PHOSPHORAMIDE AND USES THEREOF

PHOSPHORAMIDE ET UTILISATIONS CONNEXES

Description

CA 02518317 2005-09-02
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,
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CA 02518317 2005-09-02
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.
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CA 02518317 2005-09-02
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-
176
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-
2-
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
NCTCII168
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-
176
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.
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CA 02518317 2005-09-02
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.
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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).
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CA 02518317 2005-09-02
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


CA 02518317 2005-09-02
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,
50
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
7


CA 02518317 2005-09-02
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
8


CA 02518317 2005-09-02
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.
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CA 02518317 2005-09-02
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.
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CA 02518317 2005-09-02
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
11


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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
12


CA 02518317 2005-09-02
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-
176
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.
13


CA 02518317 2005-09-02
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.
14


CA 02518317 2005-09-02
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.


CA 02518317 2005-09-02
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
GAP4
l0 or GeneTool software (DoubleTwist.com), and were deposited at EMBL database
with the following IDs and accession numbers:
Designation Strain ID Acc. Number
HS 19 NCTC 12517 CJ 12517CPS BX545860
HS1 G1 CJG1CPS BX545859
HS23/36 81-176 CJ81176CPS BX545858
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
16


CA 02518317 2005-09-02
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.
17


CA 02518317 2005-09-02
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
18


CA 02518317 2005-09-02
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.
19


CA 02518317 2005-09-02
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
2
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-
4C
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°


CA 02518317 2005-09-02
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,
1181
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
21


CA 02518317 2005-09-02
(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
22


CA 02518317 2005-09-02
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
23


CA 02518317 2005-09-02
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.
24


CA 02518317 2005-09-02
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.


CA 02518317 2005-09-02
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%
26


CA 02518317 2005-09-02
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
27


CA 02518317 2005-09-02
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
28


CA 02518317 2005-09-02
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
29


CA 02518317 2005-09-02
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.


CA 02518317 2005-09-02
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
31


CA 02518317 2005-09-02
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
NCTC11168
(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
32


CA 02518317 2005-09-02
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).
33


CA 02518317 2005-09-02
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
34


CA 02518317 2005-09-02
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.

CA 02518317 2005-09-02
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
600
MHz
( H)
in
DZO
at
35C
(~
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.
CH3
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.



36


CA 02518317 2005-09-02
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
37


CA 02518317 2005-09-02
N


d
w


o >,
N
0


4, 5
~ ~ Y ;b p >,
o ,a _ O ~ o
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CA 02518317 2005-09-02
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
F1


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
B 13


C'1422c 1422 s ec GAGCTTTGATACTTTCAAGAGATGG 1356806 - 1356830
F1


Cj 1422c1422-R TTGGTGTGCCTGAGGATTATT 1358059 - 1358079


C' 1422c1422-F TGGGTATTTAAGTTGGGGAAA 1357785 - 1357805


C' 1422c1422LR GAGAGATTTTTAAAAGAAGTTGGTG 1357191 - 1357215


C' 1422c1422LF CTGGGGAAGAAAAATTCAGTAT'TTTTG 1356639 - 1356665


58


CA 02518317 2005-09-02
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


IYIDMNB7IVGDKTLFTLEKLSKILNFSSPDKNNKIFYQQLYSPLTVLLPCI


IKVNNKVKI


FVSNRFSVKNIQIMENCIDITDKFKEIFHENLIIFCSKDHFDSLINNQTLY


NVVLEYINK


FLISLKKRINVEKNKEVKVDDVLDYFKKNISVAKSYKDILDEELVYIKQHR


PDIVASWKY


YQEFERMCKELDENNQNPSLSFSNQ


59


CA 02518317 2005-09-02
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


CA 02518317 2005-09-02
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
61


CA 02518317 2005-09-02
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.
62


CA 02518317 2005-09-02
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
63


CA 02518317 2005-09-02
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.
64


CA 02518317 2005-09-02
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-
2
(60 ms). (e) 31P HSQC with 1JP,H =10 Hz, 64 transients and 240 increments. (f)
13C HSQC
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)
31P
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.


CA 02518317 2005-09-02
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.
66


CA 02518317 2005-09-02
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
67


CA 02518317 2005-09-02
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
68


CA 02518317 2005-09-02
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].
69


CA 02518317 2005-09-02
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


CA 02518317 2005-09-02
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


CA 02518317 2005-09-02
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,
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. 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.
72


CA 02518317 2005-09-02
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).
73


CA 02518317 2005-09-02
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.
74


CA 02518317 2005-09-02
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.

CA 02518317 2005-09-02
Part B: Table XV. Proton and carbon chemical shifts 8 (ppm) for C. jejuni HS:1
CPS
CPS-1 CPS-2


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).
76


CA 02518317 2005-09-02
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




CA 02518317 2005-09-02
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


78

CA 02518317 2005-09-02
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



A6 C - -


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).
79


CA 02518317 2005-09-02
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




CA 02518317 2005-09-02
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


CA 02518317 2005-09-02
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
82


CA 02518317 2005-09-02
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
83


CA 02518317 2005-09-02
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.
84


CA 02518317 2005-09-02
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
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-(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,


CA 02518317 2005-09-02
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
86


CA 02518317 2005-09-02
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
11168-H
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).
87


CA 02518317 2005-09-02
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
1H-
~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


CA 02518317 2005-09-02
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
89


CA 02518317 2005-09-02
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
100
%.
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.


CA 02518317 2005-09-02
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.
91


CA 02518317 2005-09-02



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CA 02518317 2005-09-02
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
94


CA 02518317 2005-09-02
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


CA 02518317 2005-09-02
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.
96


CA 02518317 2005-09-02
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-
4
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
H4
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,
97


CA 02518317 2005-09-02
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,
98


CA 02518317 2005-09-02
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.
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CA 02518317 2005-09-02
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
0
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.
100


CA 02518317 2005-09-02
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
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CA 02518317 2005-09-02
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
102


CA 02518317 2005-09-02
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
103


CA 02518317 2005-09-02
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.
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CA 02518317 2005-09-02
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).
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CA 02518317 2005-09-02
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)
106


CA 02518317 2005-09-02
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
5
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
107


CA 02518317 2005-09-02
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
4000 QTRAP
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.
108

CA 02518317 2005-09-02
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.
CPS-1
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.
109


CA 02518317 2005-09-02
Table XXII. NMR proton and carbon chemical shifts 8 (ppm) for
an intact enzyme purified sample of C. jejuni HS:1 CPS (CPS-2).
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.
110


CA 02518317 2005-09-02
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


111


CA 02518317 2005-09-02
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 +
H20


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
- H20


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.
112


CA 02518317 2005-09-02
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-
2
(60 ms). (e) 31P HSQC with 1JP,H =10 Hz, 64 transients and 240 increments. (f)
13C HSQC
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.
113


CA 02518317 2005-09-02
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)
31P
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.
114


CA 02518317 2005-09-02
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.
115


CA 02518317 2005-09-02
[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.
116


CA 02518317 2005-09-02
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.
31P
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.
117


CA 02518317 2005-09-02
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
14NH4C1
(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
118


CA 02518317 2005-09-02
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
15NH4C1
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
15NH4C1
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
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CA 02518317 2005-09-02
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.
120


CA 02518317 2005-09-02
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
289
mg of cells (wet pellet mass) was harvested from four lsNH4Cl enriched plates
that yielded
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CA 02518317 2005-09-02
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
11168-H
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
122


CA 02518317 2005-09-02
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
T2
attenuation of the signal. The resulting spectrum for 11168-H cells exhibited
peaks at 4.94
123


CA 02518317 2005-09-02
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).
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CA 02518317 2005-09-02
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
125


CA 02518317 2005-09-02
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.
126


CA 02518317 2005-09-02
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
127


CA 02518317 2005-09-02
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
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CA 02518317 2005-09-02
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
XY-16
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.
129


CA 02518317 2005-09-02
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
130


CA 02518317 2005-09-02
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.
131


CA 02518317 2005-09-02
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
132


CA 02518317 2005-09-02
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
111168
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
133


CA 02518317 2005-09-02
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
134


CA 02518317 2005-09-02
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
135


CA 02518317 2005-09-02
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.
136


CA 02518317 2005-09-02
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.
137

CA 02518317 2005-09-02
Table XXV. Proton and carbon chemical shifts 8 (ppm) for C. jejuhi
1421-1 capsular polysaccharide.
Atom Type 8H $~
A1
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


138


CA 02518317 2005-09-02
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:
11


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


1422-1 1422-1 + - + -
2-3


1422-13-8 1422-1 - + - +


1422-2 1422-2 + - + -
2-3


1422-2 1422-2 - + - +
3-8



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.
139


CA 02518317 2005-09-02
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
1422-
12-3, transfer onto the C4 position of the heptose is restored.
We Claim:
The invention substantially as disclosed herein.
140

CA 02518317 2005-09-02
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.
CPS-1


Atom Type 8H 8~



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


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.
141

CA 02518317 2005-09-02
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
A1


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
1P


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.



I42


CA 02518317 2005-09-02
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


143


CA 02518317 2005-09-02
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


144