identifying chemical compounds targeting persister …
TRANSCRIPT
IDENTIFYINGCHEMICALCOMPOUNDSTARGETINGPERSISTERRELATEDMECHANISMSINBACTERIA
ASeniorHonorsThesis
PresentedtotheFacultyoftheDepartmentofChemicalandBiomolecularEngineering
UniversityofHouston
InPartialFulfillmentoftheRequirementsfortheDegree
BachelorofScienceInChemicalEngineering
ByThaoVyNguyen
May2019
IDENTIFYINGCHEMICALCOMPOUNDSTARGETINGPERSISTERRELATEDMECHANISMSINBACTERIA
_____________________________________________ThaoVyNguyen
Approved: _______________________________________________________ChairoftheCommitteeMehmetOrman,AssistantProfessor,DepartmentofChemical&BiomolecularEngineering
CommitteeMembers: _______________________________________________________RichardWillson,Huffington-WoestemeyerProfessor,DepartmentofChemical&BiomolecularEngineering
_______________________________________________________FrankClaydon,Professor,DepartmentofElectrical&ComputerEngineering
____________________________________SureshK.Khator,AssociateDean,CullenCollegeofEngineering
_______________________________________________________MichaelHarold,M.D.AndersonProfessorandChairofDepartmentinChemical&BiomolecularEngineering
iv
Acknowledgements
I gratefully acknowledge the opportunity to work with Dr. Orman as well as his
mentorship throughout the duration of the project. Dr. Orman helpedme to identify the
problem,designexperiments,andunderstandthepurposeandthesignificanceoftheproject.
IacknowledgeSayedMohiuddinforbeingmymentor.Hesignificantlycontributedto
the design as well as the implementation of my experiments. His experimental data is
indispensableindrawingtheconclusionoftheproject.
IwouldalsoliketothankDr.AsmussenfortheHonorsThesisworkshopsaswellas
herguidanceincompletingmySeniorHonorsThesisproject.
IDENTIFYINGCHEMICALCOMPOUNDSTARGETINGPERSISTERRELATEDMECHANISMSINBACTERIA
AnAbstractofa
SeniorHonorsThesisPresentedto
theFacultyoftheDepartmentofChemicalandBiochemicalEngineeringUniversityofHouston
InPartialFulfillmentoftheRequirementsfortheDegree
BachelorofScienceInChemicalEngineering
By
ThaoVyNguyenMay2019
vi
Abstract
Bacterialpersistersarerare,phenotypicvariantsthataretemporarilytoleranttohigh
concentrations of antibiotics. They are generally non-growing cells and are genetically
identical to their antibiotic-susceptible kin. These cells are an important health concern
becausetheyunderlietheproclivityoftherelapseofrecurrentinfections,andtheycanserve
asareservoirfromwhichdrug-resistancemutantscanemerge.Itisbelievedthatpersister
cellssurvivequinoloneantibiotictreatmentbyactivatingtheirDNArepairmechanisms,such
asrecA,whichisanessentialproteinfortherepairofthedamagedDNA.Ithasbeenfound
that impairment of the DNA repairmechanisms could significantly decrease persistence.
Therefore, it isdesirabletodiscovermedicinallyrelevantchemicalcompoundsthattarget
bacterialDNArepairmechanismstoserveasadjuvantstoenhanceantibioticeffect.Inthis
project,wedevelopedarapidandefficientmethodtoscreenforpotentialchemicalinhibitors.
FromthePhenotypeMicroArrayschemicallibraryfromBiologInc.(Hayward,CA),wewere
abletoidentifyanumberofFDA-approvedchemicalcompoundsthatcanserveasinhibitors
of the bacterial cells’ DNA repair mechanisms, thus eliminate persistence without
independentlyeradicatingthecellcultures.
vii
TableofContents
Acknowledgements........................................................................................................................................................ivAbstract...............................................................................................................................................................................viTableofContents..........................................................................................................................................................viiListsofFigures..............................................................................................................................................................viiiChapter1–Introduction..............................................................................................................................................1BacteriaPersistence............................................................................................................................................1Persisters’associatedhealthcareconcerns...............................................................................................2Persistercells’resuscitationmechanisms.................................................................................................2
Chapter2–MaterialsandMethods........................................................................................................................4BacterialStrains....................................................................................................................................................4ChemicalsandMedia..........................................................................................................................................4CultureConditions...............................................................................................................................................5GFPExpressionInvestigationofDifferentPromoters.........................................................................5ChemicalScreeningAssay................................................................................................................................5TestingPersisterLevelsinPMPlates..........................................................................................................6gfpExpressionatVariousConcentrationsofHitChemicalCompounds......................................6PersisterLevelDeterminationatVariousConcentrationsofHitChemicalCompounds......7PersisterAssay......................................................................................................................................................7StatisticalAnalysis...............................................................................................................................................8
Chapter3–Results..........................................................................................................................................................9ChemicalScreeningAssay................................................................................................................................9PersisterLevelsinPMPlates........................................................................................................................10AnalysisofIdentifiedChemicalHits..........................................................................................................11
Chapter4–Discussion................................................................................................................................................18Chapter5–Conclusion...............................................................................................................................................21References........................................................................................................................................................................22
viii
ListsofFigures
Figure1.BiphasickillingofexponentialphaseE.colicellstreatedwithofloxacin(5µg/mL).1Figure2.SOSResponseinE.colicellsafterofloxacintreatment.Cellsharboringplasmidswith
green fluorescent protein (GFP) under the control of indicated promoters weretreatedwithofloxacininearlystationaryphase(t=5h).GFPlevelsweremonitoredusingplatereader.Control:Emptyvector......................................................................................3
Figure 3. gfp expression of cell cultures after 4 hours of treatmentwith ofloxacin in thepresenceofchemicalcompound.Dataareaveragevalues±standarderrorsfromtwobiologicalreplicates..................................................................................................................................9
Figure 4. Number of surviving cells/ well after ofloxacin treatment in the presence ofchemicalinhibitor...................................................................................................................................11
Figure5.GFPlevelsatt=4handpersisterlevelsatt=20hafterofloxacintreatmentofcellculturesinthepresenceofamitriptylinehydrochlorideatdifferentconcentrations..........................................................................................................................................................................12
Figure6.GFPlevelsatt=4handpersisterlevelsatt=20hafterofloxacintreatmentofcellcultures in the presence of trifluoperazine dihydrochloride at differentconcentrations..........................................................................................................................................13
Figure7.GFPlevelsatt=4handpersisterlevelsatt=20hafterofloxacintreatmentofcellculturesinthepresenceofhexachloropheneatdifferentconcentrations....................13
Figure8.GFPlevelsatt=4handpersisterlevelsatt=20hafterofloxacintreatmentofcellculturesinthepresenceofthioridazinehydrochlorideatdifferentconcentrations.14
Figure9.GFPlevelsatt=4handpersisterlevelsatt=20hafterofloxacintreatmentofcellculturesinthepresenceofchlorpromazinehydrochlorideatdifferentconcentrations..........................................................................................................................................................................14
Figure10.GFPlevelsatt=4handpersisterlevelsatt=20hafterofloxacintreatmentofcellculturesinthepresenceofpentachlorophenolatdifferentconcentrations.................15
Figure11.GFPlevelsatt=4handpersisterlevelsatt=20hafterofloxacintreatmentofcellculturesinthepresenceofpotassiumtelluriteatdifferentconcentrations.................15
Figure12.GFPlevelsatt=4afterofloxacintreatmentofcellcultureswithindicatedpromotersinthepresenceofthechemicalcompoundsatdifferentconcentrations.......................17
Figure13.Chemicalstructuresoftricyclicantidepressantcompounds.(A)Amitriptyline.(B)Trifluoperazine.(C)Thioridazine.(D)Chlorpromazine........................................................19
Figure 14. Chemical structures of (A) Hexachlorophene. (B) Pentachlorophenol. (C)PotassiumTellurite................................................................................................................................19
1
Chapter1–Introduction
BacteriaPersistence
Bacterial persisters are rare phenotypic variants within a susceptible isogenic
bacterialpopulationthathavetheabilitytotoleratelethalconcentrationsofantibiotics(1).
Thissmallfractionofbacterialculturecanbedetectedinanysusceptibleculturebyabiphasic
antibiotickillcurve(Figure1).Therapidkillingsegmentofthecurverepresentsthedeathof
normalcells,whiletheslowerkillingregimeindicatesthepresenceofpersisters.Persisters
are able resumegrowthupon removal of antibiotics, giving rise tonewpopulationswith
antibioticsensitivitythatisindistinguishablefromthatoftheoriginalpopulation(2).This
characteristic distinguishes bacterial persisters from drug-resistant mutants, which are
capableofdividinginthepresenceofantibiotic(3).
Figure1.BiphasickillingofexponentialphaseE.colicellstreatedwithofloxacin(5µg/mL).
1.00E-07
1.00E-06
1.00E-05
1.00E-04
1.00E-03
1.00E-02
1.00E-01
1.00E+00
0 5 10 15 20
SurvivalFraction
TimeofTreatment(h)
Rapidkillingregime
Slowkillingregime
2
Persisters’associatedhealthcareconcerns
Recurrentinfectionsaccountfor65%ofhospital-treatedinfections(4-5),andinthe
UnitedStatesaloneitisestimatedthattheyareattributedtohalfamilliondeathsandcost
the healthcare system approximately $94 billion per year (6).Many of these healthcare-
associated infections involvebacterial biofilms,whichhave the tendency to relapseupon
conclusionofantibiotictreatments(7).Bacteriathatpopulatetheseinfectionsareusuallyas
sensitive as the original population to the antibiotic used, which suggests that bacterial
persisters,ratherthandrug-resistancemutants,aretoblamefortherecurringinfections(4,
8-12).Therefore, it isdesirabletodiscovernovelcompoundsthatcanpotentiallyserveas
adjuvantstoenhancethekillingofpersisters.
Persistercells’resuscitationmechanisms
ApreviousstudybyVolzingandBrynildsendemonstratedthatpersisterssufferthe
samelevelofdamageinducedbytheantibioticascellsthatsuccumbtothetreatment.They
further verified that persister survival critically depends on its ability to repair the DNA
damageduringtheposttreatmentrecoveryperiod,whichisaftertheremovalofantibiotic
(13). This bacterial DNA repair mechanisms are initiated and maintained by the SOS
response.
Whenquinoloneantibiotic,suchasofloxacin,isintroducedintothebacterialculture,
it damages the cells’ DNA (14). Bacterial cells respond to this damage by inducing the
expressionoftheSOSresponsegenes,namelyrecAorrecN,whichplayavarietyoffunctions
relatingtotheDNArepairmechanismsofthecells.Therefore,inhibitingtheSOSresponse
canimpairtheDNArepairmechanismsofthebacterialcells,thussignificantlydecreasingor
completelyeliminatingpersisterformation.
UsinggfpreporterconstructedforthepromotersoftheSOSresponsegenes(PrecA,
PrecN, PsulA, and PtisB),we demonstrated that early-stationary-phase cultures of E. coli
3
respondedtoofloxacindamagethroughtheinductionoftheseSOSresponsegenes(Figure
2).ExpressionofrecAdemonstratedthegreatestdegreeofinductionupontheadditionof
ofloxacin. RecA performs a variety of functions, such as catalyzing DNA strand exchange
reactionstorepairdoublestrandbreaks,andstimulatingLexArepressorself-cleavageand
dissociation,leadingtotheinductionofmanygenesassociatedwiththeSOSresponseand
DNAdamagerepair.
Figure2.SOSResponse inE. coli cellsafterofloxacin treatment.Cellsharboringplasmidswithgreen fluorescentprotein (GFP)under thecontrolof indicatedpromoterswere treatedwithofloxacin inearly stationaryphase(t=5h).GFPlevelsweremonitoredusingplatereader.Control:Emptyvector.
ThisprojectisbasedontheestablishedlinkbetweentheSOSresponseandpersister
physiology, seeking to identify medically relevant chemical compounds that target DNA
repairmechanisms,thusreducesoreliminatespersisterformation.
1.000
10.000
100.000
1000.000
5 6 7 8 9
GreenFluorescent
Time(h)
ControlPsulAPtisBPrecNPrecA
4
Chapter2–MaterialsandMethods
BacterialStrains
BacterialstrainsusedinthisprojectwerederivedfromE.coliMG1655.Strainswere
generatedwithkanamycinresistancegenes,andgreenfluorescentprotein(gfp)underthe
controloffourdifferentpromoters:PrecA,PrecN,PsulA,andPtisB.Anemptyvectorstrainwasalso
usedasacontrol.
ChemicalsandMedia
Inallexperiments,weuseddistilledwaterpurifiedusingThermoFisherDIwater
system. Unless noted, all chemicals were purchased from Fisher Scientific or VWR
International.LBmediumcontained5gofyeastextract,10goftryptone,and10gofsodium
chloridein1LofDIwater.LB-agarplatesweremadefrommediumcontaining40gofLB-
agar in 1 L of DIwater. Phosphate-buffered saline (PBS) solution purchased fromFisher
Scientificwasdiluted10timesinDIwaterbeforeuse.
ThechemicallibrarychosenwasthePhenotypeMicroArrays(PM)platesfromBiolog
Inc.(Hayward,CA),whichincludes15different96-wellplates(PM-11toPM-25)containing
360chemicalsofdifferentclassesat4differentconcentrations.
OfloxacinwaspurchasedfromVWRInternational.Kanamycinwaspurchasedfrom
Fisher Scientific. Amitriptyline hydrochloride, trifluoperazine dihydrochloride,
hexachlorophene, thioridazine hydrochloride, chlorpromazine hydrochloride,
pentachlorophenol, and potassium tellurite were purchased from VWR International. All
chemicalsweredissolvedinDIwater,exceptforofloxacin,whichwasdissolvedinverydilute
sodium hydroxide (NaOH) solution, and hexachlorophene and pentachlorophenol, which
weredissolvedindimethylsulfoxide(DMSO).Allchemicalsolutionsweresterilizedusing0.2
5
µmVWRSyringeFilters,exceptforthosecontainingDMSO.AllLBbrothandLBagarwere
sterilizedbyautoclaving.
Throughouttheexperiments,finalconcentrationof5µg/mLofloxacinwasused.For
selectionpurpose,finalconcentrationof50µg/mLofkanamycinwasused.
Culture Conditions
Overnight cultures were prepared in 14 mL Falcon tube containing LB broth,
inoculatedfroma25%glycerolcellsstockat-80oCandweregrownfor24hat37oCwith
shaking(250rpm)inthepresenceofkanamycin.After24h,maincultureswerepreparedby
inoculating25mLLBbrothina250mLbaffledflaskwith1000-folddilutionoftheprepared
overnightcultures,inthepresenceofkanamycin.Themaincultureswerethengrownfor5h
at37oCwithshaking(250rpm)toreachtheearlystationaryphase.
GFPExpressionInvestigationofDifferentPromoters
Overnightandmaincultureswerepreparedasdescribedabove,usingcellstocksof
E.coliMG1655emptyvector,PrecA,PrecN,PtisB,andPsulA,allwithinduciblegfpplasmid.Early
stationaryphasemainculturesweretreatedwithofloxacin,andtheGFPlevelofeachculture
wasmonitored every hour up to 4 hours after ofloxacin treatment, using Varioskan LUX
MultimodeMicroplateReader(purchasedfromThermoFisher).Themeasurementprocess
includedplating200µLofeachtreatedcellcultureinonewellona96-wellflatbottomplate,
withexcitationandemissionwavelengthsof485nmand511nm,respectively.
ChemicalScreeningAssay
Overnightandmaincultureswerepreparedasdescribedabove,usingcellstockofE.
coliMG1655PrecAwithinduciblegfpplasmid.Earlystationaryphasemaincultureat5hwas
treatedwithofloxacinandwasimmediatelytransferredtothe96-wellPMplates,witheach
well contained 100 µL of cell culture. The GFP level of each well was monitored using
6
VarioskanLUXMultimodeMicroplateReaderatt=0h(rightafterofloxacintreatment),2h,
4h,and20h,atexcitationandemissionwavelengthsof485nmand511nm,respectively.
TheplatewascoveredwithsterileBreath-Easysealingmembraneandincubatedat37oCwith
shaking(250rpm)inbetweenGFPmeasurements.
TestingPersisterLevelsinPMPlates
Basedonthegfplevelsdataatt=4h,thewellsthatmaintainedthegfpexpression
levelsovertimefromthePMplateswereidentified.After20hoftreatmentbyofloxacinthe
presence of an identified chemical compound, the samples from thewells containing the
identifiedchemicalhitswereobtained.Thesesamplesweretobewashedoncewith900µL
ofPBSbycentrifugationat13,300rpmfor3minutes,followedbytheremovalofsupernatant.
Ten-microliterswereusedfrom100µLofwashedsamplesforserialdilutionsin90µLofPBS.
TenmicrolitersperdilutionwerespottedonLBagarplatetoenumeratecolony-formingunit
(CFU).LBagarplateswereincubatedat37oCfor16h.
gfp Expression at Various Concentrations of Hit Chemical
Compounds
Overnightandmaincultureswerepreparedasdescribedabove,usingcellstocksof
E.coliMG1655emptyvector,PrecA,PrecN,PtisB,andPsulA,allwithinduciblegfpplasmid.Early
stationaryphasemaincultureat5hwastreatedwithofloxacin in the flaskandthenwas
immediatelytransferredto14mLFalcontubes,eachcontained2mLoftreatedcellculture.
Chemicalcompoundwasthenaddedatdifferentconcentrationsintoeachrespectivetube.
Theconcentrationsofthechemicalcompoundswerearbitrarilychosen,rangingfrom0.005
mMto4mM.Differentcontrolswereused,including:untreatedcellculturesintheabsence
of chemical compound, cell cultures treated with ofloxacin in the absence of chemical
compound,untreatedcellculturesinthepresenceofdifferentconcentrationsofthechemical
7
compound. Tomaintain consistency, the same amount of DI water/DMSO as that of the
chemicalcompoundwasaddedintothefirstandsecondcontroltubes.TheGFPlevelsofthese
cell cultures were then monitored at t = 0 h (right after the addition of ofloxacin and
chemical),2h,4h,and20hbyplating200µLofcellcultureextractedfromeachrespective
tubeinawellona96-wellflatbottomplate,usingtheVarioskanLUXMultimodeMicroplate
Reader with excitation and emission wavelengths of 485 nm and 511 nm, respectively.
DuringtheGFPlevelmonitoringprocess,allthetubeswereincubatedat37oCwithshaking
(250rpm).
Persister Level Determination at Various Concentrations of Hit
ChemicalCompounds
AftermonitoringGFPlevelexpressionofcellculturesintheFalcontubes,att=20h,
1mLofcellculturewasextractedfromeachtubeandwashedtwicewith1XPBSsolutionby
centrifugationat13,300rpmfor3minutes,followedbytheremovalofsupernatantandcell
resuspension in 1 mL of PBS. After the final centrifugation 900 µL of supernatant was
removedand100µLofconcentratedcellculturewasusedforserialdilutions,with10µLof
thewashedconcentratedcellculturesdilutedin90µLofPBS.Tenmicrolitersperdilution
werespottedonLBagarplatetoenumerateCFUs.LBagarplateswereincubatedat37oCfor
16h.
PersisterAssay
Overnight and main cultures of E. coli MG1655 WT and PrecA were prepared as
described above. Early stationary phase cultures at 5 h were treatedwith ofloxacin and
immediatelytransferredto14mLFalcontubeswith2mLoftreatedcells/tube.Ateachtime
point t = 0 h (right after the addition of ofloxacin), 2 h, 4 h, and20h, 1mL samplewas
extractedfromeachtubeandwashedtwicewithPBSbycentrifugationat13,300rpmfor3
8
minutes,followedbytheremovalofsupernatantandcellresuspensionin1mLofPBS.After
thefinalcentrifugation900µLofsupernatantwasremovedand100µLofconcentratedcell
culturewasused forserialdilutions,with10µLof thewashedconcentratedcell cultures
diluted in 90 µL of PBS. Ten-microliters per dilution were spotted on LB agar plate to
enumerateCFUs.LBagarplateswereincubatedat37oCfor16h.
StatisticalAnalysis
Atleastthreebiologicalreplicateswereperformedforeachexperiment.T-testswere
performedtodeterminethereproducibilityofthereplicates.
9
Chapter3–Results
E.coliMG1655PrecAwithinduciblegfpplasmidwasusedasaprimarytestingstrain
throughout the project because of its high level of GFP expression compared to other
promotersupontheintroductionofofloxacinintothecellculture(Figure2).
ChemicalScreeningAssay
Whenofloxacinisintroducedintotheearlystationaryphasecellculture,itdamages
thebacterialcells’DNA.BacterialcellsrespondtothisdamagebyinducingtherecAgene,the
generesponsible for therepairmechanismsofDNA,whichconsequently induces theGFP
expressionofthecells.ThisresultsinthecontinuallyincreasingGFPexpressionofthecell
cultureasafunctionoftime(Figure2).Thechemicalcompoundthatsuccessfullyinhibitsthe
bacterialcells’DNArepairmechanismsbyinhibitingtheinductionofrecAgenewillmaintain
theGFPexpressionlevelofthecultureaftertheintroductionofofloxacin.
Figure 3. gfp expression of cell cultures after 4 hours of treatment with ofloxacin in the presence of chemicalcompound.Dataareaveragevalues±standarderrorsfromtwobiologicalreplicates.
0.0000
50.0000
100.0000
150.0000
200.0000
250.0000
300.0000
350.0000
400.0000
0 50 100 150 200 250 300 350
GreenFluorescent
ChemicalCompound
10
Figure3showstheGFPexpressionlevelresultsof360differentcellculturesafter4
hours of treatment with ofloxacin, in the presence of the highest concentration of the
chemicalcompounds fromthePMplates.ThecellculturesthatmaintainedtheGFP levels
lowerthan50unitsareconsideredtobeundertheinfluenceofthechemicalcompoundsthat
successfully inhibit the cells’ DNA repairmechanisms. The result shows that within 360
different chemical compounds tested, about 130 compounds have the potential to be
chemicalinhibitors.
The data shown are average values with standard errors from two biological
replicates. Although cultures with high GFP expression levels showed great fluctuation
between the replicates, potential compounds that inhibited theGFPexpression showeda
moreconsistenttrend,indicatingreproducibleresults.
PersisterLevelsinPMPlates
After20hoursofculturing,thecellculturesfromthewellscontainingthepotential
chemicalinhibitorswereassayedtodeterminethepersisterlevels.
Aseparatesetofcontrolexperimentswasalsocarriedouttodeterminethesoleeffect
ofthechemicalinhibitorsonthecellcultures.Cellcultureswereassayedafter20hoursinthe
presencethechemicalinhibitorsbutwithoutthetreatmentofofloxacin.Thepurposeofthis
controlexperimentistoconfirmtheabilityofthepotentialcompoundsaschemicalinhibitors
ofDNArepairmechanismsinsteadoftheabilityasantibioticswhichcaneradicatethecell
culture.
11
Figure4.Numberofsurvivingcells/wellafterofloxacintreatmentinthepresenceofchemicalinhibitor.
Amongst 130 previously identified potential chemical inhibitors, only about 70
chemicalinhibitorsshowedreducedpersisterlevelscomparedtothecontrol(Figure4).The
additional screening control experiments further eliminated a large number of chemical
inhibitorsthatindependentlyeradicatedthecellcultures,leaving7chemicalcompoundsthat
satisfythefollowingconditions:
1. Successfully inhibit the GFP expression levels of cell cultures after the
introductionofofloxacin
2. Resultinsignificantlyreducedpersisterlevel
3. Incapableofindependentlyeradicatingthecellculture
AnalysisofIdentifiedChemicalHits
Chemicalhitsthatarefurthertestedareasfollows:
1. Amitriptylinehydrochloride
2. Trifluoperazinedihydrochloride
1.00E+00
1.00E+01
1.00E+02
1.00E+03
1.00E+04
1.00E+05
1.00E+06
1.00E+07
1.00E+08
1 6 11 16 21 26 31 36 41 46 51 56 61 66 71 76 81 86 91 96 101
106
111
116
121
126
131
CFU/well
ChemicalCompound
12
3. Hexachlorophene
4. Thioridazinehydrochloride
5. Chlorpromazinehydrochloride
6. Pentachlorophenol
7. Potassiumtellurite
DuetothelimitationthattheconcentrationsofthesechemicalinhibitorsonthePM
plateswerenotknown, theanalysiswascarriedoutatarbitrarilyandreasonablychosen
concentrations to identify theminimum inhibitory concentration (MIC) of each chemical
compound,rangingfrom0.005mMto4mMdependingonthecompound.
Figure5.GFPlevelsatt=4handpersisterlevelsatt=20hafterofloxacintreatmentofcellculturesinthepresenceofamitriptylinehydrochlorideatdifferentconcentrations.
1.0
10.0
100.0
1000.0
1.00E+00
1.00E+01
1.00E+02
1.00E+03
1.00E+04
0mM 0.1mM 0.25mM 0.5mM 1mM 2mM 4mMGreenFluorescent
CFU/mL
ChemicalConcentration
CFU
GreenFluorescent
13
Figure6.GFPlevelsatt=4handpersisterlevelsatt=20hafterofloxacintreatmentofcellculturesinthepresenceoftrifluoperazinedihydrochlorideatdifferentconcentrations.
Figure7.GFPlevelsatt=4handpersisterlevelsatt=20hafterofloxacintreatmentofcellculturesinthepresenceofhexachloropheneatdifferentconcentrations.
1.0
10.0
100.0
1000.0
1.00E+00
1.00E+01
1.00E+02
1.00E+03
1.00E+04
0mM 0.1mM 0.25mM 0.5mM 1mM 2mM 4mM
GreenFluorescent
CFU/mL
ChemicalConcentration
CFU
GreenFluorescent
1.0
10.0
100.0
1000.0
1.00E+00
1.00E+01
1.00E+02
1.00E+03
1.00E+04
1.00E+05
1.00E+06
0mM 0.001mM 0.005mM 0.1mM 0.5mM 1mM 2mM 4mM
GreenFluorescent
CFU/mL
ChemicalConcentration
CFUGreenFluorescent
14
Figure8.GFPlevelsatt=4handpersisterlevelsatt=20hafterofloxacintreatmentofcellculturesinthepresenceofthioridazinehydrochlorideatdifferentconcentrations.
Figure9.GFPlevelsatt=4handpersisterlevelsatt=20hafterofloxacintreatmentofcellculturesinthepresenceofchlorpromazinehydrochlorideatdifferentconcentrations.
1.0
10.0
100.0
1000.0
1.00E+00
1.00E+01
1.00E+02
1.00E+03
1.00E+04
0mM 0.1mM 0.25mM 0.5mM 1mM 2mM
GreenFluorescent
CFU/mL
ChemicalConcentration
CFUGreenFluorescent
1.0
10.0
100.0
1000.0
1.00E+00
1.00E+01
1.00E+02
1.00E+03
1.00E+04
0mM 0.001mM 0.005mM 0.1mM 0.5mM 1mM 2mM 4mM
GreenFluorescent
CFU/mL
ChemicalConcentration
CFUGreenFluorescent
15
Figure10.GFPlevelsatt=4handpersisterlevelsatt=20hafterofloxacintreatmentofcellculturesinthepresenceofpentachlorophenolatdifferentconcentrations.
Figure11.GFPlevelsatt=4handpersisterlevelsatt=20hafterofloxacintreatmentofcellculturesinthepresenceofpotassiumtelluriteatdifferentconcentrations.
1.0
10.0
100.0
1000.0
1.00E+00
1.00E+01
1.00E+02
1.00E+03
1.00E+04
0mM 0.1mM 0.25mM 0.5mM 1mM 2mM 4mM
GreenFluorescent
CFU/mL
ChemicalConcentration
CFU
GreenFluorescent
1.0
10.0
100.0
1000.0
1.00E+00
1.00E+01
1.00E+02
1.00E+03
1.00E+04
0mM
0.005mM
0.0075mM
0.01mM
0.05mM
0.1mM
0.5mM 1m
M2mM
4mM
GreenFluorescent
CFU/mL
ChemicalConcentration
CFU
GreenFluorescent
16
Figures 5 to 11 show the results of GFP level expression 4 hours after ofloxacin
treatmentandthenumberofcolony-formingunits20hoursafterofloxacintreatmentinthe
presenceofdifferentconcentrationsof7chosenchemicalinhibitors.Theresultsshowthat
all chemical compounds tested were able to maintain the GFP expression levels of the
ofloxacin-treated cell cultures and eradicate the cell cultures of persisters at certain
concentrations. A consistent trend can be observed from the graphs, such that low GFP
expression levels areassociatedwith reducedpersister levels,whilehighGFPexpression
levelsareassociatedwithcultureswithsignificantpersisterlevels.Thisdemonstratesand
strengthensourhypothesisonthelinkbetweenthebacterialcells’DNArepairmechanisms
andthepersisterlevelsinthecellcultures.
These7chemicalcompoundswerefurtheranalyzedbytestingwithbacterialcultures
withinduciblegfpplasmidunderthecontrolofdifferentpromoters,includingPrecN,PtisB,and
PsulA.Theconcentrationsusedfortheaforementionedchemicalcompoundswerechosento
bethelowestworkingconcentrations(varieddependingonthechemicalcompound)based
onthepreviousexperimentresultsonPrecAstrain.TheGFPexpressionlevelsofeachstrainin
thepresenceof thechemical compounds4hoursafter the treatmentwithofloxacinwere
showninthegraphsbelow.
17
Figure12.GFPlevelsatt=4afterofloxacintreatmentofcellcultureswithindicatedpromotersinthepresenceofthechemicalcompoundsatdifferentconcentrations.
Figure17indicatedthatthe7identifiedchemicalcompoundswerenotonlyableto
inhibittheinductionofrecA,butalsotheinductionofothergenesassociatedwiththeSOS
responseofbacterialcellstoDNAdamage,suchasrecN,tisB,andsulA.
0.0
50.0
100.0
150.0
200.0
250.0
300.0
350.0
NOChemical
1.0mMAmitritylineHCl
0.5mMTrifluoperazineDi-HCl
0.1mMHexachlorophene
0.25mMThioridazineHCl
0.25mMChloropromazineHCl
0.5mMPentachlorophenol
0.0075mMPotassiumTellurite
GreenFluorescent
PrecNPtisBPsulA
18
Chapter4–Discussion
Theriseofantibiotictoleranceisoneofthemostcriticalglobalpublichealththreats
ofthe21stcentury.Underscoringtheseverityoftheproblem,theWorldHealthOrganization,
with support from the United Nations, issued the Global Action Plan on Antimicrobial
Resistance(15).Bacterialpersistenceexacerbatesthisproblembyfacilitatingtherecurrence
ofchronicinfectionsandsupplyingareservoirfortheemergenceofdrug-resistantmutants.
Todate,allanalyzedbacterialspecieshavebeenfoundtoexhibitpersisterformation,atrates
rangingfrom0.001%to1%oftheentirecellpopulation.Althoughanti-persistertherapeutic
strategies can cure chronic and recurrent infections, they are hampered by limited
knowledgeofpersisterphysiology.
Effectivechemicalinhibitorsareusuallyassociatedwithhighleveloftoxicity,which
preventstheirusageonhuman.WechoosetoscreentheBiologPMchemicallibrarybecause
it contains many FDA-approved drugs and antibiotics. This quality gives the identified
chemical inhibitorssignificantadvantageintheapprovalprocessforhumanconsumption,
whichusuallytakestheFDAabout10years.
Within the 7 identified chemical inhibitors, 4 of them belong to the tricyclic
antidepressants class, including amitriptyline hydrochloride, trifluoperazine
dihydrochloride,thioridazinehydrochloride,andchlorpromazinehydrochloride(Figure13).
19
A. B. C. D.
Figure 13. Chemical structures of tricyclic antidepressant compounds. (A)Amitriptyline. (B) Trifluoperazine. (C)Thioridazine.(D)Chlorpromazine.
Figure13showedthechemicalstructuresofthetricyclicantidepressantcompounds
(TCAs),indicatingthesimilarityintheiroverallstructures.Thesecompoundswereshownto
inhibitthebacterialATPsynthaseactivity,whichmightnegativelyimpactproteinsynthesis.
A. B. C.
Figure14.Chemicalstructuresof(A)Hexachlorophene.(B)Pentachlorophenol.(C)PotassiumTellurite.
Figure14showed thechemical structuresofhexachlorophene,pentachlorophenol
andpotassiumtellurite.Hexachloropheneisknownforinhibitingthemembrane-boundpart
of the electron transport chain and respiratory D-lactate dehydrogenase, thus induces
leakage,causesprotoplastlysisandinhibitsrespiration.Hexachlorophenehasbeenusedas
themainingredientinsomeprescriptionproductssuchasPhisohexandSapoderm.Ithas
also been used to control the outbreak of gram-positive infection. Pentachlorophenol is
knownforitsbiochemicalactivityofuncouplingofoxidativephosphorylation,anditsability
20
to inhibit mitochondrial ATP-ase activity. Pentachlorophenol is one of the antimicrobial
compoundstobeusedasdisinfectantsorantisepticsinhealthcareinaneffortofloweringthe
riskofantibioticresistanceandcontributingtomoreeffectiveantibioticuse(16).Potassium
telluritehasinhibitoryactivityagainstmostgram-positiveandgram-negativebacteriadue
toitsabilitytoactasastrongoxidizingagent.
Chemicalinhibitorsthatbelongtothetricyclicantidepressantclasshavelongbeen
usedintreatingdepression;theirappropriatedosageshavebeendeterminedandthuscan
be taken orally. On the other hand, hexachlorophene, pentachlorophenol and potassium
telluritehavenotbeenwidelyusedasprescriptiondrugs.Furtherexperimentaltrialsneed
tobeperformedonthesechemicalinhibitorstodeterminetheirtoxicityandtheappropriate
dosagesthatcanbeusedonhuman,sothattheycanbeutilizedintreatinginfections.
21
Chapter5–Conclusion
Persistence poses a serious threat to the healthcare system and exacerbates the
problemofchronicandrecurrentinfections.Therehavebeenmanyhypothesesaswellas
therapeuticapproachesaroundtheproblemofpersister.Wehypothesizethatthepersister
cells’DNArepairmechanism,initiatedbytheactivationoftheSOSresponsegenessuchas
PrecA,allowsthecellstosurviveantibiotictreatment.Thishypothesisallowsustoscreenfor
potential chemical inhibitors that inhibit the activation of these SOS response genes,
consequently impair the DNA repair mechanisms and thus suppress the formation of
persisters.Thescreeningof theBiologPMchemical libraryresults in the identificationof
seven chemical inhibitors: amitriptyline hydrochloride, trifluoperazine dihydrochloride,
hexachlorophene, thioridazine hydrochloride, chlorpromazine hydrochloride,
pentachlorophenol,andpotassiumtellurite.Thesechemicalinhibitors,afterthreeroundsof
screening,demonstratetheirabilityininhibitingthebacterialcells’DNArepairmechanism,
thus eliminate persisters from the cell cultures without independent eradicate the cell
cultures.
Furtherexperimentaltrialsshouldbecarriedoutondifferentmicroorganisms,such
as Pseudomonas aeruginosa to confirm the reproducibility of the effects of the chemical
inhibitors,aswellastodetermineeachcompound’sleveloftoxicitybeforeutilizingthemin
treatinginfectionofhuman.
Bydevelopingarapidandefficientmethodofscreeningforthechemicalinhibitors
utilizingtheinductionofGFPexpressionunderthecontroloftheSOSresponsegenes,wecan
screenotherchemicallibrariesformorechemicalinhibitorsthatcanpotentiallyaidinthe
waragainsthealthcare-associatedinfections.
22
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