Direct force measurements of specific and non-specific interactions between a single bacterium and substrate PDF
Preview Direct force measurements of specific and non-specific interactions between a single bacterium and substrate
DIRECTFORCEMEASUREMENTSOFSPECIFICANDNON-SPECIFIC INTERACTIONSBETWEENASINGLEBACTERIUMANDSUBSTRATE By JONAHDAVIDKLEIN ADISSERTATIONPRESENTEDTOTHEGRADUATESCHOOL OFTHEUNIVERSITYOFFLORIDAINPARTIALFULFILLMENT OFTHEREQUIREMENTSFORTHEDEGREEOF DOCTOROFPHILOSOPHY UNIVERSITYOFFLORIDA Copyright2002 by JonahD.Klein Thisworkisdedicatedtomyparents,whocontinuetoserveasinspirationstome;andto mylovingcompanion,Monica,whoseshowofencouragementandassurancemadethis workpossible. ACKNOWLEDGEMENTS FirstandforemostIwouldliketoacknowledgethesupportofmyadvisor.Dr. RichardDickinson,whohasbeenasteadyguidethroughoutmythesiswork. Dr. Dickinsonhasconsistentlyexemplifiedmotivation,dedication,andintegritythroughout mybuddingcareerinscientificresearch. IalsoacknowledgethesupportofmyfellowstudentswithinDr.Dickinson’s researchgroup,particularlyAaronClapp,whotaughtmethehandsonfundamentalsof opticaltrappingandevanescentwavelightscattering,andBrianBurgesswhointroduced metothefinerdetailsofautomatedlightmicroscopy. Ialsoacknowledgetheeffortsof threeundergraduatestudents,JenniferVella,IfademaOfoma,andSeungSung,who assistedwithvariouslaboratorymeasurementsandduties. Fordevelopingandsupplyingthebacterialmutantsusedinthemeasurement experiments,IacknowledgeDr.TimothyFosterandDr.OrlaHartfordfromTrinity CollegeinDublin,Ireland. Forhelpfuldiscussionsandsynthesisofpolypeptides,I acknowledgeAlfredChungoftheUniversityofFloridaInterdisciplinaryCenterfor — BiotechnologyResearch ProteinCore. Forinstructionandtheuseofthescanning electronmicroscope,IacknowledgeKerrySiebenoftheResearchCenterforParticle — ScienceandTechnologyandKarenKelley,andtheUFICBR ElectronMicroscopy Core,respectively. Forfinancialassistanceandfunding,IacknowledgetheNationalScience Foundation,theNSFEngineeringResearchCenterforParticleScienceandTechnology, IV theBiomedicalEngineeringFellowshipProgram,theUniversityofFloridaChemical EngineeringDepartment,andespeciallythesupportofmyresearchadvisorwhohas alwayshustledtosecureadequateequipmentandfundingsupport. Finally,manythankstoShirleyKelley,NancyKrell,andPeggy-JoDaugherty, DennisVince,andtotheotherstaffintheChemicalEngineeringDepartment,whose supportIwillneverforgetandwhoseeffortshavebeencrucialtomysuccess. TABLEOFCONTENTS page ACKNOWLEDGEMENTS iv ABSTRACT ix CHAPTER 1 INTRODUCTION 1 2 MEASUREMENTANDANALYSISOFBACTERIALATTACHMENT 4 MicrobialAdhesiontoSurfaces 4 BacterialAttachmenttoBiomaterials 4 BacterialAttachmentForces 6 BacterialSurfaceProperties 7 Non-SpecificMechanismsofBacterialAdhesion 8 SpecificMechanismsofBacterialAdhesion 13 MeasurementsRelevanttoBacterialAdhesion 14 QuantitativeAttachmentMeasurements 14 ForceMeasurements 17 3 THREEDIMENSIONALOPTICALTRAPPINGWITHEVANESCENTWAVE LIGHTSCATTERING 21 Introduction 21 ExperimentalProtocol 22 Apparatus 23 DataAnalysisandMeasurement 26 EvanescentWaveLightScattering 27 OpticalTrapasForceTransducer 27 CalibrationofOpticalTrapandDeterminationofEquilibriumandViscousForces 29 4 MEASUREMENTOFNON-SPECIFICFORCESBETWEENBACTERIAAND GLASS 36 ForceMeasurementsinLowElectrolyteConditions 36 ReproducibilityoftheTechnique 37 ViscousInteractionForces 38 ComparisontoDLVOTheory 40 ForceMeasurementsinPhysiologicalConditions 42 vi EquilibriumProfilesbetweenBacteriaandGlassvs.BacteriaandAdsorbedCasein 44 DirectObservationoftheAttachmentProcess 47 5 DISCUSSIONANDSOURCESOFERRORINNON-SPECIFICFORCE MEASUREMENTS 52 ParticleScatterandBackgroundScatter 52 SurfaceParticleScatterandPhotomultiplierApertureEffects 54 EvanescentWaveLightScatteringandProteinCoatedSurfaces 54 TorqueandRotationduetoShapeEffects 57 S.aureusvs.LactobacillusRhamnosus 57 CorruptionofSeparationDistanceMeasurementbyRotationofOblateEllipsoids 61 6 CONTROLOFSPECIFICINTERACTIONSANDSELECTIONOF EXPERIMENTALSYSTEM 66 Fibrinogen-ClumpingFactorInteractions 66 PreparationofDeletionMutantstoControlStalkLength 68 BiochemicalPropertiesofMutants 68 PhysicalPropertiesofMutants 70 ZetaPotentialmeasurement 70 SubstratePreparation 73 PropertiesofFibrinogen 74 PeptideConjugation 75 EvaluationofConjugatedSubstrates 77 SelectionandEvaluationofAdsorbedProteins 79 ParallelPlateFlowCellApparatusandMeasurement 80 Measurementoftheintrinsicrateconstant 81 EvaluationofBovineSerumAlbumin 86 Proteinadsorption 86 ParallelPlateFlowCelloperation 86 EvaluationofHumanSerumAlbumin,SkimMilk,Casein,andp-Casein 88 7 MEASUREMENTOFSPECIFICINTERACTIONFORCES 91 MaterialsandMethods 91 KineticRateAttachmentMeasurementsofMutantswithVaryingClumpingFactor Length 92 ForceDistanceProfilesforMutantswithVaryingClumpingFactorLength 94 DiscussionofResults 96 8 CONCLUSIONSANDSUGGESTIONSFORFUTUREWORK 99 Conclusions 99 SuggestionsforFutureWork 100 vii ImprovedOpticalTrap 101 ImproveMeasurementofSpecificInteractionsofBacteriawithCoatedSurfaces 102 APPENDIX 105 REFERENCES 114 BIOGRAPHICALSKETCH 121 viii AbstractofDissertationPresentedtotheGraduateSchool oftheUniversityofFloridainPartialFulfillmentofthe RequirementsfortheDegreeofDoctorofPhilosophy DIRECTFORCEMEASUREMENTOFTHESPECIFICANDNON-SPECIFIC INTERACTIONSBETWEENASINGLEBACTERIUMANDSUBSTRATE By JonahDavidKlein December2002 Chair: RichardDickinson MajorDepartment: ChemicalEngineering Thelong-termuseofimplantedmedicaldevicesisimpededbybacterialinfection ontheirsurfaces.Whensuchdevicesareinsertedinthebody,plasmaproteinsadsorb rapidlyontothematerial,providingasubstratethatfacilitatesthebindingandsubsequent colonizationofvariousmicroorganismsincludingStaphylococcusaureus,acommon pathogenfoundinhospitals. Theattachmentofasinglebacteriumorsmallclustersof bacteriatobiomaterialsurfacesisthenecessaryandoftentherate-limitingstepinthe pathogenesisofadevicecenteredinfection. Becausemicrobesarepresentinalmost everyaqueousenvironment,adhesiontootherman-madesurfaces(water-purification filters,airconditioningcondensers,etc.)canleadtotroublesomeandcostlyproblemsfor avarietyofindustries. Aninvestigationofboththeroleofbothnon-specificforces(i.e.,electrostatic) andspecificinteractions(i.e.,pair-wisebondformation)inthepromotionofbacterial adhesionwasperformedthroughdirectforcemeasurements. Inparticular,theeffectof IX ) MSCRAMM(microbialsurfacecomponentrecognizingadhesivematrixmolecules) lengthonattachmentkineticswasinvestigated. Experimentswereperformedto1 measuretheinteractionforcesinvolvedinS.aureusattachmentand2)determinethe kineticrateconstantofS.aureustovarioussurfacesunderwell-definedflowconditions. TheseparameterswerecontrolledthroughtheuseofspecificallyengineeredS.aureus mutantsthatexpressthemembraneprotein“clumpingfactor” (ClfA)atvaryinglengths extendingoutfromthecellsurface. ThemeasurementofinteractionforcesanddynamicsofS.aureusattachmentwas performedusinganewtechniquethatmakestheusesevanescentwavelightscattering (EWLS)inconjunctionwith3-dimensionalopticaltrapping. Tovalidatethetechnique, measurementswerefirstperformedinsimplifiedsystems,wherebaresurfacesandlow electrolyteconditionsallowedfordirectcomparisonswithestablishedtheories. Further investigationsoftheeffectofMSCRAMMlengthonbindingforcesrevealedthelength scalesatwhichspecificinteractionscanactinphysiologicalconditionsaswellasthe magnitudeoftheforcesinvolved. Thecorrespondingmeasurementofkineticrate constantsofS.aureusattachmenttosurfaceswasmadeusingaparallel-plateflow chamberwiththeaidofautomatedvideo-microscopy,theresultsofwhichwere correlatedwithdirectforcemeasurements. x
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