Direct force measurements of specific and non-specific interactions between a single bacterium and substrate

132 Pages·2002·5.1 MB·English

by  Klein, Jonah David, 1974-

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.
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