微生物耐药基础与临床之抗细菌感染策略

NovelAnti-InfectiousStrategy:FightagainstBacterialVirulenceFactors

抗细菌感染策略——抗毒力Nolongerlife-threatening: infectedwounds foodpoisoningdiseasesOnthewaytoeradication: syphilis gonorrhoeaCouldnowbecontrolled： plague choleraantibioticantibioticchallenge1:Growingantibioticresistance

抗感染+不诱发耐药性？1.Directvirulencefactors&inhibitors细菌毒力粘附素荚膜/微荚膜侵袭性物质毒素生物被膜数量感染途径（侵入部位）enzymetoxinLPSTTSSIsvirulencefactorindispensibleforbacterialsurvival?国内外使用的减毒活疫苗一览表分类名称接种途径国内使用国外使用病毒疫苗甲型肝炎减毒活疫苗皮下注射十—麻疹减毒活疫苗皮下注射十十腮腺炎减毒活疫苗皮下注射十十黄热减毒活疫苗皮下注射十十脊髓灰质炎减毒活疫苗口服十十乙型脑炎活疫苗皮下注射十—风疹减毒活疫苗皮下注射十十水痘减毒活疫苗皮下注射十十登革热减毒活疫苗皮下注射—十流感减毒活疫苗喷雾—十腺病毒减毒活疫苗肌内注射—十细菌疫苗炭疽减毒活疫苗皮上划痕十十布氏菌减毒活疫苗皮上划痕十十卡介苗皮内注射十十伤寒减毒活疫苗口服—十霍乱减毒活疫苗口服—十鼠疫减毒活疫苗皮上划痕十十致病性大肠杆菌肠产毒性大肠杆，EnterotoxingenicE.coli,ETEC肠侵袭性大肠杆菌，EnteroinvasiveE.coli,EIEC肠致病性大肠杆菌，EnteropathogenicE.coli,EPEC肠出血性大肠杆菌，EnteroheamorrhagicE.coli,EHEC肠集聚性粘附大肠杆菌，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.细菌生物被膜的基本性质细菌自身产生的外部多糖基质、纤维蛋白质、脂蛋白等包裹着的菌细胞的结构。•生物被膜是细菌的一种具有保护性的生长模式，是细胞间相互协调作用的复杂的多细胞群体，具有结构和代谢复杂性。•形成生物被膜的黏附细菌群也可以释放出生长迅速的浮游细菌，是潜在的“菌巢”。化学组成

•水分（97%）•胞外多糖（EPS）•吸附的营养物质及代谢产物、细菌及裂解物•蛋白质、DNA、RNA生物被膜的结构特点结构特点—Marchabas的成熟生物被膜模型

•由外到内依次为：①bulkofbiofilm②linkingfilm③conditioningfilm④substratum

•

细菌被大量的胞外多糖包绕形成微菌落，各微菌落之间充满水通道,是细菌获取营养和排除代谢废物的通道。细菌生物被膜广泛存在于自然环境中；可形成于各种植入医疗器械表面或体内粘膜上;具有极强的耐药性（增加10-1000倍）增强细菌的免疫逃避；是造成临床性感染的主要原因之一！DetrimentsofbiofilmsStagesofbiofilmdevelopment⒈条件膜的沉积主要涉及体液中各种糖蛋白、粘多糖、金属离子等的吸附⒉细菌的初始到达及吸附指在钠、镁等阳离子的介导下的细菌对植入物表面的吸附⒊细菌的生长繁殖指细菌的吸附、生长、繁殖及扩散⒋生物被膜的形成细菌程序性的表达并分泌EPS,不断形成微菌落，终联合成为成熟的生物被膜的过程。⒌细菌的分散及持续性感染与人类感染有关的生物膜形成菌相关疾病龋齿牙周炎中耳炎骨骼肌感染骨髓炎心内膜炎隐形眼镜所致感染缝合部位感染人工心瓣膜生物膜形成菌产酸性G+球菌口腔G-厌氧菌嗜血流感杆菌G+球菌多种细菌和真菌（混合）草绿色链球菌绿脓杆菌和G+球菌表皮葡萄球菌、金黄色葡萄球菌金黄色葡萄球菌和表皮葡萄球菌2.Howisvirulencefactorsregulated？QuorumSensing&Virulencefactor&QSinhibitors荧光细菌与海生动物共生；宿主利用其发出的光捕获食物、躲避天敌以及寻觅配偶；

V.fiscberi也获得了营养丰富的生存环境。DiscoveryofQuorum-SensingSystemNealson等在1970年首次报道了该菌的菌体密度与发光呈正相关；该发光现象受细菌本身的群体感应调节系统(Quorum-SensingSystem，QS系统)所控制。V.fischeriQuorumSensingLuxILuciferaseGenesLuxRLuxRLuxI-LuxRQuorumSensingSystemsV.fischeriP.aeruginosaA.tumefaciensE.carotovoraBioluminescenceVirulenceFactors,BiofilmsMating,TransferofMobileDNAVirulenceFactors,AntibioticSynthesis2.分类G-菌QS系统G+菌QS系统种间QS系统种内QS系统群体感应G-细菌QSAHL（Acyl-homoserinelactone)DKP（二酮哌嗪类化合物）P.aeruginosa、荧光假单胞菌(P.fluorescens)、产碱假单胞菌(P.alcaligenes)、成团肠杆菌(Enterobacteragglomerans)和弗罗因德枸橼酸杆菌(Citrobacterfreundii)p-coumaroyl-HSL(香豆酸酰高丝氨酸)光合作用细菌(Rhodopseudomonaspalustris)信号分子Acyl-HomoserineLactoneAutoinducers10%

oftheP.aeruginosagenome信号通路proteases：LasAprotease;LasBelastasehemolysinspyocyaninpililipopolysaccharidealginateT3SSeffectorproteins,ExoS,ExoT,ExoU,andExoYVirulenceFactorsAssociatedwithPseudomonasaeruginosaG+细菌QS信号分子信号通路theagrsystemregulatesofStaphyloccusaureustheagrsystemregulatesover70genesofStaphyloccusaureus;23ofwhichareknownvirulencefactors;twoclasses:thefirstclasscontainsvirulencefactorsinvolvedinattachmenttothehostandimmuneevasion;thesecondinvolvedintheproductionofexoproteinsassociatedwithinvasionandtoxinproduction.theactivationoftheagrsystemessentiallyswitchesthebacteriumfromanadhesive,colonizingcommensaltoaninvasiveandaggressivepathogen.2.3种间QS信号分子——AI-2(呋喃酰硼酸二酯类化合物)此类信号分子在G+菌和G-菌中均可存在；细菌识别AI-2分子的方式与革兰氏阳性菌中双组分识别系统一致,即双组分激酶识别AI-2分子后把磷酸化基团传递给受体蛋白并启动相关基因的表达。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