微生物耐藥基礎(chǔ)與臨床之抗細(xì)菌感染策略

NovelAnti-InfectiousStrategy:FightagainstBacterialVirulenceFactors

抗細(xì)菌感染策略——抗毒力Nolongerlife-threatening: infectedwounds foodpoisoningdiseasesOnthewaytoeradication: syphilis gonorrhoeaCouldnowbecontrolled： plague choleraantibioticantibioticchallenge1:Growingantibioticresistance

抗感染+不誘發(fā)耐藥性？1.Directvirulencefactors&inhibitors細(xì)菌毒力粘附素莢膜/微莢膜侵襲性物質(zhì)毒素生物被膜數(shù)量感染途徑（侵入部位）enzymetoxinLPSTTSSIsvirulencefactorindispensibleforbacterialsurvival?國內(nèi)外使用的減毒活疫苗一覽表分類名稱接種途徑國內(nèi)使用國外使用病毒疫苗甲型肝炎減毒活疫苗皮下注射十—麻疹減毒活疫苗皮下注射十十腮腺炎減毒活疫苗皮下注射十十黃熱減毒活疫苗皮下注射十十脊髓灰質(zhì)炎減毒活疫苗口服十十乙型腦炎活疫苗皮下注射十—風(fēng)疹減毒活疫苗皮下注射十十水痘減毒活疫苗皮下注射十十登革熱減毒活疫苗皮下注射—十流感減毒活疫苗噴霧—十腺病毒減毒活疫苗肌內(nèi)注射—十細(xì)菌疫苗炭疽減毒活疫苗皮上劃痕十十布氏菌減毒活疫苗皮上劃痕十十卡介苗皮內(nèi)注射十十傷寒減毒活疫苗口服—十霍亂減毒活疫苗口服—十鼠疫減毒活疫苗皮上劃痕十十致病性大腸桿菌腸產(chǎn)毒性大腸桿，EnterotoxingenicE.coli,ETEC腸侵襲性大腸桿菌，EnteroinvasiveE.coli,EIEC腸致病性大腸桿菌，EnteropathogenicE.coli,EPEC腸出血性大腸桿菌，EnteroheamorrhagicE.coli,EHEC腸集聚性粘附大腸桿菌，EnteroaggregativeE.coli,EAggEC炭疽白喉……ToxincyclodextrinacetamideEPECstrainsarealeadingcauseofdiarrheaindevelopingcountriesthroughouttheworld.InsomelocationsEPECisthemostcommonbacterialcauseofdiarrheaininfants.DiseaseduetoEPECisrareindevelopedcountries.TypicalEPECpossessalargeplasmidthatmediatestheabilityofthesestrainstoadheretohostcellsinapatternknownaslocalizedadherence.AtypicalEPECstrainslackthisplasmidanddonotperformlocalizedadherence.TypeIVfimbriaeOMPCMSec-(orSec-like)dependentSec-independentTypeIATPADPNCNCTypeIIITypeIITypeIVSec(AdaptedfromStathopoulusetal.(2000);providedbyERucks)ATPADPADPATPATPADPhostcellhostcellTypeVSecB11Gram-negativesecretionNCbacterialsecretionsystemGram-negativesecretionTypeI-ATP-bindingcassette(ABC)transporterTypeII-generalpathway(Sec-dependent)-majorsecretorypathwayTypeIII-contact-dependenttranslocationintoeukaryoticcellsTypeIV-(Sec-likedependent)-translocationofDNA/proteincomplexTypeV-auto-transporter(Sec-dependent)-includesb-poreformingdomain~Tat-(twinargininetransport)-movesfoldedproteinsacrossCMSRP-(signalrecognitionparticle)(Sec-dependent)-usedforCMproteinsGram-negative-TypeIIIsecretionregulonEffectsofExoSoneukaryoticcellfunctionInhibitionofDNAsynthesisCellrounding(alteredcytoskeleton)Anti-phagocytic/anti-invasiveLossofcellsurfacemicrovilliLossofadhesionorre-adhesionLossofcellviability(Fraylicketal,Infect.Immun.1999)Strain388388DExoS(1hour)Strain388(1hour)EffectsofExoSonhumanepithelialcellsEffectsofExoSGAPandADPRTactivityonmacrophages

(Rochaetal,Infect.Immun.2002)Protectionagainstsecretion-linkedvirulencefactorsBiofilm“InapaperinSciencein1999,wesaid65percentofalldiseasesinthedevelopedworldarebiofilms,”Costertonsaid.“NowtheNIHsays80percent.”J.WilliamCostertonCosterton,anativeofBritishColumbia,earnedhisPh.D.inbacteriologyin1960fromtheUniversityofWesternOntario.細(xì)菌生物被膜的基本性質(zhì)細(xì)菌自身產(chǎn)生的外部多糖基質(zhì)、纖維蛋白質(zhì)、脂蛋白等包裹著的菌細(xì)胞的結(jié)構(gòu)。?生物被膜是細(xì)菌的一種具有保護(hù)性的生長模式，是細(xì)胞間相互協(xié)調(diào)作用的復(fù)雜的多細(xì)胞群體，具有結(jié)構(gòu)和代謝復(fù)雜性。?形成生物被膜的黏附細(xì)菌群也可以釋放出生長迅速的浮游細(xì)菌，是潛在的“菌巢”。化學(xué)組成

?水分（97%）?胞外多糖（EPS）?吸附的營養(yǎng)物質(zhì)及代謝產(chǎn)物、細(xì)菌及裂解物?蛋白質(zhì)、DNA、RNA生物被膜的結(jié)構(gòu)特點(diǎn)結(jié)構(gòu)特點(diǎn)—Marchabas的成熟生物被膜模型

?由外到內(nèi)依次為：①bulkofbiofilm②linkingfilm③conditioningfilm④substratum

?

細(xì)菌被大量的胞外多糖包繞形成微菌落，各微菌落之間充滿水通道,是細(xì)菌獲取營養(yǎng)和排除代謝廢物的通道。細(xì)菌生物被膜廣泛存在于自然環(huán)境中；可形成于各種植入醫(yī)療器械表面或體內(nèi)粘膜上;具有極強(qiáng)的耐藥性（增加10-1000倍）增強(qiáng)細(xì)菌的免疫逃避；是造成臨床性感染的主要原因之一！DetrimentsofbiofilmsStagesofbiofilmdevelopment⒈條件膜的沉積主要涉及體液中各種糖蛋白、粘多糖、金屬離子等的吸附⒉細(xì)菌的初始到達(dá)及吸附指在鈉、鎂等陽離子的介導(dǎo)下的細(xì)菌對(duì)植入物表面的吸附⒊細(xì)菌的生長繁殖指細(xì)菌的吸附、生長、繁殖及擴(kuò)散⒋生物被膜的形成細(xì)菌程序性的表達(dá)并分泌EPS,不斷形成微菌落，終聯(lián)合成為成熟的生物被膜的過程。⒌細(xì)菌的分散及持續(xù)性感染與人類感染有關(guān)的生物膜形成菌相關(guān)疾病齲齒牙周炎中耳炎骨骼肌感染骨髓炎心內(nèi)膜炎隱形眼鏡所致感染縫合部位感染人工心瓣膜生物膜形成菌產(chǎn)酸性G+球菌口腔G-厭氧菌嗜血流感桿菌G+球菌多種細(xì)菌和真菌（混合）草綠色鏈球菌綠膿桿菌和G+球菌表皮葡萄球菌、金黃色葡萄球菌金黃色葡萄球菌和表皮葡萄球菌2.Howisvirulencefactorsregulated？QuorumSensing&Virulencefactor&QSinhibitors熒光細(xì)菌與海生動(dòng)物共生；宿主利用其發(fā)出的光捕獲食物、躲避天敵以及尋覓配偶；

V.fiscberi也獲得了營養(yǎng)豐富的生存環(huán)境。DiscoveryofQuorum-SensingSystemNealson等在1970年首次報(bào)道了該菌的菌體密度與發(fā)光呈正相關(guān)；該發(fā)光現(xiàn)象受細(xì)菌本身的群體感應(yīng)調(diào)節(jié)系統(tǒng)(Quorum-SensingSystem，QS系統(tǒng))所控制。V.fischeriQuorumSensingLuxILuciferaseGenesLuxRLuxRLuxI-LuxRQuorumSensingSystemsV.fischeriP.aeruginosaA.tumefaciensE.carotovoraBioluminescenceVirulenceFactors,BiofilmsMating,TransferofMobileDNAVirulenceFactors,AntibioticSynthesis2.分類G-菌QS系統(tǒng)G+菌QS系統(tǒng)種間QS系統(tǒng)種內(nèi)QS系統(tǒng)群體感應(yīng)G-細(xì)菌QSAHL（Acyl-homoserinelactone)DKP（二酮哌嗪類化合物）P.aeruginosa、熒光假單胞菌(P.fluorescens)、產(chǎn)堿假單胞菌(P.alcaligenes)、成團(tuán)腸桿菌(Enterobacteragglomerans)和弗羅因德枸櫞酸桿菌(Citrobacterfreundii)p-coumaroyl-HSL(香豆酸酰高絲氨酸)光合作用細(xì)菌(Rhodopseudomonaspalustris)信號(hào)分子Acyl-HomoserineLactoneAutoinducers10%

oftheP.aeruginosagenome信號(hào)通路proteases：LasAprotease;LasBelastasehemolysinspyocyaninpililipopolysaccharidealginateT3SSeffectorproteins,ExoS,ExoT,ExoU,andExoYVirulenceFactorsAssociatedwithPseudomonasaeruginosaG+細(xì)菌QS信號(hào)分子信號(hào)通路theagrsystemregulatesofStaphyloccusaureustheagrsystemregulatesover70genesofStaphyloccusaureus;23ofwhichareknownvirulencefactors;twoclasses:thefirstclasscontainsvirulencefactorsinvolvedinattachmenttothehostandimmuneevasion;thesecondinvolvedintheproductionofexoproteinsassociatedwithinvasionandtoxinproduction.theactivationoftheagrsystemessentiallyswitchesthebacteriumfromanadhesive,colonizingcommensaltoaninvasiveandaggressivepathogen.2.3種間QS信號(hào)分子——AI-2(呋喃酰硼酸二酯類化合物)此類信號(hào)分子在G+菌和G-菌中均可存在；細(xì)菌識(shí)別AI-2分子的方式與革蘭氏陽性菌中雙組分識(shí)別系統(tǒng)一致,即雙組分激酶識(shí)別AI-2分子后把磷酸化基團(tuán)傳遞給受體蛋白并啟動(dòng)相關(guān)基因的表達(dá)。QSinhibitorsQuorumsensinginhibitors

Natural

ProkaryoticQSIAnimalbasedQSIsPlantbasedQSIsMarineorganismbasedQSIsFungusbasedQSIsAntibodybasedQSISyntheticQSI

SignalsynthesisModificationsintheAHLsidechainModificationsintheAHLringmoietyAntagonistsofreceptorligandinteractionsAlkylDPDanaloguesQseCsignalAIP-IIsignalmodificationMetallo-complexOthersNaturalproductsApplMicrobiolBiotechnol(2010)86:813–823ProblemsHowtostrengthentheevidenceforquorumsensinginhibition?Assessmentoftoxiceffectsofputativequorumsensinginhibitors2.Two-ComponentRegulatorySystems&VirulencefactorProkaryotesregulatecellularmetabolisminresponsetoenvironmentalfluctuationsExternalsignalistransmitteddirectlytothetargetExternalsignaldetectedbysensorandtransmittedtoregulatorymachinery(Signaltransduction)Mostsignaltransductionsystemsaretwo-componentregulatorysystems1.Two-ComponentRegulatorySystems

TCS1.CompositionofTCSTwo-componentregulatorysystemsMadeupoftwodifferentproteins:Sensorkinase:(incytoplasmicmembrane)detectsenvironmentalsignalandautophosphorylatesResponseregulator:(incytoplasm)DNA-bindingproteinthatregulatestranscriptionAlsohasfeedbackloopTerminatessignalEnvironmentalsignalPhosphatase

activityTranscriptionblockedRNA

polymeraseSensor

kinaseCytoplasmic

membraneResponseregulatorDNAPromoterOperatorStructuralgenes?2012PearsonEducation,Inc.Two-ComponentRegulatorySystemsAlmost50differenttwo-componentsystemsinE.coliExamplesincludephosphateassimilation,nitrogenmetabolism,andosmoticpressureresponseSomesignaltransductionsystemshavemultipleregulatoryelementsSomeArchaeaalsohavetwo-componentregulatorysystemsTCSiswidespreadamonglivingorganismsThesesignaltransductionsystemsarefoundinallkingdomsoflife,rangingfro