Recently, the use of metabolomics permitted the unequiv-ocal characterization of unique flagellin modifications in Campylobacter, including novel legionaminic acid sugars in Campylobacter
Trang 1on Campylobacter jejuni 11168 flagellin using
metabolomics-based approaches
Susan M Logan1, Joseph P M Hui2, Evgeny Vinogradov1, Annie J Aubry1, Jeremy E Melanson2, John F Kelly1, Harald Nothaft1and Evelyn C Soo2
1 NRC-Institute for Biological Sciences, Ottawa, Canada
2 NRC-Institute for Marine Biosciences, Halifax, Canada
Campylobacter is an important human pathogen and
the most prevalent causative agent of bacterial
gastro-enteritis worldwide, with Campylobacter jejuni
repre-senting over 90% of all Campylobacter infections [1]
Most cases of Campylobacter infections are sporadic
and can be traced to the consumption of undercooked
(or the handling of) contaminated chicken, but
out-breaks, although rare, do occur mainly as a result of the consumption of contaminated sources of water or unpasteurized milk [2] Although the majority of cases
of Campylobacteriosis are self-limiting, complications can occur and these range in severity from bloody diarrhoea and fever lasting over 1 week, to more severe and life-threatening conditions, such as the
Keywords
Campylobacter; flagellin; glycosylation;
metabolomics; pseudaminic acid
Correspondence
E C Soo, MS Metabolomics Group,
NRC-Institute for Marine Biosciences,
1411 Oxford Street, Halifax, NS B3H 3Z1,
Canada
Fax: +1 902 426 9413
Tel: +1 902 426 0780
E-mail: evelyn.soo@nrc-cnrc.gc.ca
(Received 22 September 2008, revised 3
December 2008, accepted 5 December
2008)
doi:10.1111/j.1742-4658.2008.06840.x
It is well known that the flagellin of Campylobacter jejuni is extensively gly-cosylated by pseudaminic acid and the related acetamindino derivative, in addition to flagellin glycosylation being essential for motility and coloniza-tion of host cells Recently, the use of metabolomics permitted the unequiv-ocal characterization of unique flagellin modifications in Campylobacter, including novel legionaminic acid sugars in Campylobacter coli, which had been impossible to ascertain in earlier studies using proteomics-based approaches To date, the precise identities of the flagellin glycosylation modifications have only been elucidated for C jejuni 81-176 and C coli VC167 and those present in the first genome-sequenced strain C jejuni
11168 remain elusive due to lability and respective levels of individual gly-can modifications We report the characterization of the carbohydrate modifications on C jejuni 11168 flagellin using metabolomics-based approaches Detected as their corresponding CMP-linked precursors, struc-tural information on the flagellin modifications was obtained using a com-bination of MS and NMR spectroscopy In addition to the pseudaminic acid and legionaminic acid sugars known to be present on Campylobacter flagellin, two unusual 2,3-di-O-methylglyceric acid modifications of a nonu-losonate sugar were identified By performing a metabolomic analysis of selected isogenic mutants of genes from the flagellin glycosylation locus of this pathogen, these novel CMP-linked precursors were confirmed to be di-O-methylglyceric acid derivatives of pseudaminic acid and the related acetamidino sugar This is the first comprehensive analysis of the flagellar modifications in C jejuni 11168 and structural elucidation of di-O-methyl-glyceric acid derivatives of pseudaminic acid on Campylobacter flagellin
Abbreviations
HILIC, hydrophilic interaction liquid chromatography; HMBC, heteronuclear multiple bond correlation; Pse, pseudaminic acid.
Trang 2post-infection neuropathy Guillain–Barre´ Syndrome [3]
and rare malignant lymphomas of the small intestine
known as immunoproliferative small intestine disease
[4]
Flagellae comprise an important virulence factor of
many bacterial pathogens that confers motility and
allows colonization of host cells In Campylobacter, the
major structural protein FlaA must be glycosylated for
flagellar filament assembly [5] and, given the
impor-tance of motility in infectivity, there is tremendous
potential to target the flagellin glycosylation process in
the development of novel antimicrobial therapies [6]
For some time, there have been extensive studies on
flagellin glycosylation in Campylobacter in terms of the
identification of the glycosyl moieties present on the
flagellar protein and the genes involved in the
bio-synthetic process The complete genome sequence of
C jejuni11168 published in 2000 [7] revealed a
flagel-lin glycosylation locus consisting of approximately 50
genes, and included genes that displayed significant
homology to sialic acid biosynthesis genes Studies
using proteomics-based methods identified the
5,7-di-N-acetylated derivative of 5,7-diamino-3,5,7,
9-tetradeoxy-l-glycero-l-manno-nonulosonic acid
[5,7-di-N-acetyl-pseudaminic acid (Pse), Pse5Ac7Ac] and its
acetamidino derivative to be the major glycosyl
modifi-cations on C jejuni 81-176 flagellin in addition to
minor amounts of two related glycans
(Pse5Ac7A-c8OAc, Pse5Ac7Ac8O-GlnAc) [8,9]
Campylobac-ter coli VC167 flagellin was also shown to contain
Pse5Ac7Ac, as well as two other novel legionaminic
acid derivatives that were not found on flagellin from
C jejuni81-176 These novel legionaminic acid
deriva-tives were synthesized exclusively by the ptm genes,
which were present within the flagellar glycosylation
locus of this strain [10] However, due to the labile
nat-ure of many of these carbohydrate modifications and
their considerably low abundance on the flagellin
pro-tein, attempts to characterize the precise structures of
many of these observed carbohydrates using the
estab-lished proteomics-based approaches were unsuccessful
In addition, although the identification of biosynthetic
genes had been made via mutagenesis studies [5], the
functional characterization of the flagellar glycan
bio-synthetic enzymes and nonulosonate sugar pathways
was poorly described
Metabolomics has recently emerged as an
invalu-able tool for the study of poorly characterized
meta-bolic pathways In the first metabolomic study of
Campylobacter, a targeted metabolomic screen of
C jejuni 81-176 revealed the tremendous potential for
using metabolomics to identify unknown substrates
and elucidate the role of genes in the biosynthesis of
the novel flagellin glycan structures [11] This work led to expanded studies of the flagellin glycosylation locus in Campylobacter [12,13] and highlighted the innovative use of metabolomics as an alternative to proteomics-based approaches [8–10,14] to gain precise structural information on novel carbohydrate moieties that glycosylate the flagellar protein The use of hydrophilic interaction liquid chromatography (HI-LIC)-MS in these recent studies was critical because
it allowed discrimination of metabolites with the same mass-to-charge (m⁄ z) ratios, which would otherwise
be indistinguishable using MS alone The HILIC-MS method allowed the separation of complex mixtures
of CMP-linked nonulosonic acids and the large-scale purification of these novel metabolites for NMR analysis The resolution afforded by HILIC for sugar-nucleotides ultimately led to the unexpected identification of a family of legionaminic acid sugars
in C coli VC167, which had previously been thought
to be derivatives of Pse [10,13]
As noted earlier, in contrast to C jejuni 81-176 and
C coliVC167, there is much less knowledge of the fla-gellin glycosylation process and the precise nature of the flagellar glycans of genome-sequenced strain C je-juni11168 Comparative analysis of the flagellin glyco-sylation locus of these three strains shows the presence
of genes known to be involved in the biosynthesis of Pse [15] legionaminic acid [13] and related derivatives
in C jejuni 11168 It is noteworthy that the flagellin glycosylation locus of C jejuni 11168 is more complex than either C jejuni 81-176 or C coli VC167, suggest-ing a genetic potential for C jejuni 11168 to glycosy-late its flagellin with additional novel glycans Given the relative ease of gaining precise structural informa-tion on unique flagellin modificainforma-tions using metabolo-mics approaches [12,13], the present study provides a comprehensive study of the flagellar glycan structures
of C jejuni 11168 flagellin
Results
Metabolomic analysis of wild-type C jejuni 11168 The metabolome of wild-type C jejuni 11168 was screened for potential biosynthetic sugar-nucleotides relating to the carbohydrate moieties found on its fla-gellin using an established HILIC-MS method [12] During precursor ion scanning for ions characteristic
of CMP (m⁄ z 322), an intracellular pool of eight CMP-linked sugars was detected within cell lysates of the wild-type strain (Fig 1) Upon closer examination
of these CMP-linked sugars, it was observed that the retention times of six of the CMP-linked sugars and
Trang 3their corresponding m⁄ z values were consistent with
those obtained in earlier metabolomic studies of
C jejuni 81-176 and C coli VC167 [12,13],
identify-ing these metabolites as the CMP-linked
precur-sors of Pse5Ac7Ac, Pse5Ac7Am, Leg5Ac7Ac,
Leg5Am7Ac, Leg5MeAm7Ac and Neu5Ac (Leg is
5,7-diamino-3,5,7,9-tetradeoxy-d-glycero-d-galacto-non
ulosonic acid) The presence of these CMP-linked
pre-cursors in C jejuni 11168 is not surprising because
the genes known to be involved in their biosynthesis
in C jejuni 81-176 (Pse5Ac7Ac, Pse5Ac7Am and
Neu5Ac) or C coli VC167 (Leg5Ac7Ac, Leg5Am7Ac,
Leg5MeAm7Ac) are also present in the 11168 strain
However, in addition to these well-characterized
CMP-linked intermediates, two unknown CMP-linked
precursors were also observed in the metabolome of
C jejuni 11168 As shown in Fig 1, one of these
novel CMP-sugars was detected at 13.6 min as
[M) H]) ions at m⁄ z 712, whereas the second novel
CMP-sugar was observed at 17.3 min at m⁄ z 711 In
the positive mode, oxonium ions corresponding to
these novel CMP-linked sugars are produced and
these were observed as precursor ions at m⁄ z 714 (I)
and m⁄ z 713 (II) (data not shown)
MS analysis of 11168 flagellin glycopeptides
To confirm that the corresponding glycan moieties of
these novel CMP-sugars (m⁄ z 711, 389 Da; m ⁄ z 712,
390 Da) were actually present on the flagellin protein
of C jejuni 11168, flagellin glycopeptides were analysed
by LC-MS⁄ MS following enzymatic digestion of flagel-lin protein The presence of the oxonium ions m⁄ z 390 and 391, which correspond to glycans of mass 389 and
390 Da on two flagellar glycopeptides, is indicated in Fig 2 These two glycan modifications were the most abundant modification found on all flagellin glycopep-tides examined (data not shown)
Structural analysis of I and II by high resolution MS
The HILIC-MS and precursor ion scanning method provides a highly selective means for detecting
sugar-Fig 1 Intracellular CMP-sugars detected in parent strain C jejuni
11168 by HILIC-MS and precursor ion scanning for fragment ions
related to CMP (m ⁄ z 322).
Fig 2 NanoLC-MS ⁄ MS analysis of the tryptic digest of 11168 fla-gellin (A) MS ⁄ MS spectrum of the doubly charged ion at m ⁄ z 1167.1 corresponding to the glycopeptide, T 203–222 , modified with a single glycan moiety The amino acid sequence of this peptide is shown in the inset and the y-fragment ions arising from fragmenta-tion of the peptide bonds are indicated in the spectrum This pep-tide is modified with either the 389 Da or the 390 Da sugar and the low m ⁄ z region of this spectrum is dominated by their oxonium ions at m ⁄ z 390.2 and 391.2 (underlined) and related degradation products Loss of water molecules from the m ⁄ z 391.2 ion yields the strong fragment ions at m ⁄ z 373.2 and 355.2, respectively, whereas the ion at m ⁄ z 346.2 arises from the loss of CO 2 from the oxonium ion at m ⁄ z 390.2 The ion at m ⁄ z 1943.4 corresponds to the intact peptide ion (singly charged) having lost the glycan modifi-cation (B) MS ⁄ MS spectrum of the doubly charged ion at m ⁄ z 1064.5 corresponding to the glycopeptide, T 463–479 , also modified with a single glycan moiety In this instance, the peptide appears to
be modified predominantly with the 389 Da glycan as its oxonium ion and the related degradation products are dominant Regions of both spectra have been expanded to highlight some of the less abundant but informative fragment ions.
Trang 4nucleotides within the metabolome However, to derive
meaningful structural information on the novel
CMP-sugars and reduce ambiguity, it was necessary to
employ high resolution MS for subsequent
experi-ments Accurate mass measurements of I and II were
performed on a Waters Q-ToF Premier mass
spec-trometer (Waters Corp., Milford, MA, USA) and
accurate masses of the protonated [M + H]+ions of I
and II were revealed as 714.2222 and 713.2403 Da,
respectively (Fig 3A–C) A database search of the
plausible molecular formulae satisfying these mass
constraints and their isotopic patterns (see Table S1)
suggested an empirical formula of C25H41N5O17P
(the-oretical mass = 714.2235 Da, 1.8 p.p.m.) for I and
C25H42N6O16P (theoretical mass = 713.2395 Da;
1.1 p.p.m.) for II
To obtain structural information on these novel
metabolites, a series of tandem MS experiments were
also performed using high resolution MS In the
nega-tive mode, MS⁄ MS of the novel sugar-nucleotides revealed a major fragment ion at m⁄ z 322, which is consistent with the expected m⁄ z for [CMP-H]), sug-gesting that these novel metabolites are CMP-linked (data not shown) In the positive mode, MS⁄ MS of I and II revealed major fragment ions at m⁄ z 391.1723 and 390.1887, which correspond to the masses of the two novel carbohydrate moieties (Fig 3D,E) To gener-ate structural information on these carbohydrgener-ate moie-ties, further tandem MS experiments were carried out
to fragment the novel carbohydrates As shown in Fig 4A,B, the second generation product ion spectra for I and II revealed fragmentation patterns that are typically observed for nonulosonic acids [9,10,12–14] For example, characteristic and consecutive neutral losses of water, ammonia and formic acid were observed in the second generation product ion spectra
of both I and II It is noteworthy that a prominent loss
of the acetamidino functionality, CH3C(=NH)NH
Fig 3 Accurate mass measurements of unknown CMP-sugars detected in C jejuni 11168 (A) Extracted ion chromatogram of m⁄ z 714 (I) and 713 (II) (B) MS at 14.6 min showing the accurate mass of I (C) MS at 17.9 min showing accurate mass of II (D) Corresponding
MS ⁄ MS spectrum of I (E) Corresponding MS ⁄ MS spectrum of II.
Trang 5(neutral loss of 58 Da), was also observed in the second
generation product ion spectrum of II (Fig 4B) Based
on our existing knowledge of Campylobacter flagellar
glycans, it is highly plausible that II is the related
ace-tamidino derivative of I because such a feature appears
to be prevalent among the nonulosonic sugars in
Cam-pylobacter (e.g between Pse5Ac7Ac and Pse5Ac7Am
[12] and Leg5Ac7Ac and Leg5Am7Ac [13])
Interest-ingly, fragment ions relating to nonulosonic acid were
also apparent in the second generation product ion
spectra of I and II (Fig 4A,B) [9], suggesting that these
CMP-sugars may be novel derivatives of a nonulosonic
acid Accordingly, the mass differences of 74.0374 Da
observed between the oxonium ions of Pse5Ac7Ac
(C13H21N2O7; theoretical mass = 317.1349 Da) and I,
and of 74.0378 Da observed between Pse5Ac7Am
(C13H22N3O6, theoretical mass = 316.1509 Da) and II,
indicate that the novel substituent differs from acetate
by C3H6O2(theoretical mass = 74.0367 Da), thus
hav-ing the overall formula C5H9O3 To unequivocally
assign the structures of I and II, large-scale purifica-tions of the metabolites were achieved, as described previously [12,13] for NMR structural elucidation
Structural analysis of II by NMR spectroscopy
By contrast to the earlier work on C jejuni 81-176 and
C coli VC167, the novel CMP-linked metabolites I and II detected in C jejuni 11168 were rather unstable compounds MS analysis of the purified substrates revealed that approximately 20 lg of each metabolite was isolated but, upon their analysis by NMR, it was observed that degradation of the metabolites had occurred This was particularly true for I where com-plete degradation of the metabolite appeared to have taken place, and therefore it was only possible to pur-sue the structural analysis of II by NMR spectroscopy
In addition to the problem of instability, it was also observed that the sample contained appreciable amounts of impurities, including glycerol, lactic acid and glyceric acid The presence of these low molecular weight impurities presented a further challenge with respect to deducing the precise structure of II by NMR and attempts to remove these low molecular mass impurities by gel chromatography were unsuc-cessful However, despite these complications, it was possible to deduce from the NMR spectra spin-systems
of nonulosonic acid, ribose and cytosine (see Table S2) The coupling constants and chemical shifts of the nonulosonic acid residue agreed well with that observed in CMP-5-acetamido-7-ami-
dino-3,5,7,9-tetradeoxy-l-glycero-a-l-manno-nonuloson-ic acid (CMP-Pse5Ac7Am), whdino-3,5,7,9-tetradeoxy-l-glycero-a-l-manno-nonuloson-ich had been detected
in earlier metabolomic studies of the flagellin glycosyl-ation process in C jejuni 81-176 [12] Linkage of Pse
to phosphate was also confirmed by the observation of H-P coupling on H-3ax (4 Hz) The spectra also con-tained the characteristic signal of an acetamidine group (C-1 at 167.4 p.p.m., H⁄ C-2 at 2.24 ⁄ 19.5 p.p.m.) but
no signals of the acetyl group were observed, suggest-ing that one of the amino groups of Pse was acylated
by a novel acyl group The NMR spectra also con-tained signals of two methyl groups, which gave het-eronuclear multiple bond correlations (HMBC) with the signals of the CH and CH2 groups Two latter sig-nals correlated with each other in the COSY spectrum, but nothing else These data, taking into account posi-tion of 13C signals of these groups (see Table S2) cor-responded to 2,3-di-O-methyl-glycerate The COOH group signal was not observed in HMBC due to its presence in concentrations below the limit of detection; thus, the linkage of dimethylglycerate to the nonulo-sonic acid could not be directly confirmed However,
Fig 4 Second generation product ion spectrum of (A) I and (B) II.
Broken arrows indicate the possible neutral loss of the
2,3-di-O-methyl-glyceramide (C5H11NO3).
Trang 6given that the exact theoretical mass of
CMP-Pse5acy-l7Ac (where acyl is 2,3-di-O-methylglycerate) (i.e
C25H42N6O16P1) is 713.2395 Da, this proposed
deriva-tive shows a near perfect agreement with the
experi-mentally observed value of 713.2403 Da (1.1 p.p.m
error) The presence of a dimethylglycerate substituent
would also be consistent with earlier predictions from
the second generation product ion experiments on II
of a novel modification with a molecular formula of
C5H9O3, and, as indicated in the second generation
product ion spectra of I and II (Fig 4A,B), this could
be observed as the neutral loss of the
2,3-di-O-methyl-glyceramide (C5H11NO3)
The respective position of the amidine and
dimethyl-glycerate on N-5 and N-7 of the Pse could not be
experimentally confirmed The HMBC correlations
with C-1 of acyl groups were not observed due to the
low concentration of the purified metabolite II A
comparison of 13C chemical shifts of II with model
compounds bearing either two acetyl groups
(Pse5A-c7Ac) or one acetyl group and one amidino group at
N-7 (Pse5Ac7Am) showed good agreement of the
7-amidino derivative with the data for the analyzed
compound (see Table S2) The 7-N-acetyl derivative
had C-7 signal shifted more than 4 p.p.m upfield
com-pared to that of the 7-amidino derivative This
differ-ence is characteristic for the amidine substitution and
has been reported also with other sugars [16] Thus, II
is likely to be substituted with the dimethylglycerate
group at N-5 and with amidine at N-7
Metabolomic analysis of pseB and pseC Pse
biosynthetic genes
Earlier studies on Pse biosynthesis in Campylobacter
identified key roles for pseB and pseC in the
biosyn-thetic process [12,17,18] and the insertional
inactiva-tion of these two genes had led to the disappearance
of CMP-Pse5Ac7Ac and CMP-Pse5Ac7Am from the
metabolome of C jejuni 81-176 [12,17,18] The analysis
of I and II by MS and NMR analyses in the present study strongly suggests that these two CMP-sugars are novel modifications of Pse However, considering the complexity of the flagellin glycosylation locus of
C jejuni 11168 and the ability of this strain of Cam-pylobacter to synthesize both Pse and legionaminic acid sugars, it would be invaluable to obtain support-ing biological data to confirm these CMP-sugars as novel derivatives of Pse Given that pseB and pseC are exclusively involved in Pse5Ac7Ac biosynthesis, there
is the potential to employ metabolomics to explore the sugar-nucleotide complement of isogenic mutants pseB and pseC of strain 11168, and confirm whether I and
II are indeed novel modifications of Pse Accordingly, the metabolomes of isogenic mutants pseB (see Fig S1) and pseC (data not shown) of C jejuni 11168 were prepared and probed for CMP-sugars using the HILIC-MS and the precursor ion scanning method
As expected, the CMP-linked precursors of Pse5Ac7Ac and Pse5Ac7Am were absent from the metabolomes of both pseB and pseC but, in addition, the novel CMP-sugars were also no longer present, confirming that I and II are synthesized through the Pse biosynthetic pathway It is noteworthy that the biosynthesis of the CMP-precursors of the legionaminic acid sugars was not affected by the insertional inactivation of pseB and pseC, thereby providing further evidence that I and II are the dimethylglyceric acid modifica-tions of Pse and its related acetamidino derivative, respectively The absolute configuration of I or II was not determined However, the relative configura-tion of II was determined by NMR Based on the structural data obtained by MS and metabolomic screening of isogenic mutants pseB and pseC, I was identified tentatively as CMP-7-acetimidoylamino- 5-(2,3-di-O-methylglyceroyl)amino-3,5,7,9-tetradeoxy-l-glycero-a-l-manno-nonulosonic acid, and II was determined to be CMP-7-acetamido-5-(2,3-di-O-
methylglyceroyl)amino-3,5,7,9-tetradeoxy-l-glycero-a-l-manno-nonulosonic acid (Fig 5)
Fig 5 Proposed structures of I and II.
Trang 7Metabolomics has provided a unique opportunity to
highlight subtle differences in the nature of the
glyco-syl moieties that decorate the flagellin of different
Campylobacter strains through structural elucidation
of their corresponding biosynthetic intermediates
Ear-lier studies of C coli VC167 had revealed the potential
for Campylobacter to synthesize a variety of
legionami-nic acid sugars, and this capability was again utilized
in the present study to investigate C jejuni 11168 In
addition to previously well characterized glycosyl
mod-ifications, two novel carbohydrate modifications were
also detected in the metabolome of C jejuni 11168 as
their corresponding CMP-linked intermediates
Exten-sive structural analysis of these CMP-sugars using a
combination of high resolution MS and NMR
spec-troscopy identified these metabolites as the
dimethyl-glyceric acid derivatives of Pse and the related
acetamidino derivative It is noteworthy that the use of
high resolution MS in the present study was
instru-mental in elucidating the structures of the
carbohy-drate moieties This was particularly true of I, where
complete degradation of the metabolite had occurred
during NMR analysis and structural information could
only be gained using high resolution MS Although the
absolute configuration of the novel glycans could not
be determined, the metabolomic analysis of selected
genes known to have an exclusive role in Pse
biosyn-thesis clearly demonstrated a role for these genes in
the biosynthesis of the two novel metabolites, thereby
confirming them as derivatives of Pse
This is the first report of dimethylglyceric acid
deriv-atives of Pse in Campylobacter and further illustrates
the considerable capacity of Campylobacter to
synthe-size a large number of nonulosonate sugar derivatives
Although the precise biological role of these novel
derivatives has yet to be defined, a recent study
dem-onstrated that the legionaminic acid biosynthetic genes
present in the Campylobacter flagellar glycosylation
island are a genetic marker for a livestock associated
clade [19] In addition, legionaminic acid modifications
found on the flagellin of 11168 are involved in the
ability of this strain to persist in the gastrointestinal
tract of chickens (S L Howard, A Jagannathan,
E C Soo, J P M Hui, A J Aubry, I Ahmed,
A Karlyshev, J F Kelly, M A Jones, M P Stevens,
S M Logan and B W Wren, unpublished results)
By contrast, flagellins from C jejuni 81-176 are
glyco-sylated with only Pse derivatives [9] This strain was
originally isolated from a patient during an outbreak
of campylobacteriosis [20] and is highly pathogenic in
monkeys and in human trials [21–23] The precise role
of the Pse5Ac7Ac and Pse5Ac7Am glycans remains to
be established, but they have been shown to play a key role in virulence of this strain [24] The biological role
of the two novel flagellar glycans characterized in the present study can now also be explored
Campylobacter is not unique in attaching novel nonulosonate sugar derivatives to its flagellin Flagel-lins from a strain of Campylobacter botulinum, a Gram-positive spore forming anaerobe, have also been shown to be glycosylated with a novel legionaminic acid derivative, 7-acetamido-5-(N-methyl-glutam-4-yl)- amino-3,5,7,9-tetradeoxy-d-glycero-a-d-galacto-nonulo-sonic acid (Leg5GluMe7Ac) [25], whereas other strains appear to produce related structures It is not yet known whether these modifications also contribute to the colonization ability of C botulinum isolates in distinct animal hosts
Experimental procedures
Bacterial strains and growth conditions
C jejuni 11168 and isogenic mutants pseB and pseC were grown using the procedure as described previously [12]
Purification of flagellin
Flagellin was purified as previously described [26], although the solubilization step in 1% SDS was eliminated and the crude pellet after ultracentrifugation was characterized directly by MS
LC-MS/MS analysis of flagellin
Flagellin protein was digested overnight with trypsin (50–
200 lg; Promega, Madison, WI, USA) at a ratio of 30 : 1 (protein : enzyme, v⁄ v) in 50 mm ammonium bicarbonate
at 37C Protein digests were analyzed by MS as previ-ously described [10]
Construction of C jejuni 11168 pseB and pseC mutant strains
To generate pseB(Cj1293)::cat and pseC(Cj1294)::cat mutants
in C jejuni 11168, a 4052 bp fragment (dcd-Cj1295) was amplified by PCR from chromosomal DNA using oligonucle-otides Pse1 (5¢-ATTTTACACTTTGACTAGGTTGAGC-3¢) and Pse2 (5¢-ATATTATGCCAAGATTTACAAGTGG-3¢) The product was inserted into the EcoRV site of plasmid PCRscript and the SmaI site of plasmid pUC19, resulting in plasmids PCRscript(pse) and pUC19(pse) A 1.1 kb
Trang 8chloram-phenicol-resistance cassette (cat) obtained from SmaI digested
plasmid pRY109 [27] was inserted either into the unique
BseRI (for pseB) or DraIII (for pseC) site, and subsequently
treated with T4 DNA Polymerase, of PCRscript(pse) and
PCR-script(pseB::cat) and pUC19(pseC::cat) Five micrograms of
Escherichia coliDH5a derived gene knockout plasmid DNA
carrying the cat gene in a nonpolar orientation, as verified by
PCR analyses with primer Pse1 and cat-specific
oligonucleo-tides ccatB (5¢-TTCTGAAAAAACGCCTACCTG-3¢) and
ccatF (5¢-AATGTCCGCAAAGCCTAATC-3¢), was used to
transform C jejuni 11168 by electroporation, as described
pre-viously [28] Chloramphenicol-resistant transformants were
characterized by PCR with oligonucleotides ccatF⁄ 1295-2
GC-3¢) to confirm that the incoming plasmid DNA had
inte-grated by a double cross-over event
Preparation of cell lysates
Cell lysates of parent strain C jejuni 11168 and isogenic
mutants pseB and pseC were prepared and extraction of
intracellular sugar-nucleotides from the cell lysates was
achieved using ENVI-Carb (Supelco, Bellefonte, PA, USA)
solid phase extraction cartridges, as described previously
[12]
MS
Cell lysates of parent strain C jejuni 11168 and isogenic
mutants pseB and pseC were probed for intracellular
sugar-nucleotides using HILIC-MS and a precursor ion scanning
method, as described previously [12] The intracellular
sugar-nucleotides were separated by HILIC on a TSKgel
Amide80 column (inner diameter 250· 4.6 mm; Tosoh
Bio-science, Montgomeryville, PA, USA) using an Agilent 1100
Series LC system (Santa Clara, CA, USA) and detected by
precursor ion scanning on a 4000 QTRAP hybrid triple
quadrupole linear ion trap mass spectrometer (AB⁄ MDS
Sciex, Concord, ON, Canada) For large-scale purifications
of the unknown intermediates, the flow from the LC system
was split 2 : 8 v⁄ v to the mass spectrometer and the
fractions collected and pooled for subsequent structural
analyses
For high resolution MS experiments, an Agilent 1100
Series LC system was coupled to a Q-ToF Premier hybrid
quadrupole TOF mass spectrometer equipped with an
Elec-trospray Ionization (ESI) LockSpray modular source
(Waters Corp.) Calibration was performed using the
MS⁄ MS fragment ions of [Glu1
]-Fibrinopeptide B (1 pmo-lÆlL)1; Sigma-Aldrich, St Louis, MO, USA) in the positive
mode and a lock mass solution of leucine enkephelin
(100 pgÆlL)1; Sigma-Aldrich) A typical mass accuracy of
2 p.p.m or less was obtained During data acquisition, the
lock mass solution was infused continuously at a frequency
of 10 : 1 (sample-to-reference ratio) Separations were achieved using the TSKgel Amide80 column and the same mobile phase as reported previously [12,13]
The MS data were acquired using the centroid mode Tandem MS of the sugar nucleotide molecule was carried out using argon as collision gas with a collision energy
of 18 eV To obtain MS⁄ MS of the sugar molecules, the cone voltage was increased to 50 V to promote in-source fragmentation and the resulting sugar oxonium ions were selected as precursor ions for tandem MS (collision
ener-gy = 20 eV) All data acquisition was performed using masslynx, version 4.1 (Waters Corp.) Elemental compo-sition was performed using a mass tolerance of 10 p.p.m and was sorted by i-FIT score assigned by masslynx based on the isotopic patterns of the target ions The ele-ments used for searching were limited to C, H, N, O and P
NMR spectroscopy
1
H and 13C NMR spectra were recorded using a Varian Inova 600 spectrometer (Varian, Palo Alto, CA, USA) with
a cold probe in D2O (Cambridge Isotopes Laboratories Inc., Andover, MA, USA) solutions at 25C with acetone standard (2.23 p.p.m for1H and 31.5 p.p.m for13C) using standard COSY, TOCSY (mixing time 120 ms), ROESY (mixing time 200 ms), HSQC and HMBC (100 ms long-range transfer delay)
Acknowledgements
We would like to thank Dr C Szymanski, University
of Alberta, for providing pseB and pseC isogenic mutants of 11168 and Tom Devecseri, NRC-IBS, for his assistance in preparing the figures
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Supporting information
The following supplementary material is available:
Fig S1 Intracellular CMP-sugars detected in isogenic
mutant pseB by HILIC-MS and precursor ion
scan-ning for fragment ions related to CMP (m⁄ z 322)
Table S1 Elemental composition report for unknown
CMP-sugars I and II based on accurate mass
measure-ments carried out on a Waters Q-ToF Premier mass spectrometer
Table S2 NMR chemical shifts (p.p.m.) and coupling constants J (Hz) for II and model compounds CMP-Pse5Ac7Ac and CMP-Pse5Ac7Am CMP1H signals at 6.12⁄ 8.01 p.p.m
This supplementary material can be found in the online version of this article
Please note: Wiley-Blackwell is not responsible for the content or functionality of any supplementary materials supplied by the authors Any queries (other than missing material) should be directed to the corre-sponding author for the article