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.

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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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