分离与分析细胞壁组成重要

Isolation and analysis of cell wall components from Streptococcus pneumoniaeNhat Khai Bui a ,1,Alice Eberhardt a ,1,2,Daniela Vollmer a ,Thomas Kern b ,3,Catherine Bougault b ,Alexander Tomasz c ,Jean-Pierre Simorre b ,Waldemar Vollmer a ,⇑aCentre for Bacterial Cell Biology,Institute for Cell and Molecular Biosciences,Newcastle University,Newcastle upon Tyne NE24AX,UK bInstitut de Biologie Structurale,UMR5075(CEA/CNRS/UJF),38027Grenoble,France cLaboratory of Microbiology,Rockefeller University,New York,NY 10021,USAa r t i c l e i n f o Article history:Received 23September 2011Received in revised form 14November 2011Accepted 22November 2011Available online 1December 2011Keywords:Streptococcus pneumoniae Peptidoglycan Teichoic acid Muropeptides Solid-state NMRa b s t r a c tThe complex and heterogeneous cell wall of the pathogenic bacterium Streptococcus pneumoniae is com-posed of peptidoglycan and a covalently attached wall teichoic acid.The net-like peptidoglycan is formed by glycan chains that are crosslinked by short peptides.We have developed a method to purify the glycan chains,and we show that they are longer than approximately 25disaccharide units.From purified pep-tidoglycan,we released 50muropeptides that differ in the length of their peptides (tri-,tetra-,or penta-peptides with or without mono-or dipeptide branch),the degree of peptide crosslinking (monomer,dimer,or trimer),and the presence of modifications in the glycan chains (N-deacetylation,O-acetylation,or lack of GlcNAc or GlcNAc-MurNAc)or peptides (glutamic acid instead of glutamine).We also estab-lished a method to isolate wall teichoic acid chains and show that the most abundant chains have 6or 7repeating units.Finally,we obtained solid-state nuclear magnetic resonance spectra of whole insoluble cell walls.These novel tools will help to characterize mutant strains,cell wall-modifying enzymes,and protein–cell wall interactions.Ó2011Elsevier Inc.All rights reserved.The gram-positive bacterium Streptococcus pneumoniae is a ma-jor pathogen capable of causing otitis media,pneumonia,meningi-tis,and bloodstream infections [1].The cell wall protects the bacterial cell from bursting by the internal turgor and is required to maintain cell shape.The main constituents of pneumococcal cell wall are peptidoglycan and the covalently attached wall teichoic acid (WTA),4capsular polysaccharides,and cell surface proteins.More than 90serotypes with different structures in their polysac-charide capsule [2]and a strain-dependent suite of more than 30cell surface proteins [3]allow the versatile S.pneumoniae to colonize the human nasopharynx and evade the immune system.The focus of this work was on the peptidoglycan–teichoic acid cell wall complex (Fig.1A).Peptidoglycan forms a network ofglycan chains of alternating N -acetylmuramic acid (MurNAc)and N -acetylglucosamine (GlcNAc)residues that are connected by short peptides containing L -and D -amino acids and unusual amide bonds [4].In S.pneumoniae ,the newly made peptides with the se-quence L -Ala-D (c )Gln-L -Lys-D -Ala-D -Ala are efficiently used either to form crosslinks with neighboring peptides by DD -transpeptida-tion,forming dimeric,trimeric,or tetrameric structures,and/or to be trimmed to tetra-or tripeptides by carboxypeptidases [5,6].Some peptides contain L –Ser-L -Ala or L -Ala-L -Ala dipeptides linked to the e -amino group of L -Lys,and this modification is abundant in certain b -lactam-resistant strains [7,8].The glycan chains in pneu-mococcal peptidoglycan are subject to secondary modification by various degrees of deacetylation of the GlcNAc [9]residues and O-acetylation of MurNAc residues [10];both modifications con-tribute to the resistance of the cell to lysozyme,an important bac-teriolytic enzyme of the innate immune system.Although previous work has analyzed the peptides and lactoyl peptides from pneu-mococcal peptidoglycan [5,8,11],the composition of the disaccha-ride peptide subunits,the muropeptides,is not yet known.WTA is a major constituent of the cell wall in gram-positive bacteria,accounting for approximately 40to 50%of its dry weight [12].The WTA of S.pneumoniae is unusual for several reasons [13].First,it has the same structure in its repeating unit as the membrane-anchored lipoteichoic acid (LTA);in most bacteria,the WTA and LTA repeats have different structures.Second,pneumo-coccal WTA has an unusually complex primary structure,0003-2697/$-see front matter Ó2011Elsevier Inc.All rights reserved.doi:10.1016/j.ab.2011.11.026⇑Corresponding author.Fax:+441912083205.E-mail address:w.vollmer@ (W.Vollmer).1These authors contributed equally to this work.2Current address:Department of Microbiology,Tumor,and Cell Biology,Karo-linska Institutet,17177Stockholm,Sweden.3Current address:Institute of Structural Biology,Helmholtz Zentrum München,85764,Neuherberg,Germany.4Abbreviations used:WTA,wall teichoic acid;MurNAc,N -acetylmuramic acid;GlcNAc,N -acetylglucosamine;LTA,lipoteichoic acid;MS,mass spectrometry;NMR,nuclear magnetic resonance;SDS,sodium dodecyl sulfate;dH 2O,deionized water;HF,hydrofluoric acid;HPLC,high-performance liquid chromatography;ESI–MS/MS,electrospray ionization–tandem mass spectrometry;MALDI–TOF,matrix-assisted laser desorption/ionization–time-of-flight.containing the rare amino sugar 2-acetamido-4-amino-2,4,6-tride-oxy-galactose,glucose,ribitol-phosphate,N -acetylgalactosamine,and phosphocholine.Third,the presence of phosphocholine is also remarkable because it is rarely found in bacteria and pneumococci are the only bacteria known to require choline for growth [14,15],presumably due to the specificity of a teichoic acid flippase [16].Pneumococci use their WTA phosphocholine residues to bind a set of cell surface proteins,the choline-binding proteins,some of which have peptidoglycan hydrolase activity such as LytA and LytB [17].Complete LTA chains have been previously isolated and ana-lyzed by mass spectrometry (MS)[18],but WTA chains have not yet been isolated.The aim of this work was to establish novel protocols for the isolation and analysis of various pneumococcal cell wall components.We found that pneumococcal peptidoglycan glycan chains cannot be isolated by the standard cation exchange chroma-tography method due to their partial N-deacetylation.We isolated the glycan chains via size exclusion chromatography and show that they are significantly longer than those from Staphylococcus aureus or Escherichia coli .We also developed a method to determine the muropeptide composition and detected 50muropeptide structures by MS.Muropeptide analysis (but not peptide or lactoylpeptide analysis)allows one to quantify secondary glycan modifications in monomeric and crosslinked muropeptides.We also established a novel purification protocol for WTA chains and show that some chains are not fully loaded with phosphocholine residues.Finally,we established 13C labeling and 13C and 31P solid-state nuclear magnetic resonance (NMR)spectra for pneumococcal cellwall.components in S.pneumoniae and their isolation.(A)Scheme of the cell wall components and the cleavage sites of cellosyl and chains of alternating N -acetylmuramic acid (MurNAc,M)and N -acetylglucosamine (GlcNAc,G)residues.Some of the GlcNAc residues of the MurNAc residues become O-acetylated (OAc).The peptides are designated in single-letter code,with an asterisk (Ã)indicating chains are linked to the peptidoglycan via an unknown linkage unit (X),and their repeating units contain 2-acetamido-4-amino-2,4,6-trideoxy-galactose (Glc),ribitol-phosphate (ribitol-P ),N -acetylgalactosamine (GalNAc),and phosphocholine (Cho-P ).(B)Scheme for the purification indicates N-deacetylation)and wall teichoic acid (WTA)chains from pneumococcal cell wall.These novel tools will help to characterize mutant strains and the enzymology of cell wall modifications and will aid in investigating the biophysical properties and interactions of the pneumococcal cell wall.Materials and methodsMaterials,bacterial strains,and growth conditionsAll chemicals were purchased from Sigma(Dorset,UK,and Mu-nich,Germany)unless mentioned otherwise.The muramidase cel-losyl was kindly provided by Hoechst(Frankfurt,Germany).A recombinant His-tagged version of the cell wall amidase LytA was expressed in E.coli BL21(DE3)pHis-LytA and affinity purified first over Ni2+-nitrilotriacetic acid sepharose using a standard pro-tocol(Qiagen,Hilden,Germany)and then in a second purification step over DEAE sepharose as described previously[19].S.pneumo-niae laboratory strain R6[20],the penicillin-resistant strain Pen6 [21],and the mutant strains CS1[6]and R36A::pgdA[9]were grown at37°C in complex C+Y medium containing1mg/ml yeast extract[22].Strain R6was grown at37°C in C+Y medium supple-mented with2mg/L[13C]glucose for NMR analysis of the cell wall. Isolation of pneumococcal cell wallCell wall from S.pneumoniae was purified as described previ-ously[5,23,24]with modifications.Cells of a2-L culture were har-vested at an OD620of0.5by centrifugation for30min at4°C and 7500g.The cell pellet was resuspended in40ml of ice-cold 50mM Tris–HCl(pH7.0).The cell suspension was introduced dropwise into aflask with150ml of boiling5%sodium dodecyl sul-fate(SDS)solution.The solution was boiled for another30min.The cell suspension was pelleted by ultracentrifugation at25°C for 45min at130,000g.The pellet was washed twice with30ml of 1M NaCl and repeatedly with deionized water(dH2O)until it was free of SDS,confirmed by a previously published assay[25]. The pellet was resuspended in2to4ml of dH2O,1/3volume of acid-washed glass beads(diameter of0.17–0.18mm,Sigma,Mün-chen,Germany)was added,and cells were disrupted in a FastPrep machine(FP120,Thermo Scientific,Hemel Hempstead,UK).For cell disruption,2Â6pulses at maximal speed were applied for 20s,with5-min cooling periods on ice between the pulses.The glass beads were then separated from the cell lysate with a grade 4glassfilter(Winzer,Wertheim,Germany).Thefiltrate was centri-fuged at1000g for5min,and the supernatant,which contains the disrupted cell wall,was centrifuged at130,000g for45min at 25°C.The pellet was resuspended in20ml of100mM Tris–HCl (pH7.5)containing20mM MgSO4.DNase A and RNase I were added tofinal concentrations of10and50l g/ml,respectively, and the sample was stirred for2h at37°C.CaCl2(10mM)and trypsin(100l g/ml)were added,and the sample was stirred for 18h at37°C.Then,1%SDS(final concentration)was added,and the sample was incubated for15min at80°C to inactivate the en-zymes.The cell wall was recovered by centrifugation for45min at 130,000g at25°C,resuspended with20ml of8M LiCl,and incu-bated for15min at37°C.After another centrifugation(see above), the pellet was resuspended in10mM ethylenediaminetetraacetic acid(EDTA,pH7.0)and incubated at37°C for15min.The cell wall was washed with dH2O,acetone,and again with water beforefinal-ly being resuspended in2to4ml of dH2O prior to lyophilization. Cell wall samples(typically$40–60mg from1-L culture)were stored atÀ20°C.Cell walls were labeled by the addition of5l g/ml and1l Ci/ml [methyl-3H]choline to the growth medium of exponentially grow-ing cells as described previously[23].For labeling of glycan strands with[3H]GlcNAc(Amersham Pharmacia Biotech),we followed a published procedure[26].Cell walls were isolated as described above.The specific activities were 6.82Â106cpm/mg for [methyl-3H]choline-labeled cell walls and3.38Â106cpm/mg for [3H]GlcNAc-labeled cell walls(counting efficiency>90%).Preparation of peptidoglycanNext,5mg of cell wall was stirred with48%hydrofluoric acid (HF)for48h at4°C in an ultracentrifuge tube made of polyallomer tightly closed by Parafilm.The volatile,aggressive,and toxic HF was handled with great caution to avoid any contact with the skin and spillage.The peptidoglycan was recovered by centrifugation (45min,130,000g,4°C)and washed with ice-cold dH2O,100mM Tris–HCl(pH7.0),and then twice with dH2O.The pellet containing 2to2.5mg of peptidoglycan was resuspended in750l l of0.02% NaN3and stored at4°C.Preparation of peptidoglycan glycan strands[3H]GlcNAc-labeled or unlabeled peptidoglycan($0.5mg)was digested in a total volume of600l l at37°C in50mM Tris–HCl (pH7.0)with a total of90l g of recombinant and His-tagged LytA amidase for46h,whereby30-l g aliquots of the enzyme were added at time points of0,18,and28h.The sample was boiled for5min,and the clear supernatant was taken.A150-l l aliquot of LytA-digested[3H]GlcNAc-labeled peptido-glycan was N-acetylated at a pH of8.0by the addition of55l l of 100mM freshly prepared N-hydroxysuccinimido acetate(NHS ace-tate)and incubation for1h at ambient temperature.Then,Tris–HCl(pH7.5)was added to afinal concentration of100mM,and the sample was further incubated for1h.Aliquots of the N-acety-lated glycan chains were shortened by partial digestion with lyso-zyme(0.4or4l g/ml)for20min at37°C.The samples were boiled for5min and cleared by brief centrifugation.Purification of peptidoglycan glycan strandsN-acetylated glycan strands were purified on a Keystone GFS-150size exclusion column connected to a Shimadzu high-perfor-mance liquid chromatography(HPLC)system and operating at 30°C at aflow of1ml/min with dH2O.Detection occurred either with a scintillationflow-through detector for radioactive labeled material or at205nm for unlabeled glycan chains.The glycan strands eluting in the void volume were collected,and aliquots were incompletely digested with lysozyme as described above. HPLC analysis of glycan strandsGlycan strands were reduced with sodium borohydride[27]and separated by HPLC as described previously[28].Radioactive glycan strands were detected with a Canberra Packard scintillationflow-through detector.Unlabeled glycan strands were detected at 205nm.Preparation of muropeptidesMuropeptides were released from peptidoglycan with cellosyl, reduced,and analyzed by HPLC similar to the previously published procedure for E.coli muropeptides[27].Peptidoglycan suspension ($0.5mg in100l l)was incubated with10l g of cellosyl in20mM sodium phosphate(pH4.8)in a total volume of200l l for24h at 37°C while stirring.The reaction was stopped by heating the sam-ple for10min at100°C.The sample was cleared by centrifugation for10min at13,000g.For reduction,1sample volume of500mM sodium borate(pH9.0)and a few crystals of NaBH4were added,Cell wall components from S.pneumoniae/N.K.Bui et al./Anal.Biochem.421(2012)657–666659followed by30min of incubation at20°C.The reaction was stopped by adjusting the pH to4.0to5.0with20%H3PO4.For on-line electrospray–MS analysis,peptidoglycan was digested with cellosyl in20mM ammonium acetate buffer(pH 4.8)and the resulting muropeptides were not reduced[29].Separation of muropeptides by HPLCMuropeptides were separated on a reversed-phase column (Prontosil120-3-C18-AQ,250Â4.6mm,3l M,Bischoff,Germany) using an Agilent1100system operating ChemStation software. HPLC was performed with a column temperature of55°C using a linear135-min gradient from100%buffer A(10mM sodium phos-phate[pH6.0]and20l M NaN3)to100%buffer B(10mM sodium phosphate[pH6.0]and30%methanol)(Fisher Scientific,Lough-borough,UK).Muropeptide fractions detected at205nm were col-lected for electrospray ionization–tandem mass spectrometry (ESI–MS/MS)analysis.The muropeptides were quantified by inte-gration of their peak area using Laura4software(LabLogic,Shef-field,UK).The total peak area from all fractions,excluding the salt fractions(eluting before8min),was normalized to100%,and the relative area of each fraction was determined.Online and offline electrospray–MS of muropeptidesMuropeptide fractions were analyzed at the Newcastle Univer-sity Pinnacle facility by offline electrospray–MS on a Finnigan LTQ-FT FT mass spectrometer as described previously[29].Mixtures of nonreduced muropeptides were analyzed with a Waters nanoA-quity HPLC system with a self-packed column(0.5Â150mm)of Reprosil-Pur C18-AQ3l m medium(Maisch,Ammerbuch,Ger-many)with aflow rate of12l l/min connected to the same mass spectrometer.The elution occurred with0.5%acetonitrile and 0.1%formic acid for1min,followed by a60-min gradient from 0.5%acetonitrile to20%acetonitrile in0.1%formic acid.Preparation of WTA chains and their analysis by MALDI–TOF MSCell wall from S.pneumoniae R6(120mg)was stirred at37°C in 12ml of50mM Tris–HCl(pH7.0)with1.2mg of recombinant and His-tagged LytA amidase added in three aliquots(0.4mg each) after0,24,and48h for a total period of incubation of72h.The pH was adjusted with50mM sodium hydroxide to5.0,and1mg of cellosyl was added.The sample was incubated for24h at 37°C,boiled for5min,and cleared by centrifugation at25,000g for15min,and then the supernatant was lyophilized.WTA was separated from the smaller peptidoglycan fragments by size exclusion chromatography.Here,5mg of the lyophilized cell wall digest was dissolved in200l l of dH2O and applied to a BioBasic SEC-120column operated at30°C with aflow of1ml/ min of100%dH2O(300Â7.8mm,pore size of120Å,Thermo Sci-entific)using a Waters HPLC system.WTA eluted at the void vol-ume and was collected for matrix-assisted laser desorption/ ionization–time-of-flight(MALDI–TOF)MS using an ABI Voyager DE STR MALDI–TOF mass spectrometer operated in the positive ion mode.Solid-state NMR analysis of pneumococcal cell wallFor solid-state NMR analysis,a suspension of13C-labeled pneu-mococcal cell wall in50mM Hepes(pH7.5)was packed into the rotor by centrifugation[30,31].NMR experiments were performed at9.4T(1H NMR frequency at400.13MHz)and14.1T(1H NMR frequency at600MHz)using a Varian BioMAS spectrometer equipped with a3.2-mm triple resonance probe.The13C and31P experiments were done with a MAS spinning speed set to 12.5kHz and a sample temperature of285K.31P one-dimensional NMR spectra were obtained by direct31P excitation.Acquisition time was set to50ms,and SPINAL decoupling was used at a proton rffield strength of45kHz.The experimental time was5min,with an interscan delay of5s.The parameters set for recovery time and magneticfield were allowed to correlate the integral of each31P signal peak with the abundance of each phosphate group. ResultsIsolation of pneumococcal cell wall componentsWe purified the cell wall(i.e.,the peptidoglycan–WTA complex) from S.pneumoniae R6and isolated the components according to the scheme shown in Fig.1B.We then established novel methods to isolate and analyze the glycan chains from peptidoglycan and the WTA from cell wall,and we improved the method of muropep-tide analysis.We also present13C and31P solid-state NMR spectra of high-molecular-weight pneumococcal cell wall.To purify peptidoglycan from cell wall,the WTA can be frag-mented by HF,which hydrolyzes phospho mono-and diester bonds.Previous publications have used7or48%HF to remove WTA[5,9,32].To determine the efficiency of WTA removal by HF, we measured the release of radioactivity from cell wall with the la-bel either in choline,present only in WTA,or in amino sugars,pres-ent in both peptidoglycan and WTA.In addition,we measured the release of phosphate,which is present only in WTA(see Supple-mentary Table1in supplementary material).Treatment with48% HF nearly quantitatively removed choline and phosphate,which is present only in WTA,whereas only approximately70%of the radioactivity was solubilized from amino-sugar-labeled cell wall, indicating that the remaining30%of the radioactivity was present in the peptidoglycan.By contrast,treatment with7%HF was not efficient and solubilized only approximately19%of the total cho-line and5%of the total phosphate from the cell wall.Thus,treat-ment with48%HF is required to quantitatively remove WTA from pneumococcal cell walls to obtain pure peptidoglycan.In the following experiments,the peptidoglycan was digested either with cellosyl to obtain the muropeptides or with LytA amidase to isolate the glycan strands(Fig.1).Glycan chains in peptidoglycanPeptidoglycan isolated from cells grown in the presence of radioactive GlcNAc was digested with LytA amidase,and the resulting mixture of radioactive glycan strands and unlabeled pep-tides was analyzed by an HPLC system connected to a radioactivity detector.Unlike the glycan chains from E.coli or S.aureus,which separate according to their degree of oligomerization,giving rise to typical hedgehog-like HPLC elution profiles[28,33],the glycan chains from S.pneumoniae eluted in a hump of unresolved glycan material(Fig.2A,chromatogram I).We reasoned that partial deacetylation of GlcNAc residues by the enzyme PgdA[9],which occurs in S.pneumoniae but not E.coli or S.aureus,could cause het-erogeneity and short retention times of the pneumococcal glycan strands.Indeed,on chemical acetylation,the retention time of all the radioactive material shifted to the end of the chromatogram, where a methanol step elutes the high-molecular-weight glycan fraction(Fig.2A,chromatogram II)[28],indicating that virtually all glycan strand material consists of strands that are longer than approximately25disaccharide units.Consistent with thisfinding, partial or more complete digestions of the high-molecular-weight, N-acetylated glycan material with lysozyme produced the ex-pected regular pattern of oligosaccharides with1to9disaccharide units(Fig.2A,chromatograms III and IV).Thus,the peptidoglycan660Cell wall components from S.pneumoniae/N.K.Bui et al./Anal.Biochem.421(2012)657–666from S.pneumoniae contains significantly longer glycan chains than that from E.coli or S.aureus.The presence of deacetylated amino sugars prevented the puri-fication of pneumococcal glycan strands by the established method of MonoS cation exchange chromatography(data not shown)[28]. Due to their high molecular weight,we could separate the glycan strands from peptides and acetylation reagent by size exclusion chromatography(Fig.2B).The glycan strand fraction was collected and analyzed by reversed-phase chromatography with or without prior digestion with lysozyme(Fig.2C).Detection at205nm con-firmed the presence of pure high-molecular-weight glycan mate-rial,eluting in the methanol step,and the long glycan strands were digested to shorter ones with lysozyme.There were no major peaks of contaminating components such as peptides.Hence,the long pneumococcal glycan chains can be purified by size exclusion chromatography.Separation and analysis of muropeptidesPneumococcal peptidoglycan has previously been analyzed in different ways.LytA amidase or alkaline treatment released pep-tides or lactoylpeptides that were separated by HPLC[5,11].These methods fail to detect known modifications in the glycan strands such as N-deacetylation and O-acetylation.Muropeptides are the disaccharide peptide fragments of peptidoglycan;hence,their analysis provides information on both the glycan strandanalysis of peptidoglycan glycan chains.(A)Radioactively labeled glycan chains were analyzed by reversed-phase HPLC before chromatogram I indicates the partially deacetylated glycan chains that shift to the methanol step at long retention timesindicating that all glycan chains are longer than approximately20disaccharide units.Partial(III)or more complete(IV)digestion chains from1to9disaccharide units that elute between10and50min.(B)Separation of radioactively labeled,chemicallypeptides and acetylation reagent(dashed arrow)by size exclusion chromatography.Upper chromatogram:UV profile(205 Analysis of purified nonlabeled acetylated glycan chains by reversed-phase chromatography.Chromatogram I showsthe methanol step at approximately100min(arrow).Partial(II)or more complete(III)digestion with lysozyme generatesprofile of S.pneumoniae R6obtained by reversed-phase HPLC.The collected fractions(numbers)were analyzed by ESI–MS/MS(Supplementary S1shows the muropeptide profiles of different strains.The proposed muropeptide structures are shown in Supplementary Fig.S2.modifications and peptide composition.Therefore,we optimized the conditions for the analysis of pneumococcal muropeptide com-position by HPLC and LTQ–MS(Fig.3).Cell wall from pneumococ-cal strains was treated with48%HF to quantitatively remove WTA. The resulting peptidoglycan was solubilized with the muramidase cellosyl,and the muropeptides generated were reduced with so-dium borohydride prior to reversed-phase HPLC analysis.Fig.3 shows a representative muropeptide profile of strain R6.ESI–MS/ MS analysis of37fractions collected from HPLC allowed us to pro-pose50different muropeptide structures that account for71.4%of the total peak area in the muropeptide profile(Supplementary Ta-ble2).Some fractions,especially those with long retention times, contained more than one muropeptide.The large structural heter-ogeneity of pneumococcal peptidoglycan is due to combinations of (i)uncrosslinked monomeric and crosslinked dimeric and trimeric muropeptides,(ii)the different lengths of peptides(tri-,tetra-,or pentapeptides with or without an Ser-Ala or Ala-Ala branch),and (iii)the different modifications(deacetylation of GlcNAc,O-acety-lation of MurNAc,the lack of amidation of iGln,and the loss of Glc-NAc or GlcNAc-MurNAc).To aid structure assignment,we analyzed the muropeptide profiles of strains with known alterations in pep-tidoglycan structures,R36A::pgdA lacking deacetylated muropep-tides,Pen6with a high percentage of branched peptides,and CS1 with a high percentage of pentapeptides(see Supplementary Fig.S1in supplementary material).The predominant muropeptides in peptidoglycan from strain R6 were Tri(fraction3,13.0%peak area),Tri(SA)(fraction12,6.2%), TetraTri(fraction19,5.3%),and Tetra(SA)Tri(fraction23,6.6%) (Supplementary Table2);these four muropeptides contribute to 31.1%of the total peptidoglycan.35.9%of all muropeptides were monomers,25.9%were dimers and9.6%were trimers(Table1). Most peptides were efficiently trimmed to tetra-and tripeptides, as indicated by the low fraction of pentapeptides,confirming pre-vious peptide analysis[5].Modifications were not equally distrib-uted among monomers,dimers,and trimers;deacetylation of GlcNAc was most abundant in dimers(45.1%)and least abundant in trimers(14.2%).The presence of unamidated glutamate was most prevalent in monomers(12.6%)and was of low abundance in dimers and trimers.Despite the acidic treatment with HF,the acid-labile O-acetyl modification was detected in8.1%of the tri-mers.Missing GlcNAc or GlcNAc-MurNAc residues indicative of peptidoglycan hydrolase cleavage events were detected mainly in trimers(11.9or8.1%,respectively).Purification and analysis of WTA chainsIn S.pneumoniae,the membrane-anchored LTA and peptidogly-can-attached WTA have the same unusually complex repeating unit structure(Fig.1A).Recent MS analysis has led to a new model of the LTA structure[18],but WTA chains have not yet been iso-lated for analysis.Therefore,we established a method to isolate the WTA chains from cell wall after digestion of the peptidoglycan with cellosyl and LytA.The resulting WTA chains were separated from the smaller peptidoglycan fragments(peptides and disaccha-rides)on a BioBasic120size exclusion column(Fig.4A).The fraction containing the WTA was collected and analyzed by MALDI–TOF MS in the positive mode(Fig.4B).We observed a reg-ular pattern of singly charged molecular ions from WTA chains containing1to8repeating units(Fig.4B,signals I–VIII),whereby the apparently most abundant chains with6and7repeating units were also observed as double charged ions(signals VI⁄and VII⁄). Masses corresponding to peptidoglycan fragments were not de-tected,indicating that the WTA fraction contained little,if any,of these fragments.We also observed masses corresponding to WTA chains with losses of up to four phosphocholine residues per chain, indicating that some of the repeating units contain fewer than two phosphocholine residues(Fig.4B and Table2).Solid-state NMR of cell wall and peptidoglycanRecent work has used solid-state NMR spectroscopy to analyze whole peptidoglycan sacculi from E.coli[31]or the insoluble,high-molecular-weight peptidoglycan–WTA complex from Bacillus sub-tilis and S.aureus[30].Here,we established the method to13C label pneumococcal cell wall and obtained the13C–13C correlation spec-trum that offers detailed qualitative and quantitative chemical information.[13C]glucose added in the C+Y growth medium was integrated into the glycosidic parts of peptidoglycan and WTA as observed by solid-state NMR recorded on the harvested cell wall. Except for intense alanine correlations,the peptide parts of pepti-doglycan were barely observed due to the presence of unlabeled 12C carbons in the growth medium.Fig.5shows the spectrum of a typical13C–13C correlation experiment.We assigned the signals detected in the sugar C1region(90–110ppm)and in the region containing all other sugar-type carbon signals(50–80ppm).We also recorded the31P solid-state NMR spectrum of pneumococcal cell wall and found it to be similar to the published liquid state NMR spectrum of LTA[34],confirming the similarity in the chem-ical structures of the repeating units of WTA and LTA.Spectra re-corded with a long recycle delay could be used to quantify the relative proportion of the different phosphate groups.The intensity of the ribitol phosphate residues within the WTA chain was6.2-fold higher than the intensity of the terminal ribitol phosphate at the linkage unit,and the two phosphocholine signals wereTable1Modifications in monomeric,dimeric,and trimeric muropeptides from strain R6.Monomers/ oligomers (%of total)Muropeptide ormodificationRelativepercentage a(%)Monomers Tri79.9(35.9)Tetra11.8Penta8.4Unmodified43.7Deacetylated18.5Glutamate12.6Deacetylated/glutamate 5.0O-Acetylated n.d.-GlcNAc0.3-GlcNAc-MurNAc n.d.Ala-Ala8.1Ser-Ala29.7Dimers TetraTri100.0(25.9)Unmodified19.0Deacetylated45.1Glutamate 1.8Deacetylated/glutamate 1.8O-Acetylated n.d.Missing GlcNAc n.d.Missing(GlcNAc-MurNAc)n.d.Ala-Ala18.5Ser-Ala74.8Trimers TetraTetraTri97.9(9.6)TetraTetraTetra 2.1Unmodified 2.1Deacetylated14.2Glutamate n.d.Deacetylated/glutamate n.d.O-Acetylated8.1Missing GlcNAc11.9Missing(GlcNAc-MurNAc)8.1Ala-Ala31.7Ser-Ala45.6Note:n.d.,not detected.a Percentage of muropeptides or modifications in monomers,dimers,or trimers.662Cell wall components from S.pneumoniae/N.K.Bui et al./Anal.Biochem.421(2012)657–666。