Commonality and Biosynthesis of the O-MethylPhosphoramidate Capsule Modification inCampylobacter jejuni*□S
Received for publication, May 30, 2007, and in revised form, July 13, 2007 Published, JBC Papers in Press, August 3, 2007, DOI 10.1074/jbc.M704413200
David J. McNally‡1,2, Marc P. Lamoureux‡2, Andrey V. Karlyshev§, Laura M. Fiori‡, Jianjun Li‡, Gillian Thacker§,Russell A. Coleman‡, Nam H. Khieu‡, Brendan W. Wren§, Jean-Robert Brisson‡, Harold C. Jarrell‡3,and Christine M. Szymanski‡4
From the ‡Institute for Biological Sciences, National Research Council of Canada, 100 Sussex Drive, Ottawa, Ontario K1A 0R6, Canadaand the §Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine,London WC1E 7HT, United Kingdom
In this study we investigated the commonality and biosynthe-sis of theO-methyl phosphoramidate (MeOPN) group found onthe capsular polysaccharide (CPS) of Campylobacter jejuni.High resolution magic angle spinning NMR spectroscopy wasused as a rapid, high throughput means to examine multipleisolates, analyze the cecal contents of colonized chickens, andscreen a library of CPS mutants for the presence of MeOPN.Sixty eight percent ofC. jejuni strains were found to express theMeOPN with a high prevalence among isolates from enteritis,Guillain Barre, and Miller-Fisher syndrome patients. In con-trast, MeOPN was not observed for any of the Campylobactercoli strains examined. The MeOPN was detected on C. jejuniretrieved from cecal contents of colonized chickens demon-strating that the modification is expressed by bacteria inhabit-ing the avian gastrointestinal tract. In C. jejuni 11168H, thecj1415-cj1418 cluster was shown to be involved in the biosyn-thesis of MeOPN. Genetic complementation studies and NMR/mass spectrometric analyses of CPS from this strain alsorevealed that cj1421 and cj1422 encode MeOPN transferases.Cj1421 adds the MeOPN to C-3 of the �-D-GalfNAc residue,whereas Cj1422 transfers the MeOPN to C-4 of D-glycero-�-L-gluco-heptopyranose. CPS produced by the 11168H strain wasfound to be extensively modified with variableMeOPN,methyl,ethanolamine, and N-glycerol groups. These findings establishthe importance of the MeOPN as a diagnostic marker and ther-apeutic target for C. jejuni and set the groundwork for futurestudies aimed at the detailed elucidation of theMeOPN biosyn-thetic pathway.
Campylobacter jejuni is the leading cause of bacterial food-borne gastroenteritis, a causative agent of child morbidity inunderdeveloped countries and an antecedent to the Miller-Fisher and Guillain-Barre neuropathies (1–5). Furthermore, C.jejuni now surpasses Salmonella, Shigella, and Escherichia insome regions as the primary cause of bacterial gastrointestinaldisease (6–8). Because the number of reported C. jejuni infec-tions is increasing worldwide, there is growing interest to iden-tify virulence mechanisms associated with this mucosal patho-gen as a critical step toward the development of controlstrategies.The capsular polysaccharides (CPS)5 produced by C. jejuni
are known to be important virulence factors that are involved incolonization and invasion (9, 10). CPS expression was shown tobe necessary for diarrheal disease in ferrets, mediating mouseand chicken colonization, increasing resistance to humanserum, as well as increasing adherence and invasion of humanepithelial cells (9). TheCPSs produced byC. jejuni are themajorantigenic component of Penner’s serotyping system (10). Thereare nowmore than 60 serostrains described for this bacterium.Although not every strain has been examined, it is thought thateach one produces a CPS having a different structure (11, 12).Furthermore, there can be extensive phase-variable structuralmodifications such as the incorporation of methyl, ethanola-mine, and aminoglycerol groups on CPS sugars (13–15). It isthought that these extensive modifications may allow the bac-terium to evade host defenses (14, 15).The most unusual CPS modification is the O-methyl phos-
phoramidate CH3OP(O)(NH2)(OR) (MeOPN) group, which isa labile phosphorylated structure. Nitrogen-phosphorus bondsare rare in nature, and we reported the first example of such astructure produced by a bacterium in the CPS of C. jejuniNCTC11168 (HS:2) (13) (Fig. 1). Since this initial study, the G1(HS:1), NCTC12517 (HS:19) and 81-176 (HS:23/26) strains ofC. jejuni have been shown to produce MeOPN (14–16) and a
* This work was supported by the National Research Council of CanadaGenomics and Health Initiative, the Ontario Ministry of Agriculture andFood, Dow AgroSciences, the Biotechnology and Biological SciencesResearch Council, and the Leverhulme Trust. The costs of publication ofthis article were defrayed in part by the payment of page charges. Thisarticle must therefore be hereby marked “advertisement” in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.
□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Tables 1–3 and Figs. 1 and 2.
1 Recipient of funding from Dr. H. Jennings.2 Both authors contributed equally to this work.3 To whom correspondence may be addressed. Tel.: 613-993-5900; Fax: 613-
952-9092; E-mail: [email protected] To whom correspondence may be addressed. Tel.: 613-990-1569; Fax: 613-
related structure has been identified on the lipooligosaccharideof Xanthomonas campestris, a Gram-negative plant pathogen(17). Preliminary structural analyses of CPS from different C.jejuni serotypes suggested the presence of the MeOPNmodifi-cation on a diverse range of CPS sugars (18). This observationprompted us to isolate and elucidate theCPS structure from theG1 and NCTC12517 strains. The G1 CPS was subsequentlyshown to have a [-4)-�-D-Galp-(1–2)-(R)-Gro-(1-P]n repeatingunit with two labile �-D-fructofuranose branches at C-2 andC-3 of Gal. Each fructofuranose was further substituted at C-3with MeOPN groups (14). Similarly, the NCTC12517 CPS wasshown to have a [-4)-�-D-GlcA6NGro-(1–3)-�-D-GlcNAc-(1-]nrepeating unit with a labile �-L-sorbofuranose branch at C-2ofGlcA and anMeOPNatC-4 ofGlcNAc (15). For both strains,the keto sugars and MeOPN groups were found to be nonstoi-chiometric and were hypothesized to contribute to the overallstructural heterogeneity of the CPS. Furthermore, the MeOPNwas found to be variably methylated in the NCTC12517 strainthereby adding additional variability to an already structurallyheterogeneous CPS. Most recently, Kanipes and co-workers(16) demonstrated that the 81-176 strain also has a MeOPNCPS modification and provided evidence to show that it mostlikely is found at C-2 of the galactose residue.In a recent study, the CPS biosynthetic regions for selected
strains of C. jejuni were sequenced, including 176.83 (HS:41), 81-176, ATCC43456 (HS:36), CCUG10954 (HS:23),NCTC12517, and G1 (18). Comparison of the CPS sequencesfor the NCTC12517, 176.83, and G1 strains to the genome-sequenced NCTC11168 strain provided evidence for mul-tiple mechanisms of CPS variation, including exchange ofcapsular genes by horizontal transfer, gene duplication, dele-tion, fusion, and contingency gene variation. Interestingly,the study uncovered the presence of a highly conserved genecluster (cj1415–cj1420) within those strains that producethe MeOPN CPS modification (NCTC11168, NCTC12517,G1, 81-176). Because these genes have no apparent role insugar biosynthesis and because their presence coincides withthe presence of MeOPN on CPS sugars, it was hypothesizedthat they might be involved in MeOPN biosynthesis. Fur-thermore, in our recent study we demonstrated that 1H andone-dimensional 1H-31P HSQC high resolution magic anglespinning (HR-MAS) NMR can be used to quickly detectMeOPN from microliter amounts of intact bacterial cells(19).In this study, we explore the commonality of the MeOPN
modification among C. jejuni strains by using HR-MAS NMRas a rapid, high throughput means to directly examine severalanimal and human isolates from diverse clinical presentationsand geographical locations. A chicken model was then devel-oped to evaluate MeOPN expression within the natural avianhost. Finally, genes implicated in MeOPN biosynthesis wereidentified by using HR-MAS NMR to compare a library of CPSmutants for the presence of MeOPN. The identity of twoMeOPN transferases was then established using geneticcomplementation together with high resolution NMR andmass spectrometric studies of purified CPS.
EXPERIMENTAL PROCEDURES
Bacterial Strains and Growth Conditions—C. jejuniNCTC11168 (HS:2) and its motile variant, 11168H (HS:2) wereroutinely grown onMueller Hinton (MH) agar (Difco) at 37 °Cundermicroaerobic conditions (85%N2, 10%CO2, 5%O2). TheNCTC11168 strainwas originally isolated froma case of humanenteritis (20) and later sequenced by Parkhill et al. (21). Toselect for kanamycin (Kan)-resistant mutants, Kan was addedto the medium at a final concentration of 30 �g/ml. For largescale extraction of CPS, 6 liters of bacteria were grown in brainheart infusion broth (Difco) under microaerobic conditions at37 °C for 24 h with agitation at 100 rpm. Bacterial cells werethen harvested by centrifugation (9,000 � g for 20 min) andplaced in 70% ethanol. Cells were removed from the ethanolsolution by centrifugation (9000 � g for 20 min), and the bac-terial pellet was refrigerated until extraction.High Resolution Magic Angle Spinning (HR-MAS) NMR
Spectroscopy—Bacterial cells were prepared and analyzed byHR-MAS NMR spectroscopy as described previously (13, 19).HR-MAS NMR experiments were performed using a VarianInova 500-MHz (1H) spectrometer (Varian, Palo Alto, CA)equipped with a Varian 4-mm indirect detection gradientnano-NMR probe with a broadband decoupling coil. Sampleswere spun at 3 kHz, and spectra were recorded at ambient tem-perature (23 °C). HR-MAS NMR experiments were generallyperformed with suppression of the HOD signal using presatu-ration as described previously (13). 1HNMRspectra of bacterialcells were acquired using the Carr-Purcell-Meiboom-Gill pulsesequence (90-(�-180-�)n acquisition) to remove broad signalsoriginating from lipids and solid-like materials (22) and weretypically obtained using 256 transients (11min). The total dura-tion of the Carr-Purcell-Meiboom-Gill pulse (n � 2�) was 10ms with � set to 1/MAS spin rate. One-dimensional 1H-31PHSQC spectra were acquired using the standard Varian HSQCpulse sequence with one-dimensional spectra representing thefirst increment of the standardHSQC experiment. All 1HNMRspectra were referenced to an internal 3-(trimethylsilyl)propi-onic-2,2,3,3-d4 acid sodium salt standard (�H 0.00 ppm).Bacterial Colonization of Specific Pathogen-free Leghorn
Chicks—The inoculum for each chick colonization experimentwas prepared by harvesting C. jejuni 11168H cells, grown for18 h into a 0.1 M (pH 7.4) phosphate-buffered saline solution(supplemented with 0.14 M NaCl and 0.002 M KCl). One-day-old specific pathogen-free chicks were orally gavaged with 300�l of inoculum containing �3 � 1010 bacterial cells. Becausechicks typically do not consume feed during the first 48 h afterhatching (23), cecal contents were analyzed 48 h post oralgavage tominimize the amount of particulatematter within thececal contents that could potentially interfere with HR-MASNMR. Furthermore, only adherent bacteria should be presentat 48 h post oral gavage because the rate of passage through thegastrointestinal tract of leghorns is �4 h (24). All chicks wereeuthanized by cervical dislocation according to the approvedguidelines of theCanadianCouncil forAnimal Care. Cecal con-tents were then serially plated onto Karmali agar (Oxoid,Ontario, Canada) and examined for the presence of C. jejuniand for MeOPN using HR-MAS NMR spectroscopy. In total,
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the cecal contents from 39 chicks were examined (13 chicksfrom three independent experiments).Construction of C. jejuni Site-specific Mutations—Mutants
were constructed via insertion of a kanr cassette into uniquesites present within appropriate pUC18-based recombinantplasmids from a random genomic library of C. jejuni (strainNCTC11168) that was constructed during the course of thegenome sequencing project (21) (supplemental Table 1). Thekanr cassette was inserted in a nonpolar orientation, andthe derivatives were used for transformation of the C. jejuni
11168H strain via electroporation.Mutants were verified by PCR usinggene-specific and kanr-specificprimers. The cj1421/cj1422 doublemutant was created through spon-taneous insertion of the kanr cas-sette into both genes during con-struction of the cj1422 mutantbecause of the presence of identicalregions for these genes. Integrationof the kanr cassette within bothgenes was detected by PCR analysisusing cj1421 and cj1422-specificprimers and was later confirmed bysequencing.Complementation Studies of the
cj1421/cj1422 Double Mutant—cj1421 and cj1422 were PCR-ampli-fied using chromosomal DNAobtained from the 11168H strainand high fidelity Taq polymerase(Accuprime TaqHiFi, 95 °C 15 s, 30cycles of 95 °C for 15 s, 55 °C for 15 s,and 68 °C for 2 min) (Invitrogen)using the primers listed in supple-mental Table 2. PCR fragments con-taining complete gene copies were
then used for complementation studies using the pRED1 inte-grational expression vector according to Karlyshev andco-workers (25). To produce the delivery plasmids pRR1421and pRR1422, a 0.7-kb gfp-SwaI/XbaI fragment of pRED1was replaced with a 1.9-kb PCR product that was digestedwith PmeI/XbaI. As a precaution, pRR1421 and pRR1422were sequenced because cj1421 and cj1422 containhomopolymeric G tracts that are prone to length variation(18). Following electroporation of the delivery constructsinto the appropriate mutants, complemented strains were
FIGURE 1. The structure of the capsular polysaccharide produced by C. jejuni NCTC11168, 11168H, andthe isogenic mutant cj1421. A is �-D-Ribf; B is �-D-GlcpA6(NGro); C is �-D-GalfNAc; D is D-glycero-�-L-gluco-heptopyranose; and E is MeOPN.
TABLE 1Presence (�) or absence (�) of the O-methyl phosphosphoramidate (MeOPN) capsular polysaccharide modification for C. jejuni and C.coli strains as determined using HR-MAS 1H NMR and 31P decoupled, 1H-31P HSQC HR-MAS NMR experiments to screen for the presenceof the MeOPN
selected on blood agar plates and supplemented with Kan (50�g/�l) and chloramphenicol (15 �g/�l). Integration of thecmr cj1421 and cmr cj1422 gene fusions into the rRNA genecluster on the 11168H chromosome was confirmed by PCRas described previously (25).Isolation and Purification of CPS—For large scale extraction
of CPS, 6 liters of the C. jejuni 11168H wild type and the cj1421mutant were grown in brain heart infusion broth undermicroaerobic conditions at 37 °C for 24 h with agitation at 100rpm. Capsular polysaccharide was then isolated and purifiedusing a gentle enzymatic method according to McNally et al.(14).High Resolution NMR Spectroscopy of Purified CPS—For
NMR spectroscopy of CPS isolated from the C. jejuni 11168Hwild type and the cj1421 mutant, �3 mg of pure CPS was sus-pended in 200 �l of 99% buffered D2O (50 mM NH4HCO3, pD8.0) and placed in a 3-mm NMR tube. NMR experiments wereperformed using a Varian Inova 500 MHz (1H) spectrometerequipped with a Varian Z-gradient 3-mm triple resonance (1H,13C, 31P) probe, or a Varian 600 MHz (1H) spectrometerequipped with a Varian 5-mm, Z-gradient triple resonancecryogenically cooled probe (cold probe). One-dimensional 31Pspectra were acquired using a Varian Mercury 200-MHz (1H)spectrometer and a Nalorac 5-mm four nuclei probe. NMRexperiments were typically performed at 25 °C with suppres-
sion of the HOD resonance at 4.78 ppm. Standard homo- andheteronuclear correlated two-dimensional pulse sequencesfrom Varian were used for general assignments. Selective one-dimensional total correlation spectroscopy and NOESY exper-iments with a Z-filter were used for complete residue assign-ment as well as for measuring JH,H coupling constants andnuclear Overhauser enhancements (26, 27). Proton and carbonresonances were referenced to an internal acetone standard (�H2.225 ppm, �C 31.07 ppm), whereas phosphorus signals werereferenced to an external 85% phosphoric acid standard (�P0.00 ppm).Mass Spectrometric Analyses of Purified CPS—CPS isolated
from theC. jejuni 11168Hwild type and the cj1421mutantweremass-analyzed using in-source collision-induced dissociationCE-ESI/MS according to Li et al. (28) with a Prince systemcapillary electrophoresis instrument (Prince Technologies,Emmen, The Netherlands) coupled to a 4000 Qtrap spectrom-eter (Applied Biosystems/Sciex, Foster City, CA) via amicroIonspray interface. A sheath solution (isopropyl alcohol/methanol,2:1) was delivered at a flow rate of 1 �l/min. Separations were
FIGURE 2. HR-MAS NMR analysis of the cecal contents of a representativeC. jejuni colonized chick. a, HR-MAS 1H NMR spectrum of plate-grown11168H cells (256 scans). The inset shows the spectrum for the same sampleanalyzed with a 1H-31P HSQC HR-MAS NMR experiment (1024 scans, 31Pdecoupled, 1JH,P � 12 Hz). b, the HR-MAS 1H NMR spectrum of the cecal con-tents of a chick that has been colonized with C. jejuni 11168H at 48 h post-inoculation (256 scans). The inset shows the spectrum for the same cecalsample examined using the 1H-31P HSQC HR-MAS NMR experiment (1024scans). OMe is the –OCH3 group located at C-6 of residue D, and HOD is thedeuterated water resonance. All spectra were acquired at 500 MHz (1H).
FIGURE 3. Screening intact cells of a C. jejuni CPS mutant library for thepresence of MeOPN using HR-MAS 1H NMR. The inset shows the region ofthe CPS biosynthesis locus that contains putative MeOPN biosynthesis/trans-fer genes (black) and those genes that have no known role in MeOPN biosyn-thesis (white). HR-MAS 1H NMR spectrum for 11168H wild type cells (a) and forthe isogenic mutants cj1415 (b), cj1416 (c), cj1417 (d), cj1418 (e), cj1419 (f),cj1420 (g), cj1421 (h), and cj1422 (i) are shown. Shading indicates the region ofthe spectrum where –OCH3 protons of the MeOPN resonate. For all NMR experi-ments, 40 �l of cells were examined at 500 MHz (1H) using 256 scans except forcj1415 where 2816 scans were necessary to generate the spectrum shown.
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achieved on �90 cm of bare-fused silica capillary (360 �mout-side diameter � 50 �m inner diameter, Polymicro Technolo-
gies, Phoenix, AZ) using 15 mM ammonium acetate/ammo-nium hydroxide in deionized water, pH 9.0, containing 5%MeOHas the separation buffer. A voltage of 20 kVwas typicallyapplied during CE separation, and �5 kV was used as electro-spray voltage. Mass spectra were acquired with dwell times of3.0msper step of 0.1m/z�1 unit in fullmass scanmodeusing anorifice voltage of �400 V. Fragment ions formed by collisionactivation of selected precursor ions with nitrogen in the RF-only quadrupole collision cell were then mass-analyzed byscanning the third quadrupole.
RESULTS
Commonality and Specificity of the MeOPN CPS Modifica-tion in Campylobacter—Assessing the commonality and spec-ificity of MeOPN expression among Campylobacter isolates isimportant to determine the abundance of this CPS modifica-tion and its potential as a diagnostic marker and therapeutictarget. Previous studies have shown that the MeOPN is readilydetected on the surface of intact C. jejuni cells using HR-MAS1HNMR spectroscopy (13–15, 19) making this technique ame-
nable to the rapid, high throughputscreening of multiple strains. TheMeOPN can be considered as aunique NMR tag because signalsarising from its –OCH3 protons areintense compared with other CPSsignals and have a unique chemicalshift and phosphorus scalar cou-pling. To assess MeOPN preva-lence, intact cells from 63 C. jejunistrains and 18 Campylobacter colistrains were screened usingHR-MAS 1H NMR, as well as 31Pdecoupled, 1H-31P HSQC HR-MASNMR experiments. The C. jejunistrainswere comprised of human andanimal isolates; originated from arange of geographical locations thatincluded Canada, the United States,Japan, the Netherlands, Brazil, andtheUnitedKingdom; and are respon-sible for a variety of clinical presenta-tions, including asymptomatic, enter-itis, Guillain-Barre syndrome, Miller-Fisher syndrome, and septicemia(Table 1). Overall, 43 of 63 or 68% ofthe C. jejuni strains were found toexpress the MeOPN CPS modifica-tion. Interestingly, the MeOPN wasobserved for 82% of enteritis strains,80% of Guillain Barre syndromestrains, and 100% of Miller-Fishersyndrome strains. In contrast,MeOPNwas not detected for any ofthe 18 C. coli strains examined.DetectionofMeOPNfromtheCecal
Contents of Colonized Chickens—To assess the expression of theMeOPNwithin the natural avian host, a chickenmodelwas devel-opedusing1-day-old specificpathogen-free chicks thatwere inoc-ulatedwithahighdoseofC. jejuni11168Hcells. Fig. 2 is represent-ative of the 13 cecal contents that were examined using HR-MASNMR spectroscopy. The HR-MAS 1H NMR spectrum of in vitroplate-grownC. jejuni11168Hcells exhibited clear signals originat-ing fromtwopartially overlappedMeOPNsat�H3.77ppmand�H3.79 ppmwith phosphorus scalar couplings of approximately JH,P12.0 Hz (Fig. 2a, note that the partial overlap of the doublet signalfrom eachMeOPN results in the appearance of three peaks). Sig-nals originating from the anomeric protons of theCPS sugars�-D-Ribp (A1),�-D-GlcpA (B1),�-D-GalfNAc (C1), and D-glycero-�-L-gluco-heptopyranose (D1) were observed in agreement with theCPS structure reported for the NCTC11168 strain (Fig. 1 and Fig.2a) (8, 13). Two anomeric signals were observed for residue C as aresult of structural heterogeneity generated by the phase variableMeOPN group at C-3 of this sugar (13). Furthermore, a clear sin-glet was observed for the –OCH3 group (OMe) located at C-6 ofresidue D. In contrast, the HR-MAS 1H NMR spectrum of thececal contents harvested fromoneof theC. jejuni colonized chickswas complicated by signals originating from cecal matter that
FIGURE 4. High resolution NMR analysis of CPS purified from C. jejuni 11168H. a, 1H NMR spectrum (128scans). b, 1H-31P HMQC spectrum (256 scans, 32 increments, 1JP,H � 10 Hz). c, 1H-13C HSQC spectrum (128 scans,128 increments, 1JC,H � 150 Hz). *C represents �-D-GalfNAc substituted at C-3 with the MeOPN, and C is�-D-GalfNAc without the MeOPN. Spectra shown in a and b were acquired at 500 MHz (1H) whereas that shownin c was acquired at 600 MHz (1H) with a cold probe. All spectra were recorded at 25 °C in 99% buffered D2O (50mM NH4HCO3, pD 8.0).
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overlappedwithCPS signals (Fig. 2b). To specifically probe for theMeOPN, a one-dimensional, 31P decoupled, 1H-31P HSQC HR-MAS NMR experiment was used that attenuates signals fromcecal matter (15, 19). Using this experiment to examine plate-
grown intact cells, two clear signalscould be observed originating fromboth MeOPN groups at �H 3.77 and3.79 ppm (Fig. 2a, inset). In a similarfashion, analysis of the cecal contentsof C. jejuni colonized chicks revealedthat both MeOPN signals could bereadily detected in all samples exam-ined (seeFig. 2b, inset, for a represent-ative spectrum).Identification of Genes Impli-
cated in MeOPN Biosynthesis andTransfer—To identify MeOPN bio-synthesis genes, HR-MAS 1H NMRexperiments were used to rapidlyexamine a library of CPS mutantsfor the presence of MeOPN (Fig. 3,shaded region). The HR-MAS 1HNMR spectrumof 11168Hwild typecells showed the expected anomericsignals for CPS sugars, the –OCH3group located on residue D, as wellas two partially overlapped doubletsignals at �H3.77 and 3.79 ppmorig-inating from both MeOPN groupswith 31P scalar couplings of approx-imately JH,P 12.0 Hz each (Fig. 3a).In contrast, MeOPN signals werenot detected for the cj1415, cj1416,cj1417, and cj1418 mutants thatclearly implicated these genes in thesynthesis or transfer of MeOPN(Fig. 3, b–e). Of importance, all ofthe mutants examined producedCPS as indicated by the anomericresonances that are observablewithin the HR-MAS 1H NMR spec-tra (Fig. 3, labels A1–D1). HR-MAS1H NMR and one-dimensional1H-31PHSQC analyses of the cj1419and cj1420mutants revealed signalsfrom both MeOPN groups therebynegating a role for these genes inMeOPN biosynthesis (Fig. 3f). Incontrast, spectra for the cj1421mutant showed the loss of theMeOPN signal at �H 3.77 ppm,whereas that for cj1422 revealed theloss of the MeOPN signal at �H 3.79ppm thereby implicating the prod-ucts of these genes in MeOPN bio-synthesis or transfer (Fig. 3, g–i).Based on these findings, it was con-cluded that cj1415, cj1416, cj1417,
cj1418, cj1421, and cj1422 are involved inMeOPNbiosynthesisor transfer.Structure Elucidation of CPS Purified from C. jejuni 11168H—
Compared with the CPS structure reported for the genome-
FIGURE 5. Mass spectrometric analysis of CPS purified from C. jejuni 11168H. a, CE-ESI-MS total ion chro-matogram (positive ion mode, orifice voltage �400 V). b, CE-ESI-MS/MS analysis for m/z 991 representing onerepeat of the CPS containing both MeOPN modifications (positive ion mode, orifice voltage �400 V). Pentoseis �-D-Ribf, HexANGro is �-D-GlcpA6(NGro), HexNAc is �-D-GalfNAc, Hep is D-glycero-�-L-gluco-heptopyranose,and MeOPN is O-methyl phosphoramidate.
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sequenced NCTC11168 strain (8, 13), the HR-MAS 1H NMRspectrum for 11168H intact cells revealed important structuraldifferences such as a novel MeOPN modification at �H 3.79ppm (Fig. 2a). To characterize these structural differences indetail, purified CPS was investigated with NMR at 500 MHz(1H) or 600 MHz (1H) with a cold probe for optimal sensitivity(Fig. 4) and with mass spectrometry (Fig. 5).The proton spectrum for purified 11168H CPS closely
resembled that observed for intact cells in that signals originat-ing from anomeric sugars (A1–D1) and both MeOPN groupswere clearly discernible (Fig. 4a). By comparing proton andcarbon chemical shift data, as well as correlation patternsobtained using total correlation spectroscopy, correlated spec-troscopy, NOESY experiments (not shown), and an 1H-13CHSQC experiment (Fig. 4c) to those reported for NCTC11168CPS (8, 13), nearly all resonances were assigned for 11168HCPS sugars (Table 2). Two individual spin systems were identi-fied for residue C; one for C that is substituted at the 3-positionwith aMeOPNgroup (Fig. 4, labeled *C) and another where theMeOPN is absent (Fig. 4, labeled C). Because of weak signalsand spectral overlap, we were able to make only partial NMRassignments for residue *C (Table 2). These observations are ingood agreement with our previous work that showed the
MeOPN is a phase-variable CPS modification in theNCTC11168 strain (13). A 1H-31PHMQC experiment revealedthe location of the novel MeOPN group to be at C-4 of D-glyc-ero-�-L-gluco-heptopyranose (Fig. 4b). This finding is sup-ported by the proton chemical shifts for 11168H CPS sugarsthat are nearly identical to those reported for the NCTC11168CPS (8, 13) with the exception of H-4 of residue D that is down-fielded by 0.76 ppm. This downfield shift is consistent with theeffects of phosphoramidation reported for H-3 of the �-D-GalfNAc CPS sugar in the NCTC11168 strain (0.64 ppm), andH-3 of the �-D-Fruf residue in the G1 strain of C. jejuni (0.74ppm) (8, 13, 14). Resonances observed for N-glycerol, ethanol-amine, an –OCH3 group (D9) at C-6 of residue D, and a novel–OCH3 group (D8) that was determined to be located at C-3 ofresidue D (using heteronuclear multiple-bond correlation andHMQCTOXY experiments; data not shown), indicated thatthe CPS produced by the 11168H strain is structurally het-erogeneous. CE-ESI-MS/MS experiments of purified 11168HCPS were used to corroborate NMR findings, and to furthercharacterize the extent of structural heterogeneity (Fig. 5 andTable 3). Fragment ions observed at m/z 1101 and 898 con-firmed the location of the novel MeOPN and –OCH3 group onresidueD (Fig. 5a). Ions observed atm/z 283, 415, 549, 634, 664,867, 884, 947, 977, and 1180 demonstrated that both MeOPNgroups can be variably methylated, whereas those observed atm/z 533, 665, and 791 showed that at least one –OCH3 group isvariably present on residue D. Of particular interest, CE-ESI-MS/MS of m/z 991 established that at least some repeatingunits within the CPS have both MeOPN groups present (Fig.5b). Based on these results, the CPS produced by C. jejuni11168Hwas concluded to be structurally heterogeneous and tohave the same repeating unit as theNCTC11168 strain with theaddition of a novel –OCH3 group andMeOPNgroup atC-3 andC-4 of D-glycero-�-L-gluco-heptopyranose, respectively (Fig. 1).Complementation Studies of cj1421 and cj1422—Based on
BLAST searches that indicated cj1421 and cj1422 have highsequence similarity with sugar transferases, that cj1421 andcj1422 share long regions of sequence identity (18), and thephenotypes obtained for the cj1421 and cj1422mutants (Fig. 3,h and i), it seemed likely that these genes encode transferasesthat add the MeOPN to CPS sugars. To conclusively define therole of cj1421 and cj1422, complementation studies were per-formed using the 11168H cj1421/cj1422 double mutant back-ground (Fig. 6).One-dimensional, 31P-decoupled, 1H-31P HSQC HR-MAS
NMR analysis of 11168H wild type cells revealed the expectedsignals for theMeOPNgroups atC-3 of residueC (�H3.77 ppm)and the novel MeOPN at C-4 of residue D (�H 3.79 ppm) (Fig.6a, note that spectra shown in Fig. 6 were 31P-decoupled toeliminate 31P scalar couplings resulting in the appearance ofonly one peak for eachMeOPN).Mutation of cj1421 resulted inthe loss of the MeOPN signal at �H 3.77 ppm which suggestedthis gene encodes a transferase that adds theMeOPN to residueC (Fig. 6b). To ensure that other CPS structures were notaffected by thismutation, the cj1421CPSwas isolated and char-acterized using identical techniques described for the 11168Hwild type CPS (see above). NMR and mass spectrometric anal-yses of purified cj1421 CPS confirmed that it is structurally
TABLE 2NMR data for capsular polysaccharide isolated from C. jejuni 11168H(a�H, a�C) and the isogenic mutant cj1421 (b�H, b�C)
a Represents residue B that is substituted with N-glycerol.b Represents residue B that is substituted with ethanolamine. Residue A is �-D-Ribf;B is the amide of �-D-GlcpA; C is �-D-GalfNAc (for 11168H, C and *C are �-D-GalfNAc without and with the MeOPN modification, respectively), and D isD-glycero-�-L-gluco-heptopyranose. Carbon and proton chemical shifts were ref-erenced to an internal acetone standard (�H 2.225 ppm and �C 31.07 ppm). Errorfor �H is � 0.02 ppm, for �C is � 0.2 ppm and for JH,H is � 0.2 Hz.
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identical to that produced by the 11168H wild type with theexception of missing theMeOPN at the 3-position of residue C(Fig. 1, Table 2, Supplemental Table 3, and Supplemental Figs. 1and 2). In contrast, mutation of cj1422 resulted in the loss of thesignal for the novel MeOPN group at �H 3.79 ppm that indi-cated this gene encodes a transferase responsible for adding theMeOPN to C-4 of residue D (Fig. 6c). By comparing the chem-ical shifts for the anomeric protons of cj1422 CPS sugars tothose reported for NCTC11168 (13), it was concluded thatother CPS structures were not affected by this mutation. Inter-estingly,mutation of cj1421 and cj1422 resulted in the completeloss of bothMeOPNmodifications (Fig. 6d). Complementationof cj1421 in cis in the double mutant background resulted inrestoration of the MeOPN on residue C (Fig. 3e), whereascomplementation of cj1422 in cis was found to restore theMeOPN found on residue D (Fig. 3f). Based on the results ofthese complementation studies, cj1421 and cj1422 were con-cluded to encode MeOPN transferases; Cj1421 adds theMeOPN to �-D-GalfNAc, whereas Cj1422 adds the MeOPN toD-glycero-�-L-gluco-heptopyranose.
DISCUSSION
Capsular polysaccharides are surface-exposed glycans on thebacterial cell that often contribute to virulence and mediateinteractions between the pathogen, host, and the environment.
There is interest in these glycans because therapeutics directedtoward CPS have been successful in the control of bacterialinfections. For example, theNeisseria meningitidis vaccine thatis currently in use targets the conserved CPS structure of groupC organisms (29). For C. jejuni, however, where at least 60 dif-ferent serostrains have been identified to date, there does notseem to be a dominant CPS structure. In this study we haveestablished the commonality of the MeOPN CPS modificationin C. jejuni, shown that the MeOPN can be used to detect C.jejuni cells from the natural avian host, and have identified sev-eral genes implicated in its biosynthesis and transfer.The majority of the C. jejuni isolates surveyed during this
study were found to express the MeOPN CPS modification.This observation points to the commonality of the MeOPN inC. jejuni and is further corroborated bywhole genomemicroar-ray studies that showed 61 of the 111C. jejuni strains tested hadpositive hybridization reactions for genes cj1421 and cj1422(30). The fact that none of the 18C. coli strains examined in thisstudy were found to express an MeOPN indicates that theMeOPN is specific forC. jejuni. Furthermore, that theMeOPNwas readily detected from the cecal contents of colonized chicksindicates that the MeOPN is expressed by C. jejuni cells inhab-iting the avian gastrointestinal tract and thus could potentiallybe used as a diagnostic marker for C. jejuni colonization. How-
TABLE 3Positive ion CE-ESI-MS data (�400 V orifice voltage), calculated masses, and proposed fragments for CPS isolated from the 111168H strain ofC. jejuniIsotope-averaged masses of residues were used for calculation of total molecular masses based on the following proposed compositions: HexNAc (�-D-GalfNAc), 221;HexANGro (�-D-GlcpA6NGro), 267; HexANEtn (�-D-GlcpA6N-ethanolamine), 237; Hep (D-glycero-�-L-gluco-heptopyranose), 224; pentose (ribose), 150; OCH3(O-methyl group), 15; MeOPN (O-methyl phosphoramidate, CH3OP(O)(NH2)), 93.2; NGro (N-glycerol), 73.1; OPN (O-phosphoramidate), 79.0; H2O, 18.0. For thesegas-phase (IS-CID) degradation products, no H2O molecule is added to the residue unless specifically indicated.
ever, MeOPN expression is not necessary for C. jejuni coloni-zation of chicks because MeOPNmutants in both 11168H and81-176 backgrounds colonized as well as the wild type (resultsnot shown).Analysis of a library of CPS mutants resulted in the identifi-
cation of genes that are directly implicated in the synthesis or
transfer of MeOPN. The definitive role of many of these genesremains to be established, however; BLAST searches have pro-vided putative functions (Table 4). For example, Cj1416 shows32% identity to LicC, a protein found in Neisseria spp., Hae-mophilus influenzae, and Streptococcus pneumoniae (32). Thelic genes are involved in the production of phosphorylcholine, asmall phosphorus-containing molecule that decorates surfaceglycoconjugate structures. LicC is the cytidylyltransferase thatactivates phosphorylcholine, and thus it is possible that Cj1416generates a nucleotide-linked MeOPN that is then recognizedby the Cj1421 and Cj1422 transferases. Conserved domainsearches for Cj1417 identified a type 1 glutamine amidotrans-ferase. Glutamine amidotransferase activity catalyzes the trans-fer of ammonia from the amide side chain of glutamine to anacceptor substrate. Using isotope-labeled 15NH4Cl, we previ-ously showed that 11168H is able to incorporate exogenousammonia into the MeOPN moiety of its CPS (19). One couldimagine a role for Cj1417 in transferring ammonia to a phos-phorus atom thereby forming the MeOPN. Similarly, theCj1415 and Cj1418 proteins resemble phosphate kinases andmay therefore play a key role in phosphoramidate biosynthesis.Cj1421 and Cj1422 are 55% identical to each other, with
strong conservation in the N and C termini (18), which is sug-gestive of a gene duplication event. Using complementationstudies of a double cj1421/cj1422 11168H mutant, we demon-strated that these genes encode MeOPN transferases. In the11168H strain, Cj1421 adds the MeOPN to �-D-GalfNAc,whereas Cj1422 adds the MeOPN to D-glycero-�-L-gluco-hep-topyranose. The different strains that are known to express theMeOPN modification have homologues of these genes withintheir CPS loci (18). The NCTC12517 strain (MeOPN attachedat C-4 of �-D-GlcNAcp) (15) has HS19.07, which is a cj1421homologue; the G1 strain (MeOPN attached at C-3 of �-D-Frufsugars) (14) also has a cj1421 homologue, whereas the 81-176strain (MeOPN attached at C-2 of �-D-Galp) (16) has HS23/36.07, which is a cj1422 homologue. cj1421 and cj1422, as wellas other genes within the CPS biosynthesis loci, were shown toundergo phase variation because of the presence of homopoly-meric tracts (21). This phase variability explains much of thestructural heterogeneitywithin theCPS structures produced byC. jejuni and also why the NCTC11168 strain expresses onlyone MeOPN despite having the genetic potential to expressboth MeOPN groups.In this study, we have shown that the second MeOPN group
is located at C-4 of D-glycero-�-L-gluco-heptopyranose in the11168H strain. The MeOPN groups are important sources ofstructural heterogeneity because they are variably methylated
FIGURE 6. Complementation studies for MeOPN transferase genes cj1421and cj1422. A 31P-decoupled, 1H-31P HSQC HR-MAS NMR experiment (256 scans,1JH,P � 12 Hz) was used to examine intact cells for the following: a, C. jejuni11168H wild type; b, cj1421 mutant; c, cj1422 mutant; d, cj1421/cj1422 doublemutant; e, cj1421/cj1422 double mutant complemented in cis with cj1421;f, cj1421/cj1422 double mutant complemented in cis with cj1422. For all NMRexperiments, 40 �l of cells were examined at 500 MHz (1H) using 256 scans.
TABLE 4Summary of proteins identified in this study to be involved in MeOPN biosynthesis and transfer
Protein Genome re-annotation function CPS phenotype by NMR Predicted function from this studyCj1415 Putative adenylylsulfate kinase
(Adenosine 5�-phosphosulfate kinase)Loss of MeOPN Phosphate biosynthesis for MeOPN?
Cj1418 Putative transferase (pyruvate phosphate dikinase) Loss of MeOPN Phosphate biosynthesis for MeOPN?Cj1421 Putative glycosyltransferase Loss of MeOPN on GalfNAc MeOPN transferase to GalfNAcCj1422 Putative glycosyltransferase Loss of MeOPN on Hep MeOPN transferase to Hep
a GATase indicates glutamine amidotransferase.
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and because one or two MeOPN groups can be present withinone repeat of the CPS. Based on the signals originating from theMeOPN groups on the cell surface that appeared in a 1:1 ratio(Fig. 2a), it can be concluded that there is an equal number ofeach MeOPN group within the 11168H CPS. In light of thevariably methylated MeOPNs on �-D-GalfNAc and/or D-glyc-ero-�-L-gluco-heptopyranose, variable N-glycerol or ethanol-amine groups on �-D-GlcpA, and two variable –OCH3 groupson D-glycero-�-L-gluco-heptopyranose, the 11168H strain pro-duces the most decorated and structurally heterogeneous CPSreported forC. jejuni. Using the visualmolecular dynamics soft-ware (33), we constructed a model for the repeating unit of theCPS produced by the 11168H strain. As can be seen, theseextensive CPS modifications would be prominent structuralfeatures on the cell surface (Fig. 7, darker color). One may inferthat these CPS decorations contribute unique properties interms of overall cell surface charge or surface epitopes and areof significance during host-pathogen interactions. Interest-ingly, when we first identified the MeOPN modification in C.jejuni, we demonstrated that a variant expressing this modifi-cation was less reactive with the heat-labile typing sera than thewild type strain without the MeOPN (13). This suggests thateither the MeOPN group was not expressed on the surface ofthe HS:2 serostrain used for rabbit immunizations or that thepresence of the MeOPN group prevents antibody recognitionof the CPS.Because the MeOPN is the only CPS structure known to be
conserved amongC. jejuni strains, it has potential use as a diag-nosticmarker and vaccine candidate for this bacterium. Severallines of evidence now indicate an important biological role forthe MeOPN in C. jejuni; it is a prominent structural feature onthe cell surface ofmultiple strains; it is expressed by cells inhab-
iting the natural avian host; and several genes are dedicated toits biosynthesis/transfer. A link between MeOPN expressionand pathogenicity cannot be eliminated given its high preva-lence among enteritis, Guillain Barre syndrome, and Miller-Fisher syndrome strains. Future work will focus on elucidatingthe biological relevance of the MeOPN group, functional char-acterization of the enzymes involved in MeOPN biosynthesis,and exploitation of this common structure in C. jejuni.
Acknowledgments—We thankKevin Smit, Daniel Beriault, andMaryFoss for technical support and Brenda Allan, Robert Mandrell, andHubert Endtz for strains used in the MeOPN survey.
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FIGURE 7. Molecular model of the repeating unit for the CPS produced byC. jejuni 11168H. The CPS structure consists of �-D-Ribp (A), �-D-GlcpA6(NGro) (B), �-D-GalfNAc (C), and 3,6-di-O-methyl-D-glycero-�-L-gluco-heptopyranose (D). As can be seen, the variable CPS modifications (darkercolor) such as MeOPNs, –OCH3 (OMe), N-glycerol (NGro), or ethanolamine (notshown) groups that decorate the repeating unit (lighter colors) would beprominent structural features on the cell surface for this strain. OH groupshave been removed to simplify the appearance of the model.
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