Functional Characterization of the Flagellar Glycosylation Locus in Campylobacter jejuni 81–176 Using a Focused Metabolomics Approach
2006; Elsevier BV; Volume: 281; Issue: 27 Linguagem: Inglês
10.1074/jbc.m603777200
ISSN1083-351X
AutoresDavid J. McNally, Joseph P. M. Hui, Annie Aubry, Kenneth Mui, Patricia Guerry, Jean‐Robert Brisson, Susan M. Logan, Evelyn C. Soo,
Tópico(s)Cancer Research and Treatments
ResumoBacterial genome sequencing has provided a wealth of genetic data. However, the definitive functional characterization of hypothetical open reading frames and novel biosynthetic genes remains challenging. This is particularly true for genes involved in protein glycosylation because the isolation of their glycan moieties is often problematic. We have developed a focused metabolomics approach to define the function of flagellin glycosylation genes in Campylobacter jejuni 81–176. A capillary electrophoresis-electrospray mass spectrometry and precursor ion scanning method was used to examine cell lysates of C. jejuni 81–176 for sugar nucleotides. Novel nucleotide-activated intermediates of the pseudaminic acid (Pse5NAc7NAc) pathway and its acetamidino derivative (PseAm) were found to accumulate within select isogenic mutants, and use of a hydrophilic interaction liquid chromatography-mass spectrometry method permitted large scale purifications of the intermediates. NMR with cryo probe (cold probe) technology was utilized to complete the structural characterization of microgram quantities of CMP-5-acetamido-7-acetamidino-3,5,7,9-tetradeoxy-l-glycero-α-l-manno-nonulosonic acid (CMP-Pse5NAc7Am), which is the first report of Pse modified at C7 with an acetamidino group in Campylobacter, and UDP-2,4-diacetamido-2,4,6-trideoxy-α-d-glucopyranose, which is a bacillosamine derivative found in the N-linked proteinglycan. Using this focused metabolomics approach, pseB, pseC, pseF, pseI, and for the first time pseA, pseG, and pseH were found to be directly involved in either the biosynthesis of CMP-Pse5NAc7NAc or CMP-Pse5NAc7Am. In contrast, it was shown that pseD, pseE, Cj1314c, Cj1315c, Cjb1301, Cj1334, Cj1341c, and Cj1342c have no role in the CMP-Pse5NAc7NAc or CMP-Pse5NAc7Am pathways. These results demonstrate the usefulness of this approach for targeting compounds within the bacterial metabolome to assign function to genes, identify metabolic intermediates, and elucidate novel biosynthetic pathways. Bacterial genome sequencing has provided a wealth of genetic data. However, the definitive functional characterization of hypothetical open reading frames and novel biosynthetic genes remains challenging. This is particularly true for genes involved in protein glycosylation because the isolation of their glycan moieties is often problematic. We have developed a focused metabolomics approach to define the function of flagellin glycosylation genes in Campylobacter jejuni 81–176. A capillary electrophoresis-electrospray mass spectrometry and precursor ion scanning method was used to examine cell lysates of C. jejuni 81–176 for sugar nucleotides. Novel nucleotide-activated intermediates of the pseudaminic acid (Pse5NAc7NAc) pathway and its acetamidino derivative (PseAm) were found to accumulate within select isogenic mutants, and use of a hydrophilic interaction liquid chromatography-mass spectrometry method permitted large scale purifications of the intermediates. NMR with cryo probe (cold probe) technology was utilized to complete the structural characterization of microgram quantities of CMP-5-acetamido-7-acetamidino-3,5,7,9-tetradeoxy-l-glycero-α-l-manno-nonulosonic acid (CMP-Pse5NAc7Am), which is the first report of Pse modified at C7 with an acetamidino group in Campylobacter, and UDP-2,4-diacetamido-2,4,6-trideoxy-α-d-glucopyranose, which is a bacillosamine derivative found in the N-linked proteinglycan. Using this focused metabolomics approach, pseB, pseC, pseF, pseI, and for the first time pseA, pseG, and pseH were found to be directly involved in either the biosynthesis of CMP-Pse5NAc7NAc or CMP-Pse5NAc7Am. In contrast, it was shown that pseD, pseE, Cj1314c, Cj1315c, Cjb1301, Cj1334, Cj1341c, and Cj1342c have no role in the CMP-Pse5NAc7NAc or CMP-Pse5NAc7Am pathways. These results demonstrate the usefulness of this approach for targeting compounds within the bacterial metabolome to assign function to genes, identify metabolic intermediates, and elucidate novel biosynthetic pathways. The availability of bacterial genomic sequences has provided unprecedented opportunity for comparative studies. The functional analysis of each respective genome is currently under way and often involves an integrative, multidisciplinary approach that combines bioinformatics, mutagenesis, proteomics, and microarray technologies. Most recently, metabolomics-based analyses, although limited in their number, are becoming established as an additional tool to elucidate gene function (1Weckwerth W. Morgenthal K. Drug Discov. Today. 2005; 10: 1551-1558Crossref PubMed Scopus (237) Google Scholar, 2Weckwerth W. Annu. Rev. Plant Biol. 2003; 54: 669-689Crossref PubMed Scopus (537) Google Scholar). Metabolomics is the characterization of all low molecular weight compounds in a defined biological system and differs from classical metabolism studies by its greater breadth and speed of metabolite analysis. Although recent advances in mass spectrometry (MS), 2The abbreviations used are: MS, mass spectrometry; Bac, 2,4-diamino-2,4,6-trideoxy-α-d-glucopyranose (also known as bacillosamine); CE, capillary electrophoresis; ESMS, electrospray ionization-MS; HILIC, hydrophilic interaction liquid chromatography; HMBC, heteronuclear multiple bond correlation; HSQC, heteronuclear single quantum correlation; NOESY, nuclear Overhauser effect spectroscopy; Pse5NAc7NAc, 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-α-l-manno-nonulosonic acid (also known as pseudaminic acid); Pse5NAc7Am, 5-acetamido-7-acetamidino-3,5,7,9-tetradeoxy-l-glycero-α-l-manno-nonulosonic acid; TEAA, triethylammonium acetate; TOCSY, total correlation spectroscopy; UDP-4-keto-4,6-dideoxy-α-d-GlcNAc, UDP-2-acetamido-2,4,6-trideoxy-α-d-glucose-hexos-4-ulose; UDP-6-deoxy-α-d-GlcNAc4NAc, UDP-2,4-diacetamido-2,4,6-trideoxy-α-d-glucose; UDP-4-keto-4,6-dideoxy-β-l-AltNAc, UDP-2-acetamido-2,4,6-trideoxy-β-l-altrose-hexos-4-ulose; UDP-6-deoxy-β-l-AltNAc4N, UDP-2-acetamido-4-amino-2,4,6-trideoxy-β-l-altrose; UDP-6-deoxy-β-l-AltNAc4NAc, UDP-2,4-diacetamido-2,4,6-trideoxy-β-l-altrose; J, coupling constant; δ, chemical shift; SPE, solid-phase extraction; LC, liquid chromatography; Am, acetamidino group. such as the ultimate resolving capability of the Fourier-transform ion cyclotron resonance MS, and also in nuclear magnetic resonance spectroscopy (NMR) technologies, such as the development of higher magnetic field spectrometers and cryogenically cooled probes (cold probes), has greatly facilitated metabolomic analysis (2Weckwerth W. Annu. Rev. Plant Biol. 2003; 54: 669-689Crossref PubMed Scopus (537) Google Scholar), such an undertaking still presents numerous technological challenges due to the complexity of the metabolome. Therefore, to facilitate the elucidation of unknown intermediates involved in novel biosynthetic pathways, we felt it might be more useful to employ a focused approach to simplify the metabolite pool and target relevant compounds. Glycosylation of prokaryotic proteins in both N- and O-linkage with novel glycan components is now well established (3Benz I. Schmidt M.A. Mol. Microbiol. 2002; 45: 267-276Crossref PubMed Scopus (175) Google Scholar, 4Upreti R.K. Kumar M. Shankar V. Proteomics. 2003; 3: 363-379Crossref PubMed Scopus (137) Google Scholar, 5Szymanski C.M. Wren B.W. Nat. Rev. Microbiol. 2005; 3: 225-237Crossref PubMed Scopus (338) Google Scholar). Campylobacter jejuni is unique in having both a general protein glycosylation pathway that is responsible for the N-linked addition of a heptasaccharide containing N-acetylgalactosamine, glucose, and 2,4-diacetamido-2,4,6-trideoxy-α-d-glucopyranose (2,4-diacetamido-Bac) to at least 30 different proteins as well as an O-linked system, which is responsible for glycosylation of the flagellin structural protein at up to 19 sites per monomer with the novel 9 carbon sialic acid-like sugar 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-α-l-manno-nonulosonic acid (Pse5NAc7NAc) and related derivatives including an acetamidino derivative, PseAm (6Young N.M. Brisson J.R. Kelly J. Watson D.C. Tessier L. Lanthier P.H. Jarrell H.C. Cadotte N. St. Michael F. Aberg E. Szymanski C.M. J. Biol. Chem. 2002; 277: 42530-42539Abstract Full Text Full Text PDF PubMed Scopus (360) Google Scholar, 7Thibault P. Logan S.M. Kelly J.F. Brisson J.R. Ewing C.P. Trust T.J. Guerry P. J. Biol. Chem. 2001; 276: 34862-34870Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar). Each of these systems is represented by a distinct and dedicated genetic locus that contains a number of biosynthetic genes. The functional characterization of the protein glycosylation N-linked locus has revealed that the process shows considerable homology to the eukaryotic N-linked system (8Glover K.J. Weerapana E. Numao S. Imperiali B. Chem. Biol. (Lond.). 2005; 12: 1311-1315Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 9Linton D. Dorrell N. Hitchen P.G. Amber S. Karlyshev A.V. Morris H.R. Dell A. Valvano M.A. Aebi M. Wren B.W. Mol. Microbiol. 2005; 55: 1695-1703Crossref PubMed Scopus (176) Google Scholar). In contrast, the O-linked flagellar glycosylation locus is heterogeneous (involving between 25–50 genes depending on the strain) and contains a large number of hypothetical genes as well as genes implicated in glycan biosynthesis (10Guerry P. Ewing C.P. Schirm M. Lorenzo M. Kelly J. Pattarini D. Majam G. Thibault P. Logan S. Mol. Microbiol. 2006; 60: 299-311Crossref PubMed Scopus (201) Google Scholar, 11Parkhill J. Wren B.W. Mungall K. Ketley J.M. Churcher C. Basham D. Chillingworth T. Davies R.M. Feltwell T. Holroyd S. Jagels K. Karlyshev A.V. Moule S. Pallen M.J. Penn C.W. Quail M.A. Rajandream M.A. Rutherford K.M. van Vliet A.H. Whitehead S. Barrell B.G. Nature. 2000; 403: 665-668Crossref PubMed Scopus (1570) Google Scholar, 12Champion O.L. Gaunt M.W. Gundogdu O. Elmi A. Witney A.A. Hinds J. Dorrell N. Wren B.W. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 16043-16048Crossref PubMed Scopus (138) Google Scholar). Although the specific mechanism of the flagellar O-linked process is currently poorly described, the functional characterization of the biosynthetic pathway enzymes responsible for the production of Pse5NAc7NAc and related derivatives has recently received attention (13Schoenhofen I.C. McNally D.J. Vinogradov E. Whitfield D. Young N.M. Dick S. Wakarchuk W.W. Brisson J.R. Logan S.M. J. Biol. Chem. 2006; 281: 723-732Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 14Chou W.K. Dick S. Wakarchuk W.W. Tanner M.E. J. Biol. Chem. 2005; 280: 35922-35928Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Importantly, Pse5NAc7NAc and PseAm were shown to be essential for flagellar assembly and consequent motility (7Thibault P. Logan S.M. Kelly J.F. Brisson J.R. Ewing C.P. Trust T.J. Guerry P. J. Biol. Chem. 2001; 276: 34862-34870Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar, 15Schirm M. Soo E.C. Aubry A.J. Austin J. Thibault P. Logan S.M. Mol. Microbiol. 2003; 48: 1579-1592Crossref PubMed Scopus (225) Google Scholar, 16Logan S.M. Kelly J.F. Thibault P. Ewing C.P. Guerry P. Mol. Microbiol. 2002; 46: 587-597Crossref PubMed Scopus (121) Google Scholar, 17Goon S. Kelly J.F. Logan S.M. Ewing C.P. Guerry P. Mol. Microbiol. 2003; 50: 659-671Crossref PubMed Scopus (152) Google Scholar) and more recently the presence of these novel carbohydrate moieties on the flagellin was shown to play a role in pathogenesis (10Guerry P. Ewing C.P. Schirm M. Lorenzo M. Kelly J. Pattarini D. Majam G. Thibault P. Logan S. Mol. Microbiol. 2006; 60: 299-311Crossref PubMed Scopus (201) Google Scholar, 15Schirm M. Soo E.C. Aubry A.J. Austin J. Thibault P. Logan S.M. Mol. Microbiol. 2003; 48: 1579-1592Crossref PubMed Scopus (225) Google Scholar, 18Arora S.K. Neely A.N. Blair B. Lory S. Ramphal R. Infect. Immun. 2005; 73: 4395-4398Crossref PubMed Scopus (140) Google Scholar). It is believed that agents which interfere with the flagellin glycosylation process and inhibit infection would offer tremendous therapeutic potential even if these agents were not detrimental to the bacteria (19Alksne L.E. Expert Opin. Investig. Drugs. 2002; 11: 1149-1159Crossref PubMed Scopus (30) Google Scholar, 20Kauppi A.M. Nordfelth R. Uvell H. Wolf-Watz H. Elofsson M. Chem. Biol. (Lond.). 2003; 10: 241-249Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). Thus, the detailed characterization of the genetic locus as well as the elucidation of flagellar glycan biosynthetic pathways is of great significance. Recently, the entire flagellin glycosylation locus of 26 genes from C. jejuni 81–176 was characterized (10Guerry P. Ewing C.P. Schirm M. Lorenzo M. Kelly J. Pattarini D. Majam G. Thibault P. Logan S. Mol. Microbiol. 2006; 60: 299-311Crossref PubMed Scopus (201) Google Scholar). Mutational analysis revealed that a total of nine genes from the locus were involved in flagellin glycosylation, seven of which resulted in a non motile phenotype (pseB, pseC, pseE, pseF, pseG, pseH, and pseI). In addition, mutation of two other genes (pseA and pseD) resulted in flagellin glycosylated with Pse5NAc7NAc but lacking PseAm. Although this study demonstrated a role for several genes in the biosynthesis or transfer of the Pse5NAc7NAc/PseAm moiety, the precise function of these genes remains to be elucidated. In earlier work we had developed a focused metabolomics approach to examine sugar-nucleotide metabolites related to pseudaminic acid biosynthesis within the metabolome of Helicobacter pylori and C. jejuni (15Schirm M. Soo E.C. Aubry A.J. Austin J. Thibault P. Logan S.M. Mol. Microbiol. 2003; 48: 1579-1592Crossref PubMed Scopus (225) Google Scholar, 21Soo E.C. Aubry A.J. Logan S.M. Guerry P. Kelly J.F. Young N.M. Thibault P. Anal. Chem. 2004; 76: 619-626Crossref PubMed Scopus (58) Google Scholar). Using CE-ESMS and precursor ion scanning for fragment ions characteristic of CMP, we were able to identify CMP-Pse5NAc7NAc and CMP-PseAm as nucleotide-activated precursors related to the biosynthesis of Pse5NAc7NAc and PseAm in C. jejuni 81–176 (21Soo E.C. Aubry A.J. Logan S.M. Guerry P. Kelly J.F. Young N.M. Thibault P. Anal. Chem. 2004; 76: 619-626Crossref PubMed Scopus (58) Google Scholar). In addition, during precursor ion scanning for fragment ions characteristic of the UDP carrier, the accumulation of UDP-linked mono- and diacetamido trideoxyhexose intermediates was observed in two defined mutants, pseF (Cj1311) and pseI (Cj1317), which clearly implicated these two proteins in the Pse5NAc7NAc biosynthetic pathway. In this study we expand this work by including all the mutants described by Guerry et al. (10Guerry P. Ewing C.P. Schirm M. Lorenzo M. Kelly J. Pattarini D. Majam G. Thibault P. Logan S. Mol. Microbiol. 2006; 60: 299-311Crossref PubMed Scopus (201) Google Scholar) to provide new data on the role of these genes in the Pse5NAc7NAc and PseAm pathways. The metabolomes of the C. jejuni 81–176 wild-type strain and isogenic mutants were probed for nucleotide-activated monosaccharides relevant to Pse5NAc7NAc/PseAm biosynthesis using the CE-ESMS and precursor ion scanning approach. Next, nucleotide-activated metabolites of interest were purified from cell lysates using a novel hydrophilic interaction liquid chromatography (HILIC)-MS method. The identities of HILIC-MS-purified metabolites were then determined with NMR at 600 MHz (1H) equipped with a cryogenically cooled probe (cold probe) for maximal sensitivity. Bacterial Strains and Growth Conditions—Parent strain C. jejuni 81–176 and isogenic mutants described by Guerry et al. (10Guerry P. Ewing C.P. Schirm M. Lorenzo M. Kelly J. Pattarini D. Majam G. Thibault P. Logan S. Mol. Microbiol. 2006; 60: 299-311Crossref PubMed Scopus (201) Google Scholar) (see Table 1) were grown in Mueller-Hinton broth (500 ml) under microaerophilic conditions for 24 h at 37 °C. For purification of intracellular metabolites from C. jejuni 81–176 or pseC, cells from 10 liters of overnight culture were utilized.TABLE 1Metabolomic analysis of flagellin glycosylation genes from C. jejuni 81–176Strain/Cj numberaCj number refers to the gene showing highest homology in C. jejuni 1168 and in which the mutation was made in C. jejuni 81-176 (10).Gene annotationbGene annotations are from Guerry et al. (10) or Karlyshev et al. (37).Phenotype of mutantcThe phenotype of the mutant was determined on motility agar/isoelectric focusing (IEF) gels/MS structural characterization of the flagellin protein (10). A change in IEF indicates a change in glycosylation pattern. wt, wild type.Intracellular sugar-nucleotide81-176CMP-Pse5NAc7NAc, CMP-PseAmCj1293pseBmot-Nothing detectedCj1294pseCmot-UDP-229noneunknown/CjB1301mot+/wt IEFCMP-Pse5NAc7NAc, CMP-PseAmCj1311pseFmot-UDP-229, UDP-187Cj1312pseGmot-UDP-229, UDP-187Cj1313pseHmot-UDP-229, UDP-187Cj1314cprobable cyclasemot+; wt IEFCMP-Pse5NAc7NAc, CMP-PseAm, UDP-229, UDP-187Cj1315cAmidotransferasemot+; wt IEFCMP-Pse5NAc7NAc, CMP-PseAm, UDP-229, UDP-187Cj1316cpseAmot+/IEF change/PseAm-, 486 Da-CMP-Pse5NAc7NAcCj1317pseImot-UDP-229, UDP-187Cj1334maf3mot+; wt IEFCMP-Pse5NAc7NAc, CMP-PseAmCj1333pseDmot+/IEF change/PseAm-; 486 Da-; 487 Da+CMP-Pse5NAc7NAc, CMP-PseAmCj1337pseEmot-CMP-Pse5NAc7NAc, CMP-PseAmCj1341cmaf6mot+; wt IEFCMP-Pse5NAc7NAc, CMP-PseAmCj1342cmaf7 (37Karlyshev A.V. Linton D. Gregson N.A. Wren B.W. Microbiology. 2002; 148: 473-480Crossref PubMed Scopus (136) Google Scholar)mot+; wt IEFCMP-Pse5NAc7NAc, CMP-PseAma Cj number refers to the gene showing highest homology in C. jejuni 1168 and in which the mutation was made in C. jejuni 81-176 (10Guerry P. Ewing C.P. Schirm M. Lorenzo M. Kelly J. Pattarini D. Majam G. Thibault P. Logan S. Mol. Microbiol. 2006; 60: 299-311Crossref PubMed Scopus (201) Google Scholar).b Gene annotations are from Guerry et al. (10Guerry P. Ewing C.P. Schirm M. Lorenzo M. Kelly J. Pattarini D. Majam G. Thibault P. Logan S. Mol. Microbiol. 2006; 60: 299-311Crossref PubMed Scopus (201) Google Scholar) or Karlyshev et al. (37Karlyshev A.V. Linton D. Gregson N.A. Wren B.W. Microbiology. 2002; 148: 473-480Crossref PubMed Scopus (136) Google Scholar).c The phenotype of the mutant was determined on motility agar/isoelectric focusing (IEF) gels/MS structural characterization of the flagellin protein (10Guerry P. Ewing C.P. Schirm M. Lorenzo M. Kelly J. Pattarini D. Majam G. Thibault P. Logan S. Mol. Microbiol. 2006; 60: 299-311Crossref PubMed Scopus (201) Google Scholar). A change in IEF indicates a change in glycosylation pattern. wt, wild type. Open table in a new tab Materials and Reagents—The sugar-nucleotide standards used for HILIC-MS method development consisted of UDP-α-d-Glc and UDP-α-d-GalNAc (both obtained from Sigma-Aldrich) and CMP-β-d-Neu5Ac, GDP-α-d-Man, and ADP-Glc (purchased from Calbiochem). Intracellular sugar-nucleotides were extracted from the cell lysates using ENVI-Carb (12 ml, 1 g) solid phase extraction cartridges from Supelco (Bellefonte, PA). HILIC separations were performed on a TSK-gel Amide-80 column (250 × 4.6 mm inner diameter) obtained from Tosoh Bioscience (Montgomeryville, PA). High performance liquid chromatography grade acetonitrile and methanol were from Fisher, whereas 1 m triethylammonium acetate (TEAA), trifluoroacetic acid, ammonium acetate, morpholine, ammonium hydroxide, and acetic acid were from Sigma-Aldrich. De-ionized water was obtained from a MilliQ system (Millipore, Bedford, MA). For CE-ESMS experiments bare fused-silica capillaries (100 cm × 50 μm inner diameter) were purchased from Polymicro Technologies (Tucson, AZ). Preparation of Cell Lysates—Cells were harvested and washed twice with ice-cold phosphate-buffered saline. Upon re-suspension in a minimum amount of ammonium bicarbonate (50 mm; pH 8.0), cells were lysed using BugBuster (Novagen, Madison, WI), and cellular debris was removed by centrifugation (15,000 × g for 45 min). Ice-cold ethanol was added to the lysates to a final concentration of 60%, and the insoluble material was removed by a second centrifugation. The cell lysates were then evaporated to dryness on a SpeedVac SC110A concentrator (ThermoSavant, Holbrook, NY), reconstituted in de-ionized water, and filtered through Amicon 10-kDa cut-off cellulose membrane filters (Millipore, Bedford, MA) for further analysis. Extraction of Intracellular Sugar-nucleotides from C. jejuni— Solidphase extraction (SPE) is one of the most effective methods for the cleanup of biological samples and the extraction of trace-level analytes from complex biological matrices. However, nucleotide-activated sugars are highly polar molecules and, therefore, have limited affinity toward reverse-phase sorbents such as C18 and C8, which are typically found in SPE materials. Recently, Räbinä et al. (22Räbinä J. Mäki M. Savilahti E.M. Järvinen N. Penttilä L. Renkonen R. Glycoconj. J. 2001; 18: 799-805Crossref PubMed Scopus (86) Google Scholar) reported impressive recoveries of sugar-nucleotides from yeast and bacterial cells using graphitized non-porous carbon SPE material along with the ion-pairing reagent, triethylammonium acetate, in the elution solvent. Subsequent ion-pairing LC experiments permitted separation of intracellular sugar-nucleotides, and their identification was possible by matrix-assisted laser desorption ionization time-of-flight MS (22Räbinä J. Mäki M. Savilahti E.M. Järvinen N. Penttilä L. Renkonen R. Glycoconj. J. 2001; 18: 799-805Crossref PubMed Scopus (86) Google Scholar). Using commercially available ENVI-Carb SPE cartridges, intracellular sugar-nucleotides were extracted from the cell lysates of C. jejuni 81–176 and the isogenic mutant pseC using an optimized SPE method based on that of Räbinä et al. (22Räbinä J. Mäki M. Savilahti E.M. Järvinen N. Penttilä L. Renkonen R. Glycoconj. J. 2001; 18: 799-805Crossref PubMed Scopus (86) Google Scholar). Briefly, the ENVI-Carb solid-phase extraction cartridges were activated using 6 ml of acetonitrile-trifluoroacetic acid (0.1%) (80–20, v/v) followed by 4 ml of de-ionized water. Cell lysates (2 ml) were loaded on the ENVI-Carb cartridges and washed with 4 ml of water, 4 ml of 5% acetonitrile, and finally with 4 ml of triethylammonium acetate (10 mm; pH 7.0). Elution of intracellular sugar-nucleotides from the ENVI-Carb cartridges was achieved using 2 × 4 ml of acetonitrile-TEAA (25 mm; pH 7.0) (35–65 v/v). The extracts were evaporated to dryness on a SpeedVac concentrator to remove the TEAA and reconstituted in either de-ionized water for CE-ESMS analysis or acetonitrile-ammonium acetate (6.5 mm; pH 5.5) (70–30, v/v) for HILIC-MS. Ten-liter cultures of pseC and C. jejuni 81–176 were necessary to provide sufficient amounts of purified UDP-diacetamido-trideoxyhexose and CMP-PseAm for NMR analyses. CE-ESMS and precursor ion scanning for fragment ions related to UDP (m/z 323, 385, and 403) or CMP (m/z 322) were used as a rapid means to monitor the wash fractions of the SPE procedure for the loss of the metabolites and the presence of the metabolites in the elution steps. An 85% recovery was observed for the extraction procedure. CE-ESMS and HILIC-MS—Cell lysates from wild-type and isogenic mutants of C. jejuni 81–176 were probed for intracellular sugar-nucleotide intermediates using a CE-ESMS and precursor ion scanning method described earlier (21Soo E.C. Aubry A.J. Logan S.M. Guerry P. Kelly J.F. Young N.M. Thibault P. Anal. Chem. 2004; 76: 619-626Crossref PubMed Scopus (58) Google Scholar). The CE-MS instrumentation consisted of an Agilent CE system coupled to a 4000 QTRAP mass spectrometer equipped with a TurboV source via coaxial sheath flow interface (AB/Sciex, Concord, Canada). For the HILIC-MS experiments an Agilent 1100 Series LC system was coupled to the 4000 QTRAP. Mobile phases based on acetonitrile and ammonium acetate were delivered at a flow rate of 1.0 ml·min–1.5-μl injections were used during method development, and this was increased to 10 μl for sample fraction collection. Except for the precursor ion scans, all MS, MS/MS, and MS3 acquisitions were performed using the linear ion trap (LIT)-mode operating at a scan rate of 1000 a.m.u./s in negative mode and using a typical fixed LIT fill time of 20 ms. A collision energy in the range of 35–45 eV (laboratory frame of reference) was employed for MS/MS, whereas a fragmentation time of 100 ms and an excitation frequency of 150 was used for MS3. Analyst 1.4.1 software (AB/MDS Sciex) was used for data acquisition and processing. NMR Spectroscopy of Purified Metabolites—Metabolites were lyophilized, resuspended in 200 μl of 99% D2O (Cambridge Isotopes Laboratories Inc., Andover, MA), and analyzed by NMR spectroscopy. To observe exchangeable N-H protons, CMP-PseAm samples were suspended in 95% H2O (5% D2O), and the pH was lowered through the addition of dilute DCl to reduce the rate of proton exchange, pH 3.5. All samples were analyzed in 3 mm NMR tubes. Standard homo- and heteronuclear correlated two-dimensional 1H NMR, 31P NMR, 13C HSQC, 31P HSQC, HMBC, COSY, TOCSY, and NOESY pulse sequences from Varian (Varian, Palo Alto, CA) were used for general assignments. Selective one-dimensional TOCSY experiments with a Z-filter and one-dimensional NOESY experiments were used for complete residue assignments and measurement of proton coupling constants (JH,H) and nuclear Overhauser effects (23Brisson J.R. Sue S.C. Wu W.G. McManus G. Nghia P.T. Uhrin D. Jimenez-Barbero J. Peters T. NMR Spectroscopy of Glycoconjugates. 2002: 59-93Google Scholar, 24Uhrin D. Brisson J.R. Barbotin J.N. Portais J.C. NMR in Microbiology: Theory and Applications. 2000: 165-190Google Scholar). NMR experiments were performed with a Varian 600 MHz (1H) spectrometer equipped with a Varian 5-mm Z-gradient triple resonance (1H, 13C, 15N) cryogenically cooled probe (cold probe) and with a Varian Inova 500 MHz (1H) spectrometer with a Varian Z-gradient 3-mm triple resonance (1H, 13C, 31P) probe. NMR experiments were typically performed at 25 °C with suppression of the H2O or HOD resonance at 4.78 ppm. For proton and carbon experiments, the methyl resonance of acetone was used as an internal reference (δH 2.225 ppm and δC 31.07 ppm). Lysates from 15 mutants in the flagellin glycosylation locus of strain 81–176 were compared with the wild-type by CE-ESMS for sugar nucleotide intermediates of the Pse5NAc7NAc/PseAm biosynthetic pathway. This included seven non-motile mutants, three of which encode enzymes in the Pse biosynthetic pathway (pseB, pseC, and pseI) (13Schoenhofen I.C. McNally D.J. Vinogradov E. Whitfield D. Young N.M. Dick S. Wakarchuk W.W. Brisson J.R. Logan S.M. J. Biol. Chem. 2006; 281: 723-732Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 14Chou W.K. Dick S. Wakarchuk W.W. Tanner M.E. J. Biol. Chem. 2005; 280: 35922-35928Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar), one that has been tentatively assigned as CMP-Pse5NAc7NAc synthase based on significant homology to neuA, the CMP-sialic acid synthase (pseF), and two of unknown function (pseG and pseH) (15Schirm M. Soo E.C. Aubry A.J. Austin J. Thibault P. Logan S.M. Mol. Microbiol. 2003; 48: 1579-1592Crossref PubMed Scopus (225) Google Scholar). Two mutants were included that were motile but produced flagellin modified by Pse5NAc7NAc but lacking PseAm (pseA and pseD), and the remaining six mutants were fully motile with no changes in flagellin glycosylation as detected by isoelectric focusing gels (10Guerry P. Ewing C.P. Schirm M. Lorenzo M. Kelly J. Pattarini D. Majam G. Thibault P. Logan S. Mol. Microbiol. 2006; 60: 299-311Crossref PubMed Scopus (201) Google Scholar). CE-ESMS results indicated that CMP-Pse5NAc7NAc and CMP-PseAm intermediates were detected in wild-type 81–176, as previously reported (21Soo E.C. Aubry A.J. Logan S.M. Guerry P. Kelly J.F. Young N.M. Thibault P. Anal. Chem. 2004; 76: 619-626Crossref PubMed Scopus (58) Google Scholar) (Fig. 1a; Table 1). The first two enzymatic steps of Pse5NAc7NAc synthesis are encoded by pseB and pseC (13Schoenhofen I.C. McNally D.J. Vinogradov E. Whitfield D. Young N.M. Dick S. Wakarchuk W.W. Brisson J.R. Logan S.M. J. Biol. Chem. 2006; 281: 723-732Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar), as summarized in Fig. 2. A novel UDP-sugar intermediate was observed in the pseC mutant (m/z 631) (Fig. 1b), and tandem mass spectrometry experiments performed as in previous studies (21Soo E.C. Aubry A.J. Logan S.M. Guerry P. Kelly J.F. Young N.M. Thibault P. Anal. Chem. 2004; 76: 619-626Crossref PubMed Scopus (58) Google Scholar) revealed an oxonium ion corresponding to the sugar moiety at m/z 229 (data not shown), but no intermediates were observed in the pseB mutant. Mutants in the genes encoding the two remaining known enzymes in the Pse5NAc7NAc pathway (PseI and PseF) accumulated UDP-229 and UDP-187 intermediates, as did mutants in pseG and pseH, suggesting that they may encode the two missing enzymatic steps (Fig. 2). The final non-motile mutant (pseE) did not accumulate any detectable novel intermediates, and in comparable fashion to the parent strain both CMP-Pse5NAc7NAc and CMP-PseAm were present. This suggests that the pseE gene product is not involved in biosynthesis of either of the CMP-activated sugars but may be involved in either transfer of the glycan moieties to the flagellin protein or at a later stage of the assembly process.FIGURE 2Hypothetical biosynthetic pathways for CMP-pseudaminic acid (I) and UDP-6-deoxy-α-d-GlcNAc4NAc (II, UDP-2,4-diacetamido-bac). The enzymatic activities of PseB, PseC, PglF and PglE have been described by Schoenhofen et al. (13Schoenhofen I.C. McNally D.J. Vinogradov E. Whitfield D. Young N.M. Dick S. Wakarchuk W.W. Brisson J.R. Logan S.M. J. Biol. Chem. 2006; 281: 723-732Abstract Full Text Full Text
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