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1、LETTERdoi:10.1038/nature12892A new metabolic cell-wall labelling method revealspeptidoglycan in Chlamydia trachomatisG.W.Liechti1*,E.Kuru2*,E.Hall3,A.Kalinda3,Y.V.Brun4,M.VanNieuwenhze3&A.T.Maurelli1Peptidoglycan(PG),anessentialstructureinthecellwallsofthevastmajorityofbacteria,iscriticalfordivision
2、andmaintainingcellshapeand hydrostatic pressure1.Bacteria comprising the Chlamydialeswere thought to be one of the few exceptions.Chlamydiaharbourgenes for PG biosynthesis27and exhibit susceptibility to anti-PGantibiotics8,9,yet attempts to detect PG in any chlamydial specieshave proven unsuccessful
3、(the chlamydial anomaly10).We used anovelapproachtometabolicallylabelchlamydialPGusingD-aminoacid dipeptide probes and click chemistry.ReplicatingChlamydiatrachomatiswerelabelledwiththeseprobesthroughouttheirbipha-sicdevelopmentallifecycle,andtheresultsofdifferentialprobeincor-poration experiments c
4、onducted in the presence of ampicillin areconsistentwiththepresenceofchlamydialPG-modifyingenzymes.These findings culminate 50 years of speculation and debate con-cerning the chlamydial anomaly and are the strongest evidence sofar that chlamydial species possess functional PG.Chlamydia trachomatis i
5、s the leading cause of infectious blindnessand sexuallytransmitted bacterialinfection worldwide.Itis a memberof the Chlamydiae,a phylum consisting of obligate,intracellular,bac-teria that cause a wide variety of infectious diseases in humans andanimals.Their obligate intracellular nature and dimorph
6、ic life cyclehasmadestudyingChlamydiaachallengeandquestionsremainabouteventhebasicprocessesofcelldivisionandcellenvelopemaintenancein these pathogens.The infectious form of the organism,the element-arybody(EB),issmall(,0.3mm)andessentiallymetabolicallyinert11.After attachment to and infection of a h
7、ost cell,the EB undergoes atransitiontothemetabolicallyactivereticulatebody(RB),whichrepli-catesviabinaryfissionbutisincapableofattachingtoorinfectingnewhost cells.Thus,RBs must differentiate back to the EB form to com-plete the developmental cycle.Infected cells then lyse,releasing infec-tious EBs
8、that infect new host cells.Peptidoglycan(PG)is a sugar amino acid polymer that forms amesh-like sheet surrounding the cytoplasmic membrane of bacterialcells.Inthe vastmajority of free-living bacteria,PG aids in cell division1,maintenanceofosmoticpressure,andprovidesastableanchorfortrans-membranecomp
9、lexesandintegralmembraneproteins12.Bacteriamain-taintheircellshapelargelyduetothepresenceofthisrigidyetmodifiablecellwall.AsinglePGsubunitconsistsofadisaccharidebackbonecoupledto a pentapeptide chain(Fig.1a).During cell-wall synthesis,disaccha-ride pentapeptide monomers are linked together at their
10、correspond-ingsugars,creatingasugarpolymerwithpolypeptidestems,whicharecrosslinked by transpeptidation.The pentapeptide chain is assembledsequentially by a series of ligases that specifically incorporate bothL-andD-amino acids(D-glutamic acid andD-alanine)(Fig.1a).ThesetwoD-aminoacidsareuniquetobact
11、eriaandtheyarenotusedbymam-maliancells.Thus,the enzymes involved in their synthesis and incor-porationintoPGareexcellenttargetsforantibioticssuchasb-lactamsandD-cycloserine.The existence of PG in Chlamydia has long been debated.Whereasgenetic analysis and antibiotic susceptibility indicate that chla
12、mydialPG exists8,9,13,all attempts to detect or purify PG in Chlamydia havebeenunsuccessful10,1417,resultinginthechlamydialanomaly10.Ithasbeen established that the cytosolic receptor for PG,Nod1,is triggeredupon infection by various chlamydial species18.Chlamydial homolo-gues of PG biosynthetic enzy
13、mes have been extensively studied27anda growing body of literature supports the functionality of a completebiosynthesispathway.AfunctionalchlamydialUDP-N-acetylglucosa-mineenolpyruvyltransferase(MurA)hasbeendescribed2.Theproductof the MurA reaction is specifically used for the synthesis of UDP-N-ace
14、tylmuramicacid(thesugaruniquetothePGdisaccharidebackbone),suggesting the presence of the sugar component of PG in Chlamydia.DuringPGbiosynthesisinmostbacteria,D-alanine-D-alanine(DA-DA)generated byD-alanine-D-alanine ligase(Ddl)is incorporated directlyinto growing PG peptide chains through the MurF
15、enzyme(Fig.1a).Characterization of Ddl4and MurF6enzymes in Chlamydia coupledwith recent advances in the chemical modification of PG through theincorporation of singleD-amino acids19,20present an opportunity tocovalently label the PG of actively growing Chlamydia.C.trachomatiscantakeupbothD-alaninean
16、d DA-DAdipeptide4,8;however,effortstosuccessfullylabelChlamydiausingpreviouslychar-acterizedD-amino acid probes19,20were unsuccessful(Extended DataFig.1).We reasoned that this result was due to the inability of thechlamydialPGsynthesismachinerytoincorporatethemodifiedsingleD-aminoacids.Thus,wedevelo
17、pedanovelandbroadlyapplicablePG-labelling approach that bypassed the bacterial Ddl enzyme and usedDA-DA dipeptide analogues modified with alkyne or azide functionalgroups(Fig.1).Initial studies in Escherichia coli and Bacillus subtilisestablished that the alkyne and azide analogues of DA-DA(EDA-DA,D
18、A-EDA,ADA-DA and DA-ADA,respectively,where ADA,azido-D-alanine;EDA,ethynyl-D-alanine)arecapableofrescuingthegrowthof bacteria with depleted DA-DA dipeptide pools,whereas an alkyneanalogueoftheenantiomerL-alanine-L-alanine(LA-LA)isnotcapableofrescuinggrowth(ExtendedDataTable1).Inrichmedium,bacterialg
19、rowth is unaffected by the presence of DA-DA analogues(ExtendedData Fig.2).Once incorporatedinto amacromolecule such as PG,thefunctional groups of these dipeptides can be selectively captured via aclick-chemistryreaction20,21.LabellingstudiesusingDA-DAanaloguesin conjunction with clickable,modified
20、Alexa Fluor dyes confirmedD-enantiomer-specific incorporation of the modified dipeptides indiversebacterialspecies(ExtendedDataFigs2and3).PolarlygrowingStreptomyces venezuelae was grown in the presence of the previouslycharacterized,fluorescentD-aminoacidHADA(7-hydroxycoumarin-3-carboxylicacid-3-ami
21、no-D-alanine,seeref.19)forafewgenerations,and upon addition of EDA-DA for a brief period,subsequent polar-labelling confirmed that these dipeptides specifically label areas ofnew PG synthesis(Extended Data Fig.3e).The labelling of E.coliand B.subtilis was covalent and cell-wall-specific(Extended Dat
22、a*These authors contributed equally to this work.1Department of Microbiology and Immunology,F.Edward He bert School of Medicine,Uniformed Services University of the Health Sciences,4301 Jones Bridge Road,Bethesda,Maryland 20814-4799,USA.2Interdisciplinary Biochemistry Program,Indiana University,Bloo
23、mington,Indiana 47405,USA.3Department of Chemistry,Indiana University,Bloomington,Indiana 47405,USA.4Department ofBiology,Indiana University,Bloomington,Indiana 47405,USA.2 7F E B R U A R Y2 0 1 4|V O L5 0 6|N A T U R E|5 0 7Macmillan Publishers Limited.All rights reserved2014Fig.4a),and B.subtilis
24、label could be removed by treatment with thePG-digesting enzyme,lysozyme(Extended Data Fig.4b),indicatingthat the probes were incorporated in the PG.We next attempted to measure dipeptide probe uptake and incorp-oration in intracellular Chlamydia.L2 mouse fibroblast cells wereinfected for 18 h with
25、C.trachomatis serovar L2 strain 434/Bu in thepresence of 1mM EDA-DA.Cells were then fixed and permeabilized,and click chemistry was used to attach an Alexa fluorophore modifiedwith a terminal azide group to the alkyne group present on the EDA-DA probe.The probe localized within chlamydial inclusions
26、 withindividual bacteria clearly discernible(Fig.2).When co-labelled withantibody to the chlamydial major outer membrane protein(MOMP),EDA-DA labelling appeared as either a ring or a single line bisectingMOMP-labelledRBs(ExtendedDataFig.5).Thelabellingwasarrangedinadistinct,ring-likeshape,consistent
27、withacellulardivisionplaneandthe labelling bore a striking resemblance to images previously obtainedfor intracellular C.trachomatis stained with antibody generated withRibiadjuvant22.LabellingwasonlypresentinChlamydia-infectedcellsandonlyinthepresenceofprobe(ExtendedDataFig.6ac).Thisresultindicatest
28、hatthemajorityoflabelledchlamydialPGislocalizedtotheseptum of dividing RBs.However,we cannot rule out the possibilitythat low levels of PG exist elsewhere on the bacterium and are simplybelow the detection limit of fluorescence microscopy.Similar to ourresultswithB.subtilis,wefoundthatincubationwith
29、lysozymefortwohours was sufficient to remove EDA-DA labelling within chlamydialinclusions(Extended Data Fig.6d,e),supporting our conclusion thatthe dipeptide probes are incorporating into chlamydial PG.To further confirm that the modified probes were being taken upand incorporated into chlamydial PG
30、,we performed plaque assays23that allowquantification ofintracellular bacterialgrowthandinfectiv-ity.D-cycloserine(DCS)is an inhibitor of cell-wall biosynthesis thattargetsbacterialalanineracemaseandD-alanine-D-alanineligase24andprevious studies have shown that Chlamydia growth is inhibited byDCS at
31、 millimolar concentrations4,8,25.Growth inhibition is overcomeby supplementation with exogenousD-alanine or DA-DA dipeptide4,most likely owing to the exogenous singleD-amino acids outcompet-ing DCS for the binding sites of the chlamydial ligase or,in the case ofDA-DA,bypassingtheneedfortheligasealto
32、gether.VariousD-aminoacids,dipeptides and their corresponding alkyne-modified probeswere tested to determine the level of DCS rescue they conferred uponaPentapeptideTetrapeptideCrosslinksby PBPs DacADdlMurFAlrH3NHNOOOEDA-DAH3NHNOOODA-DAH3NHNON3OOADA-DAH3NHNOOODA-EDAEDAH3NOOD-AlaH3NOOADAH3NN3OOH3NHNO
33、OON3DA-ADAbL-AlaD-Glum-DAPD-AlaD-AlaL-AlaD-GluMurNAcGlcNAc MurNAcUDPm-DAPD-AlaL-AlaD-Ala-D-Ala(DA-DA)D-cycloserineD-Ala+Figure 1|Novel dipeptide PG labelling strategy.a,Biosynthesis of the terminal PG stem peptide ofGram-negative bacteria.TwoD-alanines are firstligated together byD-alanine-D-alanine
34、 ligase andthe dipeptide is subsequently added to the stemtripeptidebyMurF,resultinginapentapeptide.Thelabelling strategy relies on the inherent tolerance ofthe PG machinery to accept DA-DA analogues.Subsequent crosslinking between neighbouringpeptide stems is carried out by a series oftranspeptidas
35、es(penicillin-bindingproteins).Upontranspeptidation,a proximal m-DAP from aneighbouring peptide stem attacks the carbonylgroup between the penultimate and terminalD-alaninesofthePGstem.TheterminalD-alanineisthuscleavedfromthestempeptide,whichresultsina tetrapeptide.Another pathway for the loss ofter
36、minalD-alanine isD,D-carboxypeptidationcatalysed by enzymes such as DacA.b,Chemicalstructures ofD-Ala,DA-DA,and their derivativescarrying bio-orthogonal handles used in this study.ADA,azido-D-alanine;ADA-DA,azido-D-alanyl-D-alanine;D-Ala,D-alanine;L-Ala,L-alanine;Alr,alanine racemase;DA-DA,D-alanyl-
37、D-alanine;m-DAP,meso-diaminopimelic acid;Ddl,D-alanine-D-alanineligase;EDA,ethynyl-D-alanine;EDA-DA,ethynyl-D-alanyl-D-alanine;DacA,D-alanyl-D-alanine carboxypeptidase A;GlcNAc,N-acetylglucosamine;D-Glu,D-Glutamic Acid;MurF,UDP-N-acetylmuramoyl-tripeptideD-alanyl-D-alanineligase;MurNAc,N-acetylmuram
38、icacid;PBPs,penicillin-binding proteins.a10 m10 mbcdeFigure 2|Fluorescent labelling of intracellularC.trachomatisPG.ae,Differential interferencecontrast(DIC)(a)and fluorescent(be)microscopy of L2 cells infected for 18 h withC.trachomatis in the presence of the dipeptideprobeEDA-DA(1mM).Subsequentbin
39、dingoftheprobetoanazidemodifiedAlexaFluor488(green)was achieved via click chemistry.Antibody toMOMP(red)wasusedtolabelchlamydialEBsandRBs.DAPI(blue)was used for nuclear staining.be show a mergeof all threefluorescent channels.Boxes indicate location of chlamydial inclusions,and magnification of the
40、boxes is provided in ce.Fluorescent images are maximum intensityprojections of deconvoluted z-stacks.Three-dimensional renderings are provided inSupplementary Videos 1 and 2.RESEARCH LETTER5 0 8|N A T U R E|V O L 5 0 6|2 7F E B R U A R Y2 0 1 4Macmillan Publishers Limited.All rights reserved2014grow
41、ing C.trachomatis.We found that DA-DA dipeptide and thecorrespondingmodifieddipeptides(EDA-DAandDA-EDA)werebothcapable of rescuing chlamydial plaquing(Table 1),indicating theirsuccessfuluptakeandincorporationbyChlamydia.However,whereasunmodifiedD-alanine was capable of overcoming the growth inhib-it
42、ory effects of DCS,the corresponding chemically modified,singleD-alanine probe(EDA)was not.These results were consistent withour inability to detect fluorescent labelling of C.trachomatis throughsingle EDA probes(Extended Data Fig.1).DespiterescueofchlamydialgrowthbybothEDA-DAandDA-EDAin the DCS pla
43、que assay,we initially were not able to label chlamydialPGwithDA-EDA(ExtendedDataFig.7).Similarly,wewereunabletoobtain labelling with DA-EDA in E.coli(Extended Data Fig.2).Wereasoned that the inability to label Chlamydia with DA-EDA was duetotheremovaloftheterminal,modifiedEDAaminoacidfromthePGpenta
44、peptide stem during either transpeptidation or carboxypeptida-tion(Fig.1a).To test this hypothesis and to further validate that ourprobeswereincorporatedintoC.trachomatisPG,weconductedEDA-DA and DA-EDA labelling studies in the presence of two antibioticsthat block PG biosynthesis:DCS,a competitive i
45、nhibitor of both ala-nineracemaseandD-alanine-D-alanineligase,andampicillin,aninhib-itorofPGtranspeptidases/carboxypeptidases.Whengrownfor18hinthe presence of either antibiotic,inclusions contained enlarged,aber-rantRBs.Thepresenceoffewerbacteriaperinclusionisindicativeofapre-division block,due to t
46、he absence of transpeptidation,and is con-sistent with the literature26.In the presence of DCS and 1mM EDA-DA,fluorescentPGwasdiscerniblewithinaberrantRBs(Fig.3a).Thisresult indicates that EDA-DA was capable of partly substituting forDA-DA after depletion of the bacteriums natural dipeptide pool and
47、confirms the DCS plaquing assay results.EDA-DA labelling intensityseemed unaffected by inhibition of PG transpeptidation/carboxypep-tidation with ampicillin(Extended Data Fig.7),indicating that probeincorporation is not dependent on transpeptidation and does not occurin the periplasm in Chlamydia.Wh
48、en imaged by epifluorescence,label-lingofaberrantbodiesgrowninthepresenceofampicillinoftenappearedpunctate,owingtotheenlargedPGringstructuresthatnolongerexistwithinasinglefocalplane(seeExtendedDataFig.8).z-stackstakenoftheampicillin-treatedaberrantRBsclearlyrevealedlabelledPGseques-tered to an equat
49、orial region where the bacterial division plane wouldnormallyform(Fig.3b).FluorescencelabellingofChlamydiawithDA-EDA was only observed when transpeptidation/carboxypeptidationwas inhibited with ampicillin(Extended Data Fig.7).DA-ADA label-ling of a B.subtilisD,D-carboxypeptidase mutant(DdacA)confirm
50、edthis finding;labelling was greatly increased compared to the parental,wild-type strain and was not significantly turned over as the labelledcells were allowed to grow(Extended Data Fig.3).These observationsindicate that PG modifications(through transpeptidation and/or car-boxypeptidation)occur in