1-s2.0-S0008621513002139-maingood

NoteChemical structures of the secondary cell wall polymers (SCWPs)isolated from bovine mastitis StreptococcusuberisAnna Czaban´ska,Otto Holst,Katarzyna A.Duda ⇑Division of Structural Biochemistry,Research Center Borstel,Leibniz-Center for Medicine and Biosciences,Airway Research Center North (ARCN),Member of the German Center for Lung Research (DZL),Parkallee 4a/c,D-23845Borstel,Germanya r t i c l e i n f o Article history:Received 23April 2013Received in revised form 22May 2013Accepted 23May 2013Available online 1June 2013Keywords:MastitisStreptococcus uberis SCWPsWTA structure Rhamnan NMR analysisa b s t r a c tThe cell envelope of Gram-positive bacteria is decorated with a variety of polysaccharides.In this study wall teichoic acid (WTA)and neutral polysaccharides were isolated from the cell envelope of bovine mas-titis Streptococcus uberis .The polysaccharides were released by lysozyme treatment,and purified by hydrophobic interaction chromatography.Further separation was achieved utilizing anion-exchange chromatography which yielded two products,that is,a neutral polysaccharide with a high content of Rha and less Glc (rhamnan)and an anionic phosphate-rich one containing glycerol and Glc (WTA).The structures of these molecules were elucidated applying 1D and 2D nuclear magnetic resonance experi-ments as well as chemical analyses.In the rhamnan sample two independent molecules were identified,that is,a glucorhamnan with the structure ?2)-a -L -Rha p -(1?3)-[a -D -Glcp-(1?2)-]a -L -Rha p -(1?,and a homopolymeric rhamnan ?2)-a -L -Rha p -(1?3)-a -L -Rha p -(1?.The WTA comprised a polyphosphoglyc-erol chain substituted nonstoichiometrically with b -Glc p .Ó2013Elsevier Ltd.All rights reserved.1.IntroductionBovine mastitis,namely inflammation of the udder,is a result of intramammary infection by bacteria.1The course of the disease may be either sub-clinical or clinical depending on the factors in the host and the invading pathogen.Sub-clinical mastitis shows no obvious signs of disease,goes often unnoticed and untreated,resulting in long duration of the infection.2The clinical form of the disease is characterized by visible abnormalities in the milk (protein aggregates or clots)accompanied by pain and swelling in the affected gland and sometimes production of a secretion composed solely of aggregated protein in a serous fluid.In severe cases there may be systemic signs such as elevated temperature and loss of appetite which may lead to bacteremia,septicemia,and death of the animal.1Over 135infectious agents were reported to cause mastitis 3but most infections are due to one of the three bacterial species Strep-tococcus uberis ,4Escherichia coli,4and Staphylococcus aureus.5The cell envelope of Gram-positive bacteria is decorated with a variety of polysaccharides.Based on their structural character,they can be classified into the three groups,(i)teichoic acids,(ii)teichu-ronic acids,and (iii)other polysaccharides (neutral or acidic)which do not belong to any of the other groups.6Teichoic acids [TAs,lipoteichoic acid (LTA),and wall teichoic acid (WTA)]are phosphate-rich polyalditols which were shown to contribute to resistance to environmental stresses,7,8antimicro-bial peptides,9cationic antibiotics,and lytic enzymes produced by the host.10Non classical polysaccharides were reported to play an important role in non-covalent attachment of the S-layer to the underlying peptidoglycan (PG).11The structure of the S.uberis LTA has been published recently.12A body of literature reveals a wide structural diversity of Gram-positive bacterial WTAs which are covalently bound to O -6of muramic acid (MurNAc)of PG via a phosphodiester bond.13Their repeating unit often possesses a similar structure as that of LTA,comprising a polyglycerol-or poly-ribitolphosphate nonstoichiometrically substituted with alanine or glycosyl residues,14,15but may also show considerable differences.Structural analyses of non-classical SCWPs revealed that they are either charged 16or neutral 17heteropolysaccharides covalently bound to PG via a phosphodiester 18or pyrophosphate linkage.19The present study deals with the structures of WTA and a neutral polysaccharide isolated from the cell envelope of one of the most important mastitis pathogens,S.uberis.2.Results and discussionThe polysaccharides were released from disrupted bacterial cells of S.uberis 233by lysozyme treatment,followed by incuba-tion with RNase,DNase,and proteinase K.Hydrophobic interaction0008-6215/$-see front matter Ó2013Elsevier Ltd.All rights reserved./10.1016/j.carres.2013.05.015Corresponding author.Tel.:+4945371882490;fax:+4945371887450.E-mail address:kduda@fz-borstel.de (K.A.Duda).chromatography(HIC)was performed in order to remove possible remains of LTA.Separation of polysaccharides could be achieved by anion-exchange chromatography which yielded three fractions.Of these,thefirst and second contained the same material according to the anomeric regions of1H NMR spectra,a rhamnan,due to the high content of Rha residues.Since the quality of the2D spectra re-corded for fraction1was not sufficient for a successful assignment of the signals,further analyses were performed on fraction2.The third fraction,phosphate-rich,was identified as WTA.Compositional analysis of the rhamnan identified Rha and Glc in an approx.molecular ratio of4:1.Their absolute configurations were determined as L and D,respectively.Methylation analysis revealed the presence of2-substituted,3-substituted,and2,3-disubstituted Rha,and terminal Glc by identifying1,2,5-tri-O-acetyl-6-deoxy-3,4-di-O-methyl-[1-2H]mannitol,1,3,5-tri-O-acetyl-6-deoxy-2, 4-di-O-methyl-[1-2H]mannitol,1,2,3,5-tetra-O-acetyl-6-deoxy-4-O-methyl-[1-2H]mannitol,and1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl-[1-2H]glucitol.The WTA consisted of Gro,phosphate,and Glc.The absolute configuration of Glc was determined as D.Both,rhamnan and WTA were investigated by1D and2D NMR experiments.The1D1H NMR spectrum of the rhamnan revealed5 major signals in the anomeric region[4originating from Rha p(A–C,E)and1from Glc p(D)residues],several characteristic signals at d1.28–1.24(methyl groups of6-deoxyhexoses)and2signals at d 2.06and d2.02(methyl group of N-acetyl function)(Fig.1a).Since the latter signals were greatly diminished after further purification of the sample on TSK40(not shown)they were assigned as impu-rities.The anomeric configurations were determined on the basis of1J C-1,H-1values ranging from173to178Hz(derived from a cou-pled heteronuclear single quantum coherence1H,13C-HSQC spec-from PG constituents,namely GlcNAc and MurNAc as the samples were treated with the lysozyme.However,the complete spin sys-tems of GlcNAc and MurNAc could not be identified.Detailed analyses applying1H,1H correlation spectroscopy (COSY),total correlation spectroscopy(TOCSY),rotating frame Overhauser effect spectroscopy(ROESY),and1H,13C heteronuclear single quantum coherence(HSQC),and heteronuclear multiple bond correlation(HMBC)experiments were performed in order to establish structures of both examined molecules.The2D spectra of the rhamnan identified several minor signals in the anomeric region and those originating from short phospho-glycerol chains which were not detected by the1H NMR experi-ment.After further purification of the sample by size exclusion chromatography on TSK-40these signals could be assigned to impurities.Due to better quality of spectra,chemical shift assign-ments of strong signals corresponding to all peaks of1H spectrum were made on the non-purified sample(Table2)and only those cross-peaks are depicted in Figure2.In the HMBC experiment,con-nectivities between A,H-1/C,C-3;B,H-1/E,C-3;C,H-1/A,C-2;E, H-1/B,C-2and C,H-2/D,C-1(for A-E,see Table1)were found which were confirmed by a ROESY experiment(inter-residual NOE contacts between A H-1/C H-3,B H-1/E H-3,C H-1/A H-2,D H-1/C H-2,and E H-1/B H-2).These data suggested the existence of two rhamnan polymers,first possessing the structure?2)-B-(1?3)-E-(1?,and the second?2)-A-(1?3)-[D-(1?2)-]C-(1?] (Fig.3).No indication was obtained that both partial structures were linked(no cross connectivity between E-1and A-2was ob-served,and NOE contacts between A H-3/D H-4and A H-6/D H-2 found in the ROESY spectrum were not observed between B and D).2D NMR experiments of the WTA allowed the assignment of allFigure1.1H NMR spectra(700MHz)of(a)rhamnan and(b)WTA isolated from uberis233.The capital letters refer to the residues defined in Tables1and2.Spectra were recorded in D2O at27°C relative to external acetone(d H2.225;d C31.45).2.Excerpt of1H,13C HSQC-DEPT spectrum(700MHz)of the rhamnan isolated from S.uberis233.The spectrum was recorded in D2O at27°C relative to external acetone(d H2.225;d C31.45).A.Czaban´ska et al./Carbohydrate Research377(2013)58–6259nals originating from unsubstituted phosphoglycerol were ob-served(E0).The presence of Gro signals linked to the phosphate at O-1and at O-3free(G0)indicated that WTA chains were short. The proposed structure of the WTA is shown in Figure5.For isolation of the described polysaccharides lysozyme was used as we aimed at the elucidation of polymer structures together with their linkers to PG.This could not be achieved as we were not able to determine spin systems of PG constituents(MurNAc and GlcNAc)and thus identify the linking ually,WTAs are bound covalently to O-6of MurNAc of PG via a phosphodiester bond.13The non-classical SCWPs were reported to be either cova-lently bound to PG via a phosphodiester18or pyrophosphate linkage.19To our knowledge all published structures of non classical SCWPs indicated that these were heteropolymers.Here,novel het-ero-and homopolymers(glucorhamnan and rhamnan,respec-tively)of S.uberis were isolated and characterized.WTA of S.uberis isolated in this study was shown to be com-posed of polyphosphoglycerol substituted nonstoichiometrically A nonstoichiometrical substitution of Gro with b-Glc has been re-ported for Staphylococcus epidermidis I2,22however here the posi-tion O-2of phosphoglycerol was alternatively substituted by D-Ala.3.Experimental3.1.Bacterial growthS.uberis233was isolated from a mastitis-diseased cow in New Zealand and was cultivated as standing cultures to OD6000.8in Brain Heart Infusion(BHI)medium with and without addition of 1%agar at37°C for12h.3.2.Extraction and purification of polysaccharidesBacterial cells were harvested after12h,washed once with citric buffer(0.1M,pH4.7)and disrupted with a cell homogenizer (Vibrogen—Zellmühle Edmunt Bühler GmbH)and glass beads (0.1mM,Roth,Karlsruhe).Supernatant was separated from the glass beads by centrifugation(1000Âg,RT,5min).Then the glass beads were washed3–5Âwith citrate buffer.In order to release polysaccharides bound to the PG,an enzymatic digestion was per-formed.First,10Âbuffer for enzymatic treatment was added to the resuspended cell particles,followed by lysozyme(0.2mg/ml), DNase I and RNase(each0.1mg/ml).The sample was shaken at 37°C for24h.The insoluble cell wall fragments were removed by centrifugation(4°C,30min,10,000Âg),and the supernatant was then treated with proteinase K(0.1mg/ml)at37°C for24h. After digestion,the solution was dialyzed(3.5ku cut off)against water and lyophilized.The sample was purified by hydrophobic interaction chromatography(HIC)as described previously12in or-der to remove the remaining LTA.Fractions4–14were combined and the sample was lyophilized,and then resuspended in water,filtered,(0.45l m,PALL Life Sciences,HPLC certified)and applied on HiTrep Q Sepharose column and purified by anion-exchange chromatography.The bound material was eluted with a gradient from0to1M of NaCl of36columns volume(1column volume was5ml)with aflow rate of3ml/min.After elution the columnwas washed,first with20columns volume of1M NaCl,then with5 columns volume of2M NaCl.Fractions1–80were examined in re-gard to the phosphate content.In order to desalt the obtained sam-ples,separation on a column(2.5Â100cm)of Sephadex G-10was performed.The elution was carried out without a pump and with a buffer containing4ml pyridine and10ml glacial acetic acid in1l of water.The pyridine–acetate buffer was removed by repeated evaporation of the solvent on a rotary evaporator.Further purifica-tion of the rhamnan was performed by size exclusion chromatog-raphy on a column(1Â100cm)of TSK-40utilizing the same conditions as for Sephadex G10.positional analysesThe components of the isolated molecules were determined after methanolysis(0.5M HCl/MeOH,85°C,45min),acetylation (85°C,10min),and detection by GLC–MS[Hewlett Packard HP 5890(series II)gas chromatograph equipped with a fused-silica SPB-5column(Supleco,30mÂ0.25mmÂ0.25l mfilm thick-ness),FID and MS5989A mass spectrometer with vacuum gauge controller59827A].The temperature program was150°C for 3min,then5°C minÀ1to330°C.The hexoses were detected as alditol acetates[2M trifluoroacetic acid,100°C,2h,then reduction (NaBH4,16h in the dark)and acetylation(85°C,10min)]by GLC [HP5890(series II)gas chromatograph with FID and a column (Agilent Technologies,30mÂ0.25mmÂ0.25l mfilm thickness)]. The temperature program was150°C for3min,then3°C minÀ1to 320°C.The absolute configuration of Rha and Glc was determined by GLC,by comparison with authentic standards of the acetylated (S)-2-butanol derivative after methanolysis(2M HCl/MeOH,85°C, 45min),butanolysis(2M HCl/(S)-2-BuOH,65°C,4h),and acetyla-tion(85°C,10min).Rhamnan was methylated with CH3I after dis-solving in dry DMSO with addition of dry NaOH(room temperature,stirring,1h)and purified by chloroform–water extraction.The product was hydrolyzed(4M trifluoroacetic acid, 100°C,4h),reduced(NaBD4in MeOH/H2O1:1),acetylated D2O and27°C.All1D and2D NMR H,H COSY,TOCSY,and ROESY as well as1H,13C HSQC,coupled HSQC,and1H,31P HMQC experi-ments were recorded by a Bruker DRX Avance III700MHz spec-trometer(operating frequencies of700.75MHz for1H NMR, 176.2MHz for13C NMR and283.7MHz for31P)and standard Bru-ker software.COSY,TOCSY,and ROESY experiments were recorded using data sets(t1by t2)of4096by512points,with16scans for COSY,and32for TOCSY and ROESY.The TOCSY experiments were carried out in the phase-sensitive mode with mixing times of60 and180ms and ROESY of300ms.The1H,13C correlations mea-sured in the1H-detected mode via HSQC-DEPT with proton decou-pling in the13C domain and HMBC spectra were acquired using data sets of4096by512points and80scans for each t1value. HMBC spectra were adjusted to J coupling constant value of 145Hz and long range proton carbon coupling constant of10Hz.Chemical shifts were reported relative to external acetone(d H 2.225,d C31.45)or external phosphoric acid(d P0.0). AcknowledgmentsWe thank Pfizer Animal Health forfinancial support,Dr.Uwe Mamat for anion-exchange chromatography,Hermann Moll for help with GLC–MS,and Heiko Käßner and Dr.Nicolas Gisch for NMR recordings.References1.Leigh,J.A.Vet.J.1999,157,225–238.2.Zadoks,R.N.;Gillespie,B.E.;Barkema,H.W.;Sampimon,O.C.;Oliver,S.P.;Schukken,Y.H.Epidemiol.Infect.2003,130,335–349.3.Watts,J.L.Vet.Microbiol.1988,16,41–66.4.Leigh,J.A.;Egan,S.A.;Ward,P.N.;Field,T.R.;Coffey,T.J.Vet.Res.2010,41,63.5.Schukken,Y.H.;Günther,J.;Fitzpatrick,J.;Fontaine,M.C.;Goetze,L.;Holst,O.;Leigh,J.;Petzl,W.;Schuberth,H.-J.;Sipka,A.;Smith,D.G.E.;Quesnell,R.;Watts,J.;Yancey,R.;Zerbe,H.;Gurjar,A.;Zadoks,R.N.;Seyfert,H.-M.Vet.Immunol.Immunopathol.2011,144,270–289.6.Schäffer,C.;Messner,P.Microbiology2005,151,643–651.7.Vergara-Irigaray,M.;Maira-Litran,T.;Merino,N.;Pier,G.B.;Penades,J.R.;Lasa,I.Microbiology2008,154,865–877.8.Oku,Y.;Kurokawa,K.;Matsuo,M.;Yamada,S.;Lee, B.-L.;Sekimizu,K.J.Bacteriol.2009,191,141–151.Table21H and13C NMR data of the WTA and deacylated LTA isolated from S.uberis233Residue1(a)1b23(a)3b456a6bA01H 5.16 3.68 3.86 3.49 3.69 3.71 3.81 2-a-D-Glc p13C97.176.673.171.073.061.8B01H 5.09 3.60 3.77 3.46 3.93 4.11 4.05 t-a-D-Glc p13C97.372.373.970.773.166.0C01H 4.47 3.31 3.51 3.40 3.46 3.90 3.77 t-b-D-Glc p13C103.374.476.770.876.961.9D01H 3.83 3.51nd 3.67 3.60Gro-A013C70.0nd63.1E01H 3.93 4.02 4.06 3.93 4.02–P-Gro-P–13C67.570.367.5F01H 4.11 4.03 4.22 4.11 4.03–P-Gro-(C0)P–13C66.178.566.1G01H 3.90 3.96 3.91 3.60 3.685.Proposed structure of WTA of S.uberisA.Czaban´ska et al./Carbohydrate Research377(2013)58–62619.Peschel,A.J.Biol.Chem.1999,274,8405–8410.10.Peschel,A.;Vuong,C.;Otto,M.;Götz,F.Antimicrob.Agents Chemother.2000,44,2845–2847.11.Schäffer,C.;Messner,P.Biochimie2001,83,591–599.12.Czaban´ska, A.;Neiwert,O.;Lindner, B.;Leigh,J.;Holst,O.;Duda,K. A.Carbohydr.Res.2012,361,200–205.13.Araki,Y.;Ito,E.Crit.Rev.Microbiol.1989,17,121–135.14.Sánchez Carballo,P.M.;Vilen,H.;Palva,A.;Holst,O.Carbohydr.Res.2010,345,538–542.15.Endl,J.;Seidl,H.P.;Fiedler,F.;Schleider,K.H.Arch.Microbiol.1983,135,215–223.16.Schäffer, C.;Kählig,H.;Christian,R.;Schulz,G.;Zayni,S.;Messner,P.Microbiology1999,145,1575–1583.17.Steindl,C.;Schäffer,C.;Wugeditsch,T.;Graninger,M.;Matecko,I.;Müller,N.;Messner,P.Biochem.J.2002,368,483–494.18.Steindl,C.;Schäffer,C.;Smrecki,V.;Messner,P.;Müller,N.Carbohydr.Res.2005,340,2290–2296.19.Schäffer,C.;Müller,N.;Mandal,P.K.;Christian,R.;Zayni,S.;Messner,P.Glycoconjugate J.2000,17,681–690.20.Kessler,R.E.;van de Rijn,I.;McCarty,M.J.Exp.Med.1979,150,1498–1509.21.Kurokawa,K.;Gong,J.-H.;Ryu,K.-H.;Zheng,L.;Chae,J.-H.;Kim,M.-S.;Lee,B.L.p.Immunol.2011,35,835–839.22.Archibald,A.R.;Baddiley,J.;Shaukat,G.A.Biochem.J.1968,110,583–588.62 A.Czaban´ska et al./Carbohydrate Research377(2013)58–62。