+09 +

to Table XXIV

Part E, Section 2 Extended structural determination for the capsular polysaccharide of Campylobacter,jejuni 11168- Demonstration of phosphoramidate modification on the capsular heptose Sequential inactivation of the cps biosynthetic genes in Campylobacter jejuni followed by phosphoramidate filter analysis has identified multiple genes encoding enzymes involved in the biosynthesis of the phosphoramidate modification (OMePN). In particular, cj1421 c and cj1422c are thought to be taransferases and would play an important role in adding the phosphoramidate to CPS sugars. In C. jejuni 11168 where the OMePN was first described, cj1422c is predominantly off, while cj1421 c is mostly on and is thought to add the OMePN to C-3 of GaljNAc. There is disclosed herein the function of cj1422c. This was determined by determining the structure of the CPS for C. jejuni 1421-1, a mutant where cj1421c has been inactivated and cj1422c is on. To do so, CPS was extracted using a gentle enzymatic method (see Part D) from a large scale growth of C. jejuni 1421-1. The structure of the CPS was then determined using homo- and heteronuclear high resolution NMR experiments.
Media and growth conditions The C. jejuni 1421-1 strain was routinely maintained on Mueller Hinton (MH) agar (Difco, Kansas City, USA) plates under microaerophilic conditions (10% C02, 5%
02, 85%
N2) at 37 °C. For large scale extraction of CPS, 6 L of C. jejuni 1421-1 was grown in Brain Heart Infusion (BHI) broth (Difco, Kansas City, USA) under microaerophilic conditions at 37 °C for 24 h with agitation at 100 rpm. Bacterial cells were then harvested by centrifugation (9,000 X g for 20 min) and placed in 70% ethanol. Cells were removed from the ethanol solution by centrifugation (9000 X g for 20 min) and the bacterial pellet was refrigerated until extraction.
Enzymatic isolation of CPS
An enzymatic method of isolating CPS from C. jejuni 1421-1 cells was used based on the methodologies of Darveau and Hancock (1983), Huebner et al. (1999) and Hsieh et al. (2003). Bacterial cells harvested from 6 L of BHI broth were suspended in PBS
buffer (pH 7.4). Lysozyme was then added to a final concentration of 1 mg~mL-1 (Sigma, Oakville, Canada) prior to the addition of mutanolysin to a final concentration of 67 U~mL-1 (Sigma, Oakville, Canada). The bacterial cell suspension was then incubated for 24 h at 37 °C with agitation at 100 rpm. The mixture was emulsiflexed twice (21000 psi) to lyse cells, and DNAse I and RNAse (130 pg~mL-1 DNAse I
and RNAse, Sigma, Oakville, Canada) was added prior to being incubated for 4 h at 37 °C
with agitation at 100 rpm. Following digestion with nucleases, pronase and protease was added to a final concentration of 200 ~,g~mL-1 (Sigma, Oakville, Canada) before being incubated at 37 °C overnight with agitation at 100 rpm. The crude CPS extract was then dialyzed against running water for 72 h (MWCO 12 kDa, Sigma, Oakville, Canada), ultracentrifuged for 2 h (140,000 X g, 15 °C) and the supernatant, containing crude CPS, was lyophilized to dryness. Crude CPS was then re-suspended in H20 and purified using a Sephadex~ superfine G-50 column (Sigma, Oakville, Canada) equipped with a WatersTM differential refractometer (model 8403, Waters, Mississauga, Canada). 1H NMR at 400 MHz (Varian, Palo Alto, 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 H20 and purified using a Gilson liquid chromatograph (model 306 and 302 pumps, 811 dynamic mixer, 802B manometric module, Gilson, Middleton, uSA) with a Gilson UV
detector (220 nm) (model UV/Vis-151 detector, Gilson, Middleton, USA) equipped with a tandem QHP HiTrapTM ion exchange column (Amersham Biosciences, Piscataway, 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 Sephadex~
superfine G-15 column (Sigma, Oakville Ont.) 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.
High resolution NMR spectroscopy A 3 mg sample of enzyme purified CPS was suspended in 150 ~t.L of non-buffered 99%
D20 (pD 2.2) (Cambridge Isotopes Laboratories Inc, Andover, USA) and placed in a 3 mm NMR tube (Wilmad, Buena, USA). NMR experiments were performed using a Varian Inova 500 MHz spectrometer equipped with a Varian Z-gradient 3 mm triple resonance (lH, 13C, 3iP) probe (Varian, Palo Alto, USA). The methyl resonance of acetone was used as an internal reference (8H 2.225 ppm and ~ 31.07 ppm). The 1D 31P spectra were acquired using a Varian Mercury 200 MHz (1H) spectrometer and a Nalorac 5 mm four nuclei probe. For all 31P
experiments, spectra were referenced to an external 85% phosphoric acid standard (8P 0 ppm).
NMR experiments were typically performed at 25 °C with suppression of the deuterated HOD
resonance at 4.78 ppm. 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.
The results of high resolution homo- and heteronuclear NMR experiments showed the CPS
of C. jejuni 1421-1 to have a [2)-(3-D-Ribf (1-5)-~3-D-GalfNAc-(1-4)-a-D-GlcpA6(NGro)-(1]n repeating unit, with a 2,6-O-Me-D-a-L-glcHepp side branch attached at C-3 of GlcpA and an OMePN modification at C-4 of the glcHepp residue (Fig. 44a). "n" may be any positive integer but is preferably 4 or greater. In some instances n is 10-100, 100-1000, 1000-10,000, 10,000-100,000, 100,000-1,000,000. The 1H NMR spectrum of the purified CPS showed sharp signals originating from the anomeric protons of the CPS sugars, as well as O-methyl protons from the NAc group, both OCH3 modifications and the OMePN (Fig. 44b). The 31P NMR
spectrum showed that the chemical shift of the OMePN phosphorus atom was 13.68 ppm (1JP,H=11 Hz) and is consistent with what has been reported for the OMePN modification for C. jejuni NCTC11168 and HS:1. The 31P HMQC spectrum showed that H-4 of the glcHepp residue was strongly correlated to the phosphorous atom of the OMePN modification and therefore indicated that for C. jejuni 1421-l, the OMePN is located at C-4 of glcHepp (Fig. 44c). The 31P
HMQCTOXY spectrum revealed a strong 31P-1H correlation between the OMePN and H-4 of glcHepp as well as H-3 and H-5 of glcHepp and therefore confirmed the attachment of the OMePN at C-4 of the glcHepp (Fig. 44d). The 1~C HSQC spectrum revealed clear 13C-iH
correlations for the purified CPS and permitted the assignment of carbon and proton chemical shifts (Fig. 44e, Table XXV). Two and three-bond 13C-1H correlations observed using a HMBC
experiment was used to assign the quaternary B-6 and C-7 carbons as well as D-8 and D-9 O-methyl groups.
The NMR analyses of the purified CPS for C. jejuni 1421-1 presented herein indicates that the OMePN is located at C-4 of a 2,6-O-Me-D-a-L-glcHepp side branch. For instance, the carbon chemical shifts determined for the GalfNAc residue for the CPS of 1421-1 during this study are highly similar to those reported for the CPS of NCTC11168 without OMePN
indicating that for 1421-1, the OMePN is not located on this CPS sugar.
Furthermore, compared to the chemical shifts reported for NCTC11168 CPS, H-4 of the glcHepp side branch of 1421-1 CPS is downfielded by 0.76 ppm. This downfield shift is consistent with the effects of phosphoramidation reported for H-3 of GalfNAc residue in NCTC11168 (0.64 ppm) and H-3 of a Fruf CPS sugar in HS:l (0.74 ppm). In addition, the results of 31P HMQC
and 31P
HMQCTOXY NMR experiments show that the phosphorous atom of the OMePN was strongly correlated to H-4 of this CPS sugar.
The finding that OMePN is located on a glcHepp residue in C. jejuni 1421-1, indicates that cj1422c has a similar function to that reported for cj1421 c and acts as a glycosyltransferase responsible for transferring the OMePN to CPS sugars. Interestingly, this is the first time that the OMePN modification has been shown to exist on a pyranose sugar since in the two CPS
structures reported to date, NCTC11168 and HS:1, it was found on furanose sugars. In contrast to cj1421 c that adds OMePN to furanose sugars (among others), the results of the work presented here indicates that cj1422c adds this modification to pyranose CPS
sugars (among others). Thus, there is provided herein the use of cj 1422c as a OMePN
transferase.

Figure Captions Figure 44. a) The structure of the capsular polysaccharide for Campylobacter jejuni 1421-1 showing the novel OMePN modification located at C-4 of D-glycera-a-L-gluco-heptopyranose. b) 1H NMR spectrum for the purified CPS showing anomeric resonances, OMePN modification, O-methyl groups and NAc protons. c) 31P HMQC spectrum for the purified CPS showing the correlation between the OMePN methyl group and H-4 of D-glycero-a-L-gluco-heptopyranose (128 scans, 32 increments, IJp,H = 8 Hz, 4 h).
d) 31P
HMQCTOXY spectrum for the purified CPS showing the correlation between the OMePN
methyl group and H-4, H-3 and H-5 of D-glycero-a-L-gluco-heptopyranose (256 scans, 32 increments, 1JP,H = 8 Hz, 60 ms TOCSY mixing time, 9 h). e) 13C HSQC spectrum showing proton-carbon correlations for the purified CPS (64 scans, 256 increments, IJc,H = 150 Hz, 11 h). Abbreviations: A=~i-D-Ribf B=a-D-GlcpA6(NGro); C=(3-D-GalfNAc; D=D-glycero-a-L-gluco-heptopyranose; OMePN=O-methyl phosphoramidate.

Table XXV. Proton and carbon chemical shifts 8 (ppm) for C. jejuhi 1421-1 capsular polysaccharide.
Atom Type 8H $~

CH 5.36 106.1 A2 CH 4.18 81.0 A3 CH 4.32 70.7 A4 CH 4.13 83.9 AS/5' CHZ 3 . 8 8/3 .70 63 .0 B1 CH 5.11 98.7 B2 CH 3.93 73.1 B3 CH 4.07 73.2 B4 CH 3.92 76.0 BS CH 4.32 72.4 B6 C - 171.4 B7 CH 4.05 53.9 B8/8' CH2 3.72/3.66 61.1 C1 CH 5.01 104.3 C2 CH 4.10 62.1 C3 CH 4.22 73.8 C4 CH 4.14 82.2 CS CH 3.86 78.5 C6/C6' CH2 3.89/3.77 61.8 C7 C - 175.1 C8 CH3 2.05 22.9 D1 CH 5.57 97.8 D2 CH 3.63 82.1 D3 CH 3.64 71.7 D4 CH 4.33 74.6 DS CH 4.42 70.8 D6 CH 3.78 78.1 D7/D7' CHZ 3.87 62.6 D8 CH3 3.61 60.7 D9 CH3 3.53 59.2 E CH3 3.76 54.6 Part E, Section 3 Analysis of Cj1421 and Cj1422 complements in the Campylobacter jejuni 11168 Cj1421'/Cj1422- mutant background S
Complementation of Cj 1421 and Cj 1422 was investigated in the Cj 1422-1 (cj 1421::kan-r/cj 1422::kan-r) and Cj 1422-2 (cj 1421::kan-r/cj 1422::kan-r) mutants which have both Cj 1421 and Cj I422 knocked-out. Triplicate clones of complement strains were provided by Brendan Wren's laboratory. (London School of Hygiene and Tropical Medicine, UK) Complemented mutants include: 1422-1 2-3 which is the Cj 1422-1 mutant complemented with Cj 1421; 1422-1 3-8 which is the Cj 1422-1 mutant complemented with Cj 1422; 1422-2 2-3 which is the Cj 1422-2 mutant complemented with Cj 1421; and 1422-2 3-8 which is the Cj 1422-2 mutant complemented with Cj 1422.
The motility of all complements was subsequently analyzed and results indicate that there is no significant difference in motility between the complements and the wild type UKH11168. Complement strains were further analyzed by High Resolution Magic Angle Spinning (HR-MAS) NMR using both a CPMG filter, allowing the detection of the total CPS proton spectra, and with an HSQC
filter, allowing specific detection of the phosphoramidate on the 3 position of GalfNAc and the 4 position of the heptose. A detailed summary of the HR-MAS NMR results is presented in Table XXVI.
Table XXVI: Detailed summary of the results obtained following analysis of POMe from the complements by HR-MAS NMR:
presei~ee'o~P~3e'siri~let si a~ from:

CotlaplementaMutant 1421 1422 C3 posit'lion~4 position, _ = status'- of = of ''-Back round statusGal Ac ' he tose 1422-1 1422-1 + - + -1422-13-8 1422-1 - + - +

1422-2 1422-2 + - + -1422-2 1422-2 - + - +

a'Three clones of each complement were received and analysed by HR-MAS NMR
using an HSQC
filter for POMe detection. Each clone in a set showed similar results.

A comparison of the HR-MAS results using a CPMG filter and an HSQC filter for the 1422-2 2-3 complement and for select POMe mutants is shown in Figure 45.
Note the reappearance of the downfield resonance in the HSQC spectrum for the complement confirming that the product of Cj 1422 is responsible for the addition of POMe to the C4 of heptose. Also, note the reappearance of the upfield resonance (~5.02ppm) in the CPMG spectrum for the complement indicative of the return of phosphoramidation on the heptose residue. A representative HSQC spectrum of complements 1422-1 3-8 and 1422-2 2-3 compared to the wild type UKH11168 is shown in Figure 46 and clearly illustrates obvious differences between the phosphoramidate profiles of the complements. It also demonstrates that by complementing 1422-2 3-8 with Cj 1422, restoration of the transferase activity onto the C3 position of GalfNAc is observed while complementing with Cj 1421 into 12-3, transfer onto the C4 position of the heptose is restored.
We Claim:
The invention substantially as disclosed herein.

Table XXVII. NMR proton and carbon chemical shifts 8 (ppm) for an auto-hydrolyzed enzyme purified sample of C.
jejuni HS:1 CPS (CPS-1) and corresponding hydrolysis products.

Atom Type 8H 8~

CH 5.20 98.9 A2 CH 3.87 70.4 A3 CH 3.98 69.9 A4 CH 4.54 75.5 AS CH 4.17 71.5 A6/A6' CH2 3.74/3.74 61.6 B1/B1' CH2 4.11/4.05 65.2 B2 CH 3.97 77.9 B3/B3' CH2 3.76/3.76 62.1 Frufl/1' CH2 3.56/3.64 63.4 FrufZ C - 105.1 Fruf3 CH 4.10 76.1 Fruf4 CH 4.10 75.2 FrufS CH 3.82 81.4 Fruf6/6' CH2 3.67/3.79 63.2 Frupl/1' CH2 3.55/3.70 64.5 Frup2 C - 98.8 Frup3 CH 3.79 68.3 Frup4 CH 3.88 69.3 FrupS CH 4.02 69.4 Frup6/6' CH2 3.70/4.02 64.1 The 31P chemical shift for the monophosphate diester linkage was 8P 0.49 ppm.

Table XXVIII. NMR proton and carbon chemical shifts 8 (ppm) for an intact enzyme purified sample of C. jejuni HS:l CPS (CPS-2).
Atom Type 8H 8C

CH 5.40 98.8 CH 4.29 68.5 A2a A2b CH 4.28 68.3 A3a CH 4.33 69.4 A3b CH 4.40 68.7 A4a CH 4.74 77.2 A4b CH 4.69 77.3 AS CH 4.16 72.0 A6/A6' CH2 3.76/3.76 61.6 B 1B 1' CHz 4.15/4.11 64.5 B2 CH 4.02 77.1 B3B3' CHz 3.84/3.76 61.6 Cl/Cl' CH2 3.78/3.63 62.4 C2 C - 104.1 C3 CH 4.84 79.7 C4 CH 4.52 73.2 CS CH 3.85 81.2 C6/C6' CHZ 3.86/3.77 62.5 MeOPN CH3 3.81 54.9 *Cl/Cl' CH2 3.78/3.63 62.4 *C2 C - 104.1 *C3 CH 4.12 77.0 *C4 CH 4.12 76.6 *CS CH 3.75 81.5 *C6/C6' CH2 3.86/3.77 62.5 - *Chemical shift8 (pp for unsubstituted data m) ~i-D-fructofuranoside (MeOPN is absent).~ chemical for the monophosphate The shifts diester linkages nd b were of Gal H-4a 8P 0.40 a ppm and 0.49 ppm, respectively. chemicalshift for The 31P the MeOPN
groups was 14.67 ppm, and a scalar of 11.1 coupling 3JP,H Hz was observed.

Table XXIX. CE/MS/MS analysis of C. jejuni HS:Ol purified CPS (-200V orifice voltage). Analysis performed in negative ionization mode. For real masses, add one mass unit to the observed and calculated masses.
Mass (Da) Calculate Structure Observed Difference d 110.3 110.1 0.2 Gro + H20 153.1 153.1 0.0 GroP - HZO

171.3 171.1 0.2 GroP

205.0 205.1 0.1 Hex + P - (H20)3 223.3 223.1 0.2 Hex + P - (H20)2 240.3 241.1 0.8 Hex + P - (HZO) 254.8 254.2 0.6 Hex + OMePN - H20 259.0 259.1 0.1 Hex + P

273.0 273.2 0.2 Hex + OMeP

297.3 297.2 0.1 Hex + GroP - (H20)2 315.3 315.2 0.1 Hex + GroP - H20 333.5 333.2 0.3 Hex + GroP

377.5 377.2 0.3 Hex + GroP + P - (H20)2 385.3 385.2 0.1 (Hex)2 + P - (H20)2 395.3 395.2 0.1 Hex + GroP + P - H20 398.3 398.2 0.1 (Hex)2 + OMePN - (H20)Z

407.5 407.3 0.2 Hex + GroP + Gro 416.8 416.3 0.5 (Hex)Z + OMePN - H20 453.3 453.3 0.0 (Hex)2 + OMePN + H20 459.3 459.5 0.2 (Hex)2 + GroP - (H20)Z

469.3 469.3 0.0 Hex + (GroP)2 - HZO

477.3 477.3 0.0 (Hex)2 + GroP - H20 487.3 487.3 0.0 Hex + (GroP)Z

490.8 490.4 0.4 (Hex)Z + Gro + OMePN - H20 495.5 495.4 0.1 (Hex)2 + GroP

509.6 509.4 0.2 (Hex)Z + Gro + OMePN

539.3 539.3 0.0 (Hex)2 + GroP + P - (H20)2 551.5 551.4 0.1 (Hex)Z + GroP + Gro - HZO

557.5 557.3 0.2 (Hex)Z + GroP + P - HZO

570.3 570.4 0.1 (Hex)2 + GroP + OMePN - H20 578.8 578.4 0.4 (Hex)3 + OMePN - H20 613.5 613.4 0.1 (Hex)Z + (GroP)2 - (H20)2 621.5 621.5 0.0 (Hex)2 + GroP - (H20)2 631.3 631.4 0.1 (Hex)Z + (GroP)2 - H20 639.3 639.5 0.2 (Hex)3 + GroP - H20 652.3 652.5 0.2 (Hex) + Gro + OMePN - HZO

667.5 667.4 0.1 (Hex)2 + (GroP)2 + H20 Mass (Da) Calculate Structure Observed Difference d 701.5 701.4 0.1 (Hex)3 + GroP + P - (H20)Z

723.3 723.5 0.2 (Hex)2 + (GroP)2 + Gro 732.3 732.5 0.2 (Hex)3 + GroP + OMePN - H20 775.5 775.5 0.0 (Hex)3 + (GroP)2 - (HZO)2 793.5 793.5 0.0 (Hex)3 + (GroP)2 - H20 793.3 793.5 0.2 (Hex)2 + GroP + P + OMePN - (H20)2 829.5 829.6 0.1 (Hex)3 + (GroP)Z + H20 855.3 855.5 0.2 (Hex)3 + (GroP)2 + P - (H20)2 886.8 886.6 0.2 (Hex)3 + (GroP)Z + OMePN - H20 894.5 894.6 0.1 (Hex)4 + GroP + OMePN - HZO

929.8 929.6 0.2 (Hex)3 + (GroP)3 - (HZO)2 947.5 947.6 0.1 (Hex)3 + (GroP)3 - H20 987.5 987.7 0.2 (Hex)4 + GroP + (OMePN)Z - H20 1048.5 1048.7 0.2 (Hex)4 + (GroP)Z + OMePN - HZO

1109.5 1109.7 0.2 (Hex)4 + (GroP)3 - HZO

Claims