Missouri State Masters & Speciailst Programs in Educational Administration -- Joplin Cohort
Annual Joint Meeting of the Missouri and Missouri Valley ... · Ph.D., Masters, and undergraduate...
Transcript of Annual Joint Meeting of the Missouri and Missouri Valley ... · Ph.D., Masters, and undergraduate...
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Annual Joint Meeting of the Missouri and Missouri Valley Branches of the American Society of Microbiology
March 9-10, 2018
Hosted by
MissouriValleyBranch
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Missouri Branch Officers
President(through6/30/19)
Lea M. Daniels Email: [email protected] President-Elect: TBD Councilor (through 6/30/19) Anna Oller University of Central Missouri Email: [email protected] Meeting Coordinator Curtis V. Smith Ph.D. Professor of Biological Sciences Interim Dean of Mathematics, Science, Business, and Technology Kansas City Kansas Community College 7250 State Avenue Kansas City, Kansas 66110 Office Phone 913-288-7314 Fax 913-288-7677 Administrative Assistant's 913-288-7518; or .7627
MissouriValleyBranchOfficers
President(through5/31/19)
JulieShafferUniversityofNebraskaatKearneyDepartmentofBiologyBHS335b240111thAveKearney,[email protected]
President-Elect(through5/31/19)
TravisBourretMedMicro,CrissII,Rm514CreightonUniversity2500CaliforniaPlazaOmaha,NE,[email protected]
Secretary-Treasurer(through5/31/19)
DonaldRowenUniversityofNebraska,OmahaDepartmentofBiology6001DodgeStreetOmaha,NE68164(402)554-2143FAX:(402)[email protected]
Councilor
FranklinR.Champlin,Ph.D.OklahomaStateUniversityCenterforHealthSciencesDepartmentofBiochemistryandMicrobiology1111West17thStreetTulsa,[email protected]
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A virtual map can be found at http://www.kckcc.edu/explore-kckcc/campus/maps-directions/map
Take State Ave to Campus Blvd (just west of 72nd and State). Follow to Quindaro Ln. Parking between Argentine Ln and Armourdale Ln. Enter the Henry Louis Social Science Building. Go downstairs and to the back which takes you into the lower level of Jewell.
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Keynote Speakers
Dr.MelanieR.Mormile,AssociateProvostatMissouriUniversityofScienceandTechnology,ASMDistinguishedLecturerDr.Mormile’sspecialtyiswiththemetaboliccapabilitiesofthemembersoftheOrderHalanaerobialesandtheirpotentialbiotechnologicalapplications.Shehasresearched:1)theabilityofahaloalkaliphilicbacteriumisolatedfromSoapLake,WashingtontogenerateelectricityatpH11.0and7%salinity:2)AstreamlinedstrategyforbiohydrogenproductionwithHalanaerobiumhydrogeniformans,analkaliphilicbacterium;3)activity-basedmetagenomicscreeningandbiochemicalcharacterizationofbovinerumenprotozoanglycosylhydrolases;4)activity-basedmetagenomicsscreeningandbiochemicalcharacterizationofbovineruminalprotozoanglycosidehydrolases;and5)characterizationofamoderatelyhalo-acidophilicbacteriumisolatedfromLakeBrown,WesternAustralia;and6)theimpactofelevatedCO2concentrationsoncarbonatemineralprecipitationabilityofsulfate-reducingbacteriaandimplicationsforCO2sequestration.Shehas3patents:1)conversionofglycerolto1,3propanediolunderhaloalkalineconditions;2)acombinedfossilfreeprocessoflignocellulosispretreatmentwithbiologicalproduction;and3)fossilfuel-freeprocessofLignocellulosicPretreatmentwithBiologicalHydrogenProduction.
Dr.KarlKlose,PhD,Professor,UniveristyofTexasatSanAntonio,ASMDistinguishedLecturerDr.KarlKlosereceivedhisPh.D.inMicrobiologyatUCBerkeley,andperformedpostdoctoralstudiesatHarvardMedicalSchool.HewasthenhiredasafacultymemberattheUniversityofTexasHealthScienceCenterinSanAntonio(UTHSCSA),andlatermovedtotheUniversityofTexasatSanAntonio(UTSA),andisthefounderoftheSouthTexasCenterforEmergingInfectiousDiseases.Klose’sresearchfocusesonunderstandingbacterialpathogenesis.HislaboratorystudiesVibriocholeraeandthepotentialbioweaponFrancisellatularensis.Kloseisanauthoronmorethan90publications,andhasreceivedfundingfromnumeroussources.HehasmentoredmanyPh.D.,Masters,andundergraduatestudents.HeservedasthePresidentoftheTexasBranchoftheAmericanSocietyforMicrobiology(2001-2003),andhasbeenanorganizerofmultiplenationalandinternationalmeetings.HehastwicebeenarecipientofASMVisitingProfessorships,inKolkata,IndiaandinValparaiso,Chile.Hereceivedthe2002PresidentialJuniorResearchScholarawardatUTHSCSAandthe2009President’sDistinguishedResearchAchievementAwardatUTSA.KlosehasgivenaTEDxtalkonantibiotic-resistantbacteriathatisavailableon
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YouTubeandthathasreceivedover40,000views(https://www.youtube.com/results?search_query=karl+klose).KlosewasrecentlyelectedafellowoftheAmericanAcademyofMicrobiology.FeaturedBranchSpeakers
Dr.HeatherSeitz,Ph.D.AssociateProfessorofScience,JohnsonCountyCommunityCollegePULSELeadershipFellow-MidwestGreatPlainsRegionalNetworkDr.SeitzisthefirstauthorandleaderofthetaskforcethatcreatedtheMicrobiologyforHealthSciencesConceptInventory(MHSCI)andhasworkedcloselytoanalyzedthedataonstudentmisconceptions.Shehasdatatosharefromover3,000studentsat15institutionsintheUnitedStates.Helpingtouncoverstudentmisconceptionsisincrediblyimportantinimprovingunderstanding.Validatedandreliableconceptinventoriesaredesignedtouncoverstudentmisconceptions.Recently,theAmericanSocietyofMicrobiologysupportedthedevelopmentoftwoconceptinventoriesthatwillbediscussed:theMicrobiologyConceptInventoryandtheMicrobiologyforHealthSciencesConceptInventory.TheconceptinventoriesthathaverecentlybeenpublishedarealignedwiththeASMCurriculumGuidelines.Thedatafromthedevelopmentoftheseconceptinventorieswasusedtohelpuncoverthemostcommonmisconceptionsinundergraduatemicrobiology.Theprocessusedtocreatetheconceptinventoriesandnationaltrendsinthedatacollectedwillbehighlighted.Examplesofhowtheconceptinventoriescanbeimplementedintocoursedesign,assessment,andfacultydevelopmentwillalsobeshared.
Dr.MarkHoffman,Ph.D.,servesastheChiefResearchInformationofficerforChildren’sMercyandtheChildren’sResearchInstitute.Dr.Hoffman’sroleistoaccelerateandimprovealltypesofresearchattheChildren’sResearchInstitutethroughsuchasdata,applicationsandtechnology.In2013,hejoinedthefacultyattheUniversityofMissouriatKansasCity(UMKC)intheDepartmentsofBiomedicalandHealthInformaticsandPediatrics.AtUMKChelaunchedheCenterforHealthInsightsandbroughtnewInformaticscapabilitiestotheUniversity,IncludingREDCap,i2b2andtheCernerHealthFactsdata.Duringhis16yearsatCernerand3yearsatUMKChehadtheopportunitytomeetawidevarietyofclinicalresearchorganizationsaroundtheworld,learningabouttheirsuccessesandchallenges.In2016Dr.HoffmanjoinedChildren’sMercywhereheistheprimaryinvestigatoronaCDCgranttoutilizeclinicalandlaboratorydatawarehousestoinformqualityimprovementandhecontinuestoserveonthefacultyatUMKC.HehasdeliveredaTEDtalkonthe“Envrome”andwonthe
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iThermometercategoryintheGooglewearabledevicesinhealthcarechallengein2015.Heisaninventorof19issuedpatentsandamemberoftheAmericanAcademyofInventors.
Dr.ErikaLutter,PhD,AssistantProfessor,OklahomaStateUniversityMylabisinterestedinmechanismsofhost-cellexit,transmissionandmolecularpathogenesisoftheobligate-intracellularpathogenC.trachomatis.C.trachomatiscausesinfectionsthathavesignificantglobalmedicalandeconomicimpact.Itistheleadingcauseofinfectiousblindness(trachoma)worldwideandthemostreportedbacterialsexuallytransmittedinfectionintheUnitedStates.Asanobligateintracellularpathogenwithacomplexbi-phasicdevelopmentallifecycle,thewaysinwhichC.trachomatisutilizeshostsignallingprocessesforsurvivalandtransmissionarepoorlyunderstood.Untilrecently,C.trachomatiswasgeneticallyintractablebutthelatestadventoftransformationmethodshasenabledthedevelopmentofgenetictoolstomutate,complementandexpressgeneticconstructswithinC.trachomatis.OurresearchusesacombinationofthesenewlydevelopedgenetictoolsandcellularbiologytoaddressmygoalofdecipheringtheinteractionofChlamydialproteinsandmyosinphosphataseandtheirroleinhost-cellexitandthetransmissionofC.trachomatis.
Dr.AnnaSelmecki,PhD,AssistantProfessor,CreightonUniversityMyresearchisfocusedonunderstandingthemechanismsthatcontrolgenomestability.MylabatCreightonUniversityMedicalSchoolusesexperimentalevolutionandcomparativegenomicstoidentifymutationsthataltergenomestabilityandgeneexpressioninbothpathogenicandnon-pathogenicmicrobes.Weusemolecularbiology,biochemistryandfluorescentmicroscopytodeterminethemechanisticroleofnovelgenomestabilizingmutations.Ourexperimentsaddressthegeneticandenvironmentalcausesofincreasedratesofchromosomeaneuploidyinyeast.Finally,wedevelopmathematicalmodelstounderstandhowgenomestabilityimpactstheadaptationatthecellularandpopulationleveloverdifferenttimescales.Myvisionisthattheseapproacheswillgenerateamorecomprehensiveandmechanisticunderstandingofgenomestabilityanditsroleinadaptationthanhasbeenpossiblewithtraditionalgeneticapproaches.
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ScheduleMarch95:00-5:15p.m. Welcome LowerLevelJewell5:15-5:45p.m. HeatherSeitz,JCCC—evaluatingnationaldataon
studentmisconceptionsinmicrobiology
5:45-6:15p.m. MarkHoffman,—Medicaldatawarehousestoinformqualityimprovements
6:30-7:30p.m. Dinner Adjacentdiningroom7:30-8:45p.m. Keynotespeaker:MelanieMormile—SoapLake:
MicrobialStudiesfromaSaltyAlkalineLake
8:45-9:00p.m. Closingannouncements March108:00-9:30a.m. PosterSessionI(Undergraduates) LowerLevelJewell9:30-11:00a.m. OralPresentationsI
3roomswithpresentationsGeneralSession1,Room2325Medical,Room2326UndergraduateSession1,Room2335
11:00a.m.-12:00p.m.
OralSessionII3roomswithpresentations
GeneralSession2,Room2325UndergraduateSession2,Room2326UndergraduateSession3,Room2335
12:00-1:30p.m. LunchandKeynotespeaker:KarlKlose—KillerBunniesandBioweapons:TularemiaandBiodefense
LowerLevelDiningRoom
1:30-2:00p.m. StudentsmeetwithKeynotespeakers Room23252:00-3:00p.m. PostersessionII
(GraduateStudents)LowerLevelJewell
3:00-3:30p.m. ErikaLutter,OklahomaStateUniversity--Chlamydia
3:30-4:00p.m. AnnaSelmecki,CreightonUniversityMedicalCenter—yeastevolution
4:00-4:15p.m. Break 4:15-4:45p.m. BranchBusiness Missouri—Room2325
MissouriValley—Room23264:45-5:00p.m. AwardsandClosingremarks LowerLevelJewell
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GeneralMicrobiologyGraduateStudentOralPresentationsSession1:Jewell23259:30a.m. NirakarAdhikari D-motifMutationincAMPPhosphodiesterase,RegA,Leadsto
DevelopmentalPhenotypeDefectinDictyosteliumdiscoideum9:45a.m. BirajKayastha TheRoleofβ-carbonicanhydrasesinCalciumDepositionby
Pseudomonasaeruginosa10:00a.m. JorgeLightfoot MetabolicallyEngineeringAspergillusnidulansusingRNA
Interference10:15a.m. NickKuburich InvestigatingaPhosphodiesteraseInvolvedinAmoeboid
MulticellularDevelopmentandSignalingSession2:Jewell232511:00a.m. MarielDegadoCruz ExploringthePotentialRoleofVps1asaFusionProtein11:15a.m. M.M.King CarPisaCa2+RegulatedPutativePhytasethatisUniqueto
PseudomonadsandisHighlyConservedinClinicalIsolates11:30a.m. JaredSmothers MyosinmediatesproteinrecyclingtowardtheGolgi11:45a.m. WesShort SearchingforRecruitmentDomainsandResiduesofVps1toTarget
MembraneMedicalMicrobiology/ImmunologyGraduateStudentOralPresentationsSession1:Jewell2326
9:30a.m. AbbyRigsbee EffectoftheOuterMembranePermeabilizerCompound48/80onIntrinsicResistancetotheHydrophobicBiocideTriclosaninOpportunisticSerratiaSpecies
9:45a.m. JeffShaw ThreeTransketolasesinSalmonellaentericaContributetoDefendingAgainstOxidativeandNitrosativeStresses
10:00a.m. RobertTodd SegmentalAneuploidiesFlankedbyInvertedRepeatsCauseAzoleResistanceintheFungalPathogenCandidaalbicans
10:15a.m. AmandaZalud ROS/RNSInducedChangesinBorreliaburgdorferiOuterSurfaceProteins
10:30a.m. MacyOlson DeterminationoftheRoleofChlamydialInclusionMembraneProteinsinInclusionGrowthandDevelopmentbyProximityLabelingofInteractingPartners
10:45a.m. WilliamBoyle TheBorreliaburgdorferibb0168-EncodedDnaKSuppressorEnhancespHDependentLipoproteinExpression
UndergraduateorHighSchoolOralPresentationsSession1:Jewell2335
9:30a.m. LeaKafer UtilizingGalleriamellonellatoDeterminetheRoleofCalciuminPseudomonasaeruginosaVirulence
9:45a.m. AlisonGuyer RoleofcentromereheterogeneityinclinicalisolatesofCandidaalbicans
10:00a.m. NathanielHigdon AnalyzingProteinInteractionsofTheHerpesSimplexType1UL34Protein
10:15a.m. KeeganMcGill Determining the Localization of the Hypothetical Membrane ProteinCBU_1651fromCoxiellaburnetii
10:30a.m. SamKoshy Characterizationoftheroleofredox-activecysteinesintheregulatoryfunctionofDksAintheLymediseasespirocheteBorreliaburgdorferi
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Session2:Jewell232611:00a.m. AmburD.Robertson IdentifyingIntracellularTick-borneIllnessesinBison,Bisonbison,via
BloodCellStainingandPCR11:15a.m. DanielMcLeod
GeneratingDeletionandPointMutationstoStudyaNovelPhytase-likeProtein,CarP,ThatProtectsPseudomonasaeruginosafromElevatedCalcium
11:30a.m. GeorgieO.Tauber SoilMicrobeIsolationSurroundingNativeandInvasiveGrassestoTestforAntimicrobialPropertiesagainstE.S.K.A.P.E.relatives
11:45a.m. MakaylaNemecek LongevityAnalysisofGerm-freeDrosophilamelanogasterSession3:Jewell2335
11:00a.m. CullenHorstmann SilverNanoparticlesonYeastViabilitywithBioinformaticsAnalysis11:15a.m. RyanWindish SiteDirectedMutagenesisofKnownVps1UbiquitinationSites11:30a.m. NicholasWood InitialCharacterizationoftheTwoClpPIsoformsofChlamydia
trachomatisSuggestsIndependentFunctionalityforEach11:45a.m. ChristopherJohnson
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Abstracts PosterSession1(UndergraduateStudents)1. ImpactofFluconazoleonGenomeCopyNumberChangesinCandidaalbicans.AnnBraverman
(Undergraduate)*,RobertTodd,AnnaSelmecki.CreightonUniversityMedicalSchool,Omaha,Nebraska
Candidaalbicansisanopportunisticfungalpathogenthatcausesdebilitatinginfectionsinimmune-compromisedindividuals.Fluconazole,afungistaticanti-fungaldrug,isthemostcommonlyprescribedtreatmentforCandidiasisduetoitsrapidbio-availabilityandlowoccurrenceofsideeffects.However,duetothefungistaticnatureandcommon,widespreaduse,fluconazoleresistancehasbecomeamajorthreattopublichealth.Onemechanismassociatedwithantifungaldrugresistanceistheamplificationofimportantdrug-responsegenes.Thisincreaseingenecopynumbercanariseduetoabnormalgenomereplicationorsegregation,andmaycausechangesinwholegenomecopynumber(ploidy).Infungi,significantfitnessincreasescanbeattributedtoploidyincreases(Selmeckietal.2015;Gersteinetal.,2009;Adamsetal.1974).Here,wehaveoptimizedaflowcytometrybasedassaytorapidlydetectploidychangesinyeast-formfungiwhenexposedtofluconazole.Ourploidyassaycoupledwithanin-vitroevolutionexperimentalsystemallowsustoaddresshowfluconazoleexposurealtersgenomestability.2. SegmentalAneuploidiesFlankedbyInvertedRepeatsCauseAzoleResistanceintheFungalPathogen
Candidaalbicans.RobertTodd,TylerWikoff(undergraduate)*,ShilpaNair(undergraduate)*,CurtisFocht,RobertThomas,AnnaSelmecki.CreightonUniversitySchoolofMedicine,Omaha,Nebraska.
Candidaalbicansisthemostprevalentfungalpathogeninimmunocompromisedindividuals.Currently,treatmentofCandidainfectionsreliesheavilyonazoles,afamilyoffungistaticdrugsthatdisruptthebiosynthesisofthefungal-specificsterol,ergosterol.Aneuploidy,amplificationorlossofachromosome,isacommongenomicfeaturefoundin50%ofazole-resistantC.albicans.PreviousworkfromtheSelmeckiLabhasshownthataspecificsegmentalaneuploidy,anamplificationoftheleftarmofchromosome5,canconferazoleresistanceduetotheamplificationoftwodrug-responsegenes.Currently,littleisknownabouthowsegmentalaneuploidiesform.Usingnext-generationsequencingtechnology,chromosomekaryotyping,andlong-rangeDNAsequencingwedescribeseveralnovelsegmentalaneuploidiesfoundinazoleresistantCandidastrainsfromdiversegeneticbackgrounds.Thesesegmentalaneuploidiesconsistofamplifiedregionsofthegenome,someofwhichcanundergocopynumberincreasesofgreaterthan12copies.Theseamplificationscontainimportantdrugresistancegenesandcorrelatewithasignificantincreaseinazoleminimalinhibitoryconcentration.Here,wedescribethesenovelsegmentalaneuploidiesthatconferazole-resistanceandidentifygeneticfeaturesthatelucidateacommonmechanismofformation.3. TheEffectofGarliconEscherichiacoliandStaphylococcusaureus.AmberMason(undergraduate)*Kansas
CityKansasCommunityCollege,KansasCity,Kansas.Theactiveingredientingarlicthatinhibitsbacteriahasbeenshowntobeallicin.Theonlywaythesesubstanceworkisintherawuncookedformofgarlic,orgarlicextractasusedintheseexperiments.Concentrationsof1ml,1.5ml,and2mlwereusedtoinvestigatetheinhibitoryeffectsonE.coliandStaphaureus.Bothmicrobeswerestreakedonseparateplatesforconfluentgrowthandinhibitoryeffectsrecordedafter48hoursincubation.TheresultsdemonstratedthatE.coliwasinhibitedmoreeffectivelywithincreasingconcentrationsthanStaphaureuswhereafewresistantcolonieswereevident.
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4. TheRoleofApigenininTreatingLeukemia.RemingtonWilkerson(undergraduate)*,KansasCityKansasCommunityCollege,KansasCity,Kansas.
Apigenin-4’,5,6trihydroxyflavone,aninitiatorofcaspase-9,hasbeenshowntoinduceleukemiacellapoptosis.Itisfoundinrelativelylargequantitiesincelery,parsley,cilantro,oranges,onion,andchamomiletea.Chemicallyengineeredsupplementshavebeendevelopedfromthechamomileplant.Apigenindemonstratesantioxidantbenefitsthathelpmetabolizecelldamagingfreeradicalsanddisruptionofmetastasizingtumorcellsingeneral.Apigeninhasalsobeenshowntohaveanti-inflammatorypropertiesthathelpreduceinflammationandautoimmunediseases.ThisisareviewoftheliteratureposterthatfindsevidencefromtheNationalCancerInstituteandelsewhereshowingthatapigenincanprolongthelifeofleukemiapatients.5. GeneticKnockdownSystemforChlamydiatrachomatis.EmilyGietzen,PrakashSahandErikaLutter,
OklahomaStateUniversity,StillwaterOKChlamydiatrachomatisisanobligateintracellularpathogenthatiscommonlysexuallytransmittedamonghumans.UntilrecentlyChlamydiahasbeengeneticallyintractable,therebylimitinggeneticapproaches.However,recentdevelopmentshaveallowedforthedevelopmentofnovelgenetictoolswhichcanbeusedtomutatespecificgenes.Unfortunately,geneinactivationbytargetronorantibioticcassetteinsertioncanresultinpolareffectsofneighboringgenesmakingitdifficulttostudythegeneswithinoperons.ThisstudyfocusesondevelopinganovelknockdownstrategybyexpressingthereversecomplementspecificChlamydiagenesonashuttleplasmid.Oncecloned,theplasmidsaretransformedbackintoChlamydiaandthegenesexpressedintranswillbetranscribedandbindtheRNAofthecorrespondinggeneproducingdoublestrandedRNAwhichisdegraded.Thismethodwillallowustolookatindividualgenesinanoperonwithoutthepolareffectsofmutations.Thisstrategyisbeingusedonanoperoncontaining6genes.Afterthereversecomplementofeachgeneisexpressed,thedecreasedexpressionofthetargetgenewillbeassessedbyreversetranscriptionPCR.TheseexperimentswillbethefirsttoutilizeagenespecificknockdownstrategyinChlamydia.6. CharacterizationElizabethkingiaursingiiMutantsExpressingElevatedVancomycinResistance.BradenLanier
(undergraduate)*,WilliamL.Johnson,andJohnE.Gustafson.OklahomaStateUniversityDepartmentofBiochemistryandMolecularBiology,Stillwater,Oklahoma.
TheGram-negativeElizabethkingiabacterialgenusexhibitclinically-relevantintrinsicmultipleantibioticresistance.ClinicalreportssuggestthatthepeptidoglycanbiosynthesistargetingantibioticvancomycinwhichiscommonlyusedtotreatGram-positiveinfections,canalsobeusedtotreatElizabethkingiainfections.Inthisstudy,weinitiallydeterminedthevancomycinMICsofsixgenomically-characterizedElizabethkingiaspeciesfollowingCLSIguidelines,whichrangedfrom2to64mg/L.TheseMICssuggestthatallElizabethkingiaspeciesarevancomycin-resistant,andtherefore,refractorytothebactericidalandgrowthinhibitoryactionsofthisdrug.ElizabethkingiaursingiidemonstratedthelowestMIC(2mg/L)whileElizabethkingiameningosepticademonstratedthehighestMIC(64mg/L).WenextisolatedsingleE.ursingiicoloniesthatsurvivedonheartinfusionagarsupplementedwithvancomycin(12,14,16,18and20mg/L)andnotedthatthenumberofsurvivingcolonieswasreducedasthevancomycinconcentrationwasincreased.ThefiveisolatesselectedoffthevancomycincontainingHIAplatesdisplayedMICsrangingfrom32to256mg/L,whicharemuchhigherMICsthanthatexhibitedbytheparentstrain.TheseresultssuggestthatElizabethkingiacanacquireelevatedvancomycinresistancefollowingbriefvancomycinexposure.ThesefindingsdonotsupportstudiesthatsuggestvancomycintherapyiseffectiveagainstElizabethkingiainfections.
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7. DrosophilamelanogasterNoravirusORF1ProteinisLocalizedtotheNucleus.LarissaK.Attema(undergraduate)*,AlexisM.Page,BrandonE.Luedtke,BradL.EricsonandKimberlyA.Carlson.DepartmentofBiology,UniversityofNebraskaatKearney,Kearney,Nebraska.
Noravirusisapicorna-likevirusthatistransmittedviathefecal-oralroute.Thegenomeoftheviruscontainsfouropenreadingframes(ORFs)knownasORF1,ORF2,ORF3,andORF4. ORF1wasthefocus inthisstudy,and it isbelievedtoencodeanRNAiinhibitor.WhenweperformedasequenceanalysisofORF1,wediscoveredaputativebipartitenuclearlocalizationsignal(NLS),whichisasequenceofaminoacidsthatdirectsthetransportofproteinsintothenucleiofcells.ThegoalofthisprojectwastoverifythatthisNLSwastransportingORF1intothenucleusofthe cell. We created anORF1-GFP construct, as well as a mutant construct that deleted the NLS from ORF,transfectedtheseintoS2cells,andobservedusingflorescentmicroscopy.Theresultssuggestnuclearlocalization,astheORF1-GFPstainingoverlapswithDAPIstaininginnucleioftheS2cells,andthemutantconstructshowedGFPstaininginthecytoplasmwithnooverlapwithDAPIinthenuclei.Toourknowledge,thisisthefirstexampleofanRNAvirusthatspecifiesanRNAiinhibitorthattranslocatestothenucleus.8. THEMASSEMERGENCEOF17-YEARCICADAACCELERATESLITTERDECOMPOSITION.Ismert,K.,Reazin,C.,
Morris,S.,Jumpponen,A.,Sikes,B.,Zeglin,L.1KansasStateUniversity,Manhattan,KS(KPBS)2UniversityofKansas,Lawrence,KS(KU)
Magicicadaseptendecimaccumulatenutrientsduringtheirfeedingandprovideanutrientpulseastheyemergetomate.Tounderstandhowthematingandsubsequentmassivedepositofcicadaimpactlitterdecompositioninsoil,weconductedalitterbagexperimentintwofieldsitesinthestateofKansas.Wetargetedburoak(QuercusmacrocarpaMichx.)andhackberry(CeltisoccidentalisL.)leaflitter,withandwithoutcicadacarcasses.Atbothsites,wecomparedlitterbagswith10gofhackberryorburoaklitterwithorwithoutanadditional2gofcicada.The30µmmeshlitterbagswereincubatedinsituandsampledatthetimeofdeployment(T0),onemonth(T1),twomonths(T2),fourmonths(T3),orsix(KPBS)andtwelve(KU)monthslater(T4).Ourdatashowthat(i)bothlittertypesdecomposedfasterwithcicadaand(ii)althoughtherecalcitrantlitterdecomposedfasterinthecicada-amendedtreatment,itstillretainedgreaterproportionoftheoriginalmassatT4thantheeasilydecomposablelitter.Theseresultshighlightnutrientpulsesfrominsectpopulationdynamicsmayalternutrientcyclinganddecomposition.Analysesoftheseexperimentscontinueandadditionaleffortsaimtodissectbacterialandfungalcommunitiesandtheirdynamicsovertime.9. UsingInversePCRandSequencingtoIdentifyGenesinPseudomonasaeruginosathatContributeto
SusceptibilitytotheAntimicrobialPeptideDASamp2.MatthewM.Froid(Undergraduate)*,DonaldRowen,PhD.UniversityofNebraska-Omaha,DepartmentofBiology,OmahaNebraska
Increasedresistancetoconventionalantibioticsismakingthetreatmentofbacterialinfectionsmoredifficult.Duetothisdilemma,newalternativestotraditionalantibioticsarecurrentlybeingsought.DASamp2isapromisingnewantimicrobialpeptidethathasbeenexperimentallyshowntobeeffectiveagainstbothGrampositiveandnegativebacteria.InanefforttodeterminethetargetofDASamp2andtodeterminetheeaseatwhichbacteriacandevelopresistancetothiscompound,wepreviouslyisolatedmutantPsuedomonasaeruginosastrainswithshowedincreasedresistancetoDASamp2.Ideterminedthatmutationinonemutant,(RMB1)waslocatedinthegenemexTbyusinginversePCRandsequencing.Thegene,mexT,isbelievedtoencodeatranscriptionalregulatorinvolvedintheproductionofextracellulareffluxpumps.However,theregulationoftheproductionofeffluxpumpsinP.aeruginosaiscomplexandpoorlyunderstood.FurthercharacterizationofthisgeneregulatorynetworkisvaluableduetothelargeincreaseinresistancetoDASamp2(8foldorhigher)seenintheRMB1strain.SuchasignificantincreaseinresistanceishighlysuggestivethattheeffluxpumpsregulatedbymexTplayaroleindeterminingthesensitivityofP.aeruginosacellstoDASamp2.
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10. IsolationandCharacterizationofHalo-AcidophilicMicroorganismsfromLakeGneiss,WesternAustralia.AshleySegobiano(Undergraduate)*,MelanieR.Mormile,andSarahStewartJohnson.MissouriUniversityofScienceandTechnology,Rolla,Mo.
WesternAustralialakes,locatedintheYilgronCraton,maybethebestterrestrialanaloguetopreviousMartianconditionsastherearemanygeochemicalsimilarities.Wehypothesizethathaloacidophilicbacteriaresideintheselakes.LakeGneissisamongthemostextremeoftheselakes,withapHofaround1.4andsaturatedsaltconditions.Thepurposeofthepresentstudywastodevelopenrichmentsfortheisolationandcharacterizationofhaloacidophilestogainabetterunderstandingoftheorganismsthatresideintheseharshenvironments.Mediumdesignedtosimulatelakeconditions,microscopicobservation,andmolecularanalyseswereemployedtoachievetheaforementionedgoals.Aftersixmonthsofgrowth,thefirstenrichmentofLakeGneisshadturbidityanddevelopedapHof0.5.DNAextractionwasperformedonthefirstenrichmentofLakeGneissandagenomiclibrarywasgenerated.OurresultssupportourhypothesisthatbacteriacanexistatextremelowpHsandsaltmolaritiesandprovideshintsofthekindoflifethatcouldhavepreviouslyexistedonMars.11. Detectionof theNoravirusRegulatedProteins,Vir-1andVago, inDrosophilamelanogasterHemolymph.
IsaacJ.Lee(Undergraduate)*,AmandaMacke,DarbyJ.Carlson,andKimberlyA.Carlson.DepartmentofBiology,UniversityofNebraskaatKearney,KearneyNE68849
Research into the innate immune response ofDrosophilamelanogaster against virusesmay help identify theirfunctionsinhumans.Twoviralregulatedproteins,VirusinducedRNA-1(Vir-1)andVago,arecandidatesforanalysisbecausetheyarebiosyntheticproductsoftheinnateimmunesystem.Asofyet,thefunctionoftheseproteinsisuncharacterizedinNoravirusinfection.ThepathologyofNoravirusisunknown,butacognitivelocomotordefecthasbeenidentifiedinourlab.OurhypothesisisthatifNoravirusinfectioniscausingthelocomotordefect,thenthemostlikelyrouteoftransmissionfromtheguttothebrainwouldbethroughthehemolymph.WesternblotanalysisofNoravirusinfectedfliesdemonstratesthepresenceofNoravirus,Vir-1,andVagowithinthehemolymph.SinceNoraviruswaspreviouslythoughttoonlybelocatedwithinthegutofD.melanogaster,thisisanewfindingthatmayindicateinfectioninothertissues.MoreresearchmustbeconductiononVir-1andVago,butitisnowpossiblyidentifiedaspartoftheproteomecomprisingthehemolymphofNoraviruspositiveflies.12. IsolationofSoilMicrobestoTestagainstE.S.K.A.P.E.RelativesforAntimicrobialProperties.SaraNansel
(Undergraduate)*andClaudiaCarvalho.FortHaysStateUniversity,Hays,Kansas.TheE.S.K.A.P.E.pathogens,(Enterococcusfaecium,Staphylococcusaureus,Klebsiellapneumoniae,Acinetobacterbaumannii,Pseudomonasaeruginosa,andEnterobacterspecies)areagroupofbacteriathathavedevelopedmultipleresistancestoantibiotics(Boucheretal.).Antibioticresistanceoccurswhenbacteriadevelopthecapabilitytoadaptandmultiplyinthepresenceofantibiotics.Thispresentsaworld-widehealthconcernasantibioticsthatwerecommonlyusedtotreattheseinfectionsarenolongereffective.BytestingbacteriaobtainedfromsoilsamplescollectedaroundtheHays,KSareaagainstrelativesoftheE.S.K.A.P.E.pathogens,thegoalistofindnewantimicrobialpropertiesthatwillactagainsttherelativesandthen,potentially,theE.S.K.A.P.E.pathogens.Fromeachsoilsample,16isolateswereselectedbasedonmorphologicaldifferencestotestagainsttheE.S.K.A.P.E.relativestoobserveinhibitoryeffects.Afterselectingsixpureisolatesexhibitingthisproperty,acombinationofstainingtechniques,biochemicaltesting,aswellasgeneticanalysiswasperformedforbacterialidentificationandcharacterization.TheisolateswerefoundtobebothGramnegativeandGrampositiveandoftheBacillusandPseudomonasgenera.Organicextractioniscurrentlybeingperformedtoisolateandpurifytheinhibitorycomponentofeachisolate.
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13. ChlamydiatrachomatisManipulationofHostKinases.NickNelson(undergraduate)*,PrakashSah,andErikaLutter,OklahomaStateUniversity,StillwaterOK
Chlamydiatrachomatisisanintracellularpathogenthatcausesanestimated3millioninfectionseachyearintheUnitedStates.Infectionscanleadtofurthercomplicationssuchasinfertilityandectopicpregnancy.Duringinfection,C.trachomatislivesinaparasitophorousvacuoletermedaninclusion.Fromwithintheinclusionthebacteriamanipulatehost-cellfunctionsinordertoaidinitssurvival.Manyproteins,includingkinases,arerecruitedtotheinclusionmembrane.Withinthisstudywefocusedonhostkinasesandphosphorylatedkinasesubstrates.Recruitmentofkinasesandkinasesubstratestotheinclusionduringinfectionwasmonitoredbyimmunofluorescenceusingkinasespecificantibodies.TheoverallproteinlevelsandlevelsofphosphorylatedproteinsubstrateswereanalyzedbySDS-PAGEandwesternblot.TheanalysisofphosphorylatedsubstratesforMAPK,CDKandPKAshowedalteredphosphorylationastheC.trachomatisinfectionprogressedover48hours.BoththeMAPK-CDKandPKApathwayscontrolandplayasignificantroleincellproliferationandapoptosiswhichwouldaidC.trachomatisinitssurvivability.ManipulationofhostkinaseactivitymanipulationbyC.trachomatisisessentialtodecipheringhost-pathogeninteractionsandmayleadtofuturenoveltherapeutictargets.14. RelationshipBetween Locomotor Function andNoraVirus Infection inDrosophilamelanogaster.Amanda
McCown(undergraduate)*,AbigailBenz,&KimberlyA.Carlson,DepartmentofBiology,UniversityofNebraskaatKearney,Kearney,Nebraska.
NoravirusisamemberofthepicornavirusfamilythatinfectsDrosophilamelanogasterwithnopublishedpathogeniceffects.ApreviouslyunstudiedpathogeniceffectofNoravirusislocomotorability.Inthisstudy,theeffectNoravirushasonlongevityandlocomotorabilityisbeingexamined.Locomotionisexaminedusinggeotaxis,whereflieshaveoneminutetocrossathresholdoneinchfromthetopofthecage.TreatmentgroupsincludeNoravirusinfected,uninfectedandDrosophilaCVirus(DCV)infectedflies.LongevitycurvescreatedusingaStudent’st-testdemonstratethatNoravirusinfectedfliesaresignificantlyslowerintheirclimbingabilitiescomparedtouninfectedflies,whichsupportsarelationshipbetweengeotaxisandlocomotordysfunctionininfectedflies.NosignificantdifferencewasseeninlocomotorabilitybetweenDCVinfectedandNoravirusinfectedflies.NoravirusviralloadwasdeterminedutilizingqRT-PCRwithresultsthatdemonstrateabimodalcurveforNoravirusinfection.ThisdatasuggeststhatNoravirusdoeseffectlocomotorfunctionandcanbeclassifiedasapathogeniceffectofthevirus.15. InvestigatingtheroleofinflammatorycytokinesduringinfluenzaAvirusandAspergillusfumigatus
coinfectionsinvivo.MeaganD.Rippee-Brooks(Undergraduate)*,ChristopherR.Lupfer.MissouriStateUniversityDepartmentofBiology,Springfield,Missouri.
BacterialcoinfectionswithinfluenzaAvirus(IAV)areextremelyseriousandlife-threatening.However,thereexistslimiteddatagatheredabouttheimportanceoffungalinfectionswithIAV.Clinicalcasereportsindicatethatfungalcoinfectionsdohappenandsuggestthepandemic2009IAVhasapropensitytopredisposepatientstosecondaryfungalinfectionsmorethanpreviousIAVstrains.However,thefactorsinvolvedremaintobedetermined.Wehavedeveloped an in vitromodel using primary mouse bone marrow derived macrophages infected with IAV andcoinfectedwiththeopportunisticfungalpathogenAspergillusfumigatus.OurresultsindicatethatIAVandfungalcoinfectionssynergisticallyenhancecellsignalingandcytokineproduction.Weproposethisexacerbatedimmuneresponse during IAV andA. fumigatus predisposes clinical patients tomore severe pneumonia, andwe seek toidentifythepathwaysresponsiblefortheheightenedcytokineresponsesandtheirsignificanceinvivo.
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16. PrevalenceofCulturableEndophyticBacteriainaVarietyofSeedSpecies.FaithHiggins*(Undergraduate),GiannaMorrie*(Undergraduate),AshtenGrabill*(Undergraduate),HeatherM.Seitz.JohnsonCountyCommunityCollege,OverlandPark,Kansas.
Antibioticresistanceisaseriousthreattotheworld,anduntreatableinfectionsarenolongerapredictionforthefuture,theyarehappeningrightnow.Unfortunately,thepaceofantibioticdiscoveryisnotkeepingupwiththerapidevolutionofresistancetomicrobes.Fewnewantibioticshavebeendiscoveredinthepast30years(McIntosh,2016).Endophyticbacteriacanbedefinedasthosebacteriathatcolonizetheinternaltissueoftheplantshowingnoexternalsignofinfectionornegativeeffectontheirhost(Holliday,1989&Boyle,2006).Endophyticbacteriaareabletolessenorpreventthedeleteriouseffectofcertainpathogenicorganismsinplants(Ryan,2008).Theinvestigationofendophyticstrainsfornovelantimicrobialmetabolitesisanuntappedsourcewithminimalpreviousresearch.Wearefocusingonfindingandclassifyingendophyticbacteriathatproducenovelantibioticsthatcantreatpathogens.Inthisresearchwewilldemonstrateourapproachtoidentifyingendophyticbacteriaandsharetheprevalenceofculturablestrainsofbacteriafoundonavarietyofspecies.Furtherwehavedocumentedtheantimicrobialpropertiesoftheendophyticbacteriafrommultiplespecies.
17. TranslesionSynthesisProteinAbundanceinProliferatingCells.JazmineSnow(Undergraduate)*,SebastianWendel,NicholasWallace.DivisionofBiology,KansasStateUniversity,Manhattan,KS
HumanPapillomavirus(HPV)causesnearlyeverycervicalcancer,withapproximately14millioninfectionsannuallyintheUnitedStates.ThesecancersaretheresultofHPVoncogenes(HPVE6andE7)manipulatingthehostcells.Thesechangeswerecharacterizedbytranscriptomeanalysisoftumorandcontrolcervicalsamplesandfoundincreasedexpressionofgenesneededfortranslesionsynthesis(TLS).ThisledtothehypothesisthathighlyproliferatingcellsencountermoreDNAdamageresultinginaninductionofTLS,aDNAdamagetolerancepathway.Totestthishypothesis,wedeterminedwhethertheproliferationofhumanforeskinfibroblasts(HFFs)couldbemodulated.WegrewHFFsinagradientoffetalbovineserum(FBS)concentrations.Whenthecellsnearedconfluence,theywereharvestedandcountedonahemocytometer.ThisdatashowedanincreaseincellproliferationcorrelatingwithincreasingFBSconcentration(R2=0.87).HarvestingproteinsfromparalleltreatedcellsdeterminedthatTLSproteinlevelsmirroredproliferationrates.OurinvitrodatashowthatHPVoncogenespreventcervicalcancercellsfrominducingexpressionofPolƞ,aTLSpolymerase.IwilldetermineifHPVoncoproteinspreventproliferation-inducedincreasesinTLSproteinlevelsbyrepeatingtheseexperimentsinHFFsexpressingHPVoncogenes.18. IdentificationofCandidateCellDivisionProteinsinPlanctomyceslimnophilusUsingtheBACTHTwo-Hybrid
System.BriannaSteiert(Undergraduate)*,Dr.LilahRahn-Lee.WilliamJewellCollege,Liberty,Missouri.Understandingcelldivisionmechanismsinbacteriaisimportantbecausecelldivisionisfundamentalforbacterialreproductionandsurvival.MostbacteriadividebybinaryfissionandareassistedbyproteinssuchasFtsZthatassemblesintoaringatmid-cellduringdivision.Thisresearchusesplanctomycetes,agroupofbacteriathatareunusualbecausetheydonotuseFtsZfordivision.Planctomyceteshavemanydistinctivephenotypicfeaturesincludingthecompartmentalizationofcellswithinternalmembranesandamembrane-boundnucleoid.ThepurposeofthisresearchistostudythedivisionmechanismbyscreeningaPlanctomyceslimnophilusgenomicDNApreylibraryagainstseveralcelldivisionproteinbaitsusingtheBACTHtwo-hybridsystem.Throughbioinformaticswork,planctomycetesgeneswerecomparedtogenesinotherbacterialgenomesknowntobeinvolvedincelldivisiontoidentifythebaitsofFtsKandFtsI.Baitandpreyinteractionswillbescreenedthroughco-transformedintoDHM1cells,andpreythatinteractwithbaitwillbecharacterizedtodeterminethePlanctomyceslimnophilussequenceintheprey.Thesegeneswillbecandidatesformembersoftheplanctomyceteuniquecelldivisionmechanism.
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19. Theconstructionofageneticdevicetoinvestigatetheregulationofploidyincyanobacteria.SekiK.Anderson(Undergraduate)*andDr.LilahRahn-Lee.WilliamJewellCollege,Liberty,Missouri.
Cyanobacteriaisabacterialphylumknowntoexhibitpolyploidy;differentcyanobacteriaspecieshavedifferentcopynumbers,anddependingonthestrain,ploidyrangesfromaslowasthreecopiestoashighas218genomecopies.Bacterialstrainsaredefinedbytheirdifferencesingeneticmakeup,sowehypothesizethereisageneticfactorthatplaysaroleindeterminingthenumberofchromosomecopieswithinacyanobacteriaspecies.Weareinterestedinstudyingtwostrainswithinthesamegenusthataredifferentingenomecopynumber;Anabaenacylindricahasanaverageof25genomecopiesandAnabaenavariabilishasanaverageof8genomecopies.Tolookforgeneticdeterminantsofcopynumber,weareconstructingabistablesensorthatdetectsthenumberofchromosomecopiesastrainofcyanobacteriahas.Oncethegeneticdeviceismadeandhasbeentuned,weplantotransformagenomiclibraryfromonespeciestotheotheranduseourbistablegeneticdevicetoscreenforindividualswithchangedcopynumber.Thisresearchwillexpanduponourunderstandingofcelldivisionandbacterialdiversity,asweaimtodiscoverthemechanismscyanobacteriausetocountandregulatetheirchromosomecopynumber.20. Prevalenceof antibiotic resistant strains ofEnterococcus spp. andAcinetobacter spp. in community household
environment. Rebekah Elliott (Undergraduate)*, Jada Caplinger, and Anuradha Ghosh. Department of Biology,PittsburgStateUniversity(Pittsburg,KS)
Withincreasingprevalenceofantibioticresistancethreats,thereisanupsurgeintheoccurrenceofcommunity-acquiredinfections.ThepurposeofthisstudyistoassesstheecologyandprevalenceofEnterococcusspp.andAcinetobacterspp.(thatarewell-knownantibioticresistantnosocomialpathogens)inthehouseholdenvironment.Eachhouseholdsamplingkitcontained5swabsforeachofshoebottom,restroom,cleaningsupply,kitchentop,anddoorstep/handleaswellasademographicdatasheettobefilledup.Atotalof30suchkits(n=150)havebeenprocessed.Theswabsweresubjectedtoenrichmentusingselectivemediafortestbacterialspecies.TotalnumberkitspositiveforgrowthofsuspectedenterococciandAcinetobacterspp.weredetermined.Onlyfewcleaningsuppliesshowedgrowthforenterococciwhereasthekitchentopshowedmorefrequententerococcalcontamination.AlthoughmajorityofthelocationsswabbedwerecontaminatedwithsuspectedAcinetobacterspp.,doorstep/handleswerefreeofanyselectedmicrobe.Overall,mostoftheswabbedlocationswerecontaminatedwithbiochemicallyconfirmedAcinetobacterspp. incontrasttofewerwithenterococci.Apanelofantibioticsweretestedfortheirsusceptibility.FurtherPCRamplificationofselectivegeneswillbecarriedouttoconfirmatthespecieslevel.Theantibiotic-resistantisolateswillbegenotypedandcomparedtotheirrelativenosocomialstrains. The community will be outreached with recommended cleaning protocol and stewardship in antibioticconsumptionandresistance.Theoutcomeofthisstudymayhelpfacilitateeffectiveandappropriateantibiotictreatmentofcommunity-acquiredinfections.GeneralMicrobiologyGraduateStudentOralPresentations1. D-motifMutationincAMPPhosphodiesterase,RegA,LeadstoDevelopmentalPhenotypeDefectinDictyostelium
discoideum.NirakarAdhikari(doctoralstudent)*,NickKuburich,JeffHadwiger.OklahomaStateUniversity,Stillwater,Oklahoma.
CyclicnucleotidephosphodiesterasemoleculeshaveclinicalsignificancebecausetheydownregulatesecondmessengercAMPorcGMPincell.InDictyosteliumdiscoideumtheMAPkinase(MAPK),ERK2,down-regulatesthephosphodiesterase,RegA.RegAlowersthelevelofcAMP,animportantregulatorofcellaggregationanddifferentiation.RegAhasaMAPKdockingsite(D-motif)thatmightbeimportantininteractionswithMAPKs.CharacterizingthefunctionofthisD-motifinRegAwill likely provide insights on the interactionsbetweenRegAandMAPKsduring thedevelopmental life cycle ofDictyostelium.Thesefindingscanleadtoabetterunderstandinginothereukaryotesincludingmammals.Intheabsence
ofD-motif,littleornointeractionisexpectedtooccurbetweenRegAD-
andErk2.Thismightpreventthedown-regulation
ofRegAD-
andcausereducedlevelsofcAMP.LowlevelsofcAMPareexpectedtodelaytheaggregationanddifferentiation
ofDictyosteliumcells.RegAD-motifwasmutagenizedandthecorrespondingmutantgene(regAD-
)hasbeenexpressed
intoregA-
mutantcells.ThecloneswithstablephenotypesshowedthattheseregAD-
cloneswereslowerinoverallrateofdevelopment and have defect in their developmental stage. Western blot analysis will be used to determine if any
interactionsoccurbetweenRegAD-
andErk2.
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2. TheRoleofβ-carbonicanhydrasesinCalciumDepositionbyPseudomonasaeruginosa.BirajB.Kayastha(Doctoral)*1,ShalakaLotlikar1,ErinGallaway1,ClaudiuT.Supuran2,andMariannaPatrauchan1,1OklahomaStateUniversity,Stillwater,OK,2UniveristyofFlorence,Florence,Italy
Pseudomonasaeruginosaisanopportunistichumanpathogencausinglife-threateninginfectionsinpatientswithcysticfibrosisandinfectiveendocarditis,whichcanbeassociatedwithcalcificationatlaterstages.EarlierresultsshowedthatP.aeruginosaproducesextracellulardepositsofcalcium(Ca2+)whengrownatelevatedCa2+levels.Weidentifiedandcharacterizedthreeβ-carbonicanhydrases(CAs):psCA1,psCA2,andpsCA3.WhilethedeletionofallthreeencodinggenesdelayedP.aeruginosagrowthatambientairby4hours,itcausednoimpactonstaticgrowthat5%CO2.MeasuringbothdepositedandfreeCa2+inliquidmediumforthewildtypeshowedthatstaticgrowthconditionsand5%CO2favorCa
2+deposition.AnalysesofCAmutantsdeterminedthatpsCA1isthemajorcontributortocalciumdeposition.DeletionofpsCA1alonecaused~2folddecreaseinCa2+depositionandalmostabolishedfreeCa2+removalfromthemediumatboth5and10mMCa2+.TheresultsshowthatpsCA2alsocontributetoCa2+depositionat10mMCa2+,butpsCA3playsnoroleinthisprocess.CurrentlywearetestingtheeffectofCAinhibitorsontheβ-CA-inducedCa2+depositionandtheroleofβ-CA-dependentcalcificationinbiofilmformationandvirulenceofP.aeruginosa.3. MetabolicallyEngineeringAspergillusnidulansusingRNAInterference.JorgeLightfoot(Doctoral)*,Rolf
Prade.OklahomaStateUniversity,Stillwater,OklahomaThefilamentousfungi,A.nidulans,canproducenearly100gramsperliterofindustriallyrelevantproteinsunderoptimalconditions.However,manyoftheseproteinsaredegradedorproducedalongsideotherproteins,whichdrasticallyreducetheirefficacyinacellulosefermentationreaction.Weproposeanovelmechanism,utilizingRNAinterference,tocombinatoriallysilencegenes,whichdegradeorcontaminateclientproteins.Usingdualpromoters,wewillflankasequencecontaining30or40bpcomplementarysequencesformultipleclientgenes.ThiswillinducedoublestrandedRNAproduction,inturnloadingtheseindividualcomplementarysequencesintotheArgonautecomplex,silencingthemessengerRNAforeachtargetgene.WehavealsoutilizedLC-MS/MStoexaminechangesintheproteomeofoursilencedstrains.Wehaveseenmarkeddecreasesinourtargetgenesequencesaswellastheinductionofnewproteins,actingasacompensationmechanismforthefungus.Oursilencedstrains,whentransformedtoproduceclientproteins,havealsohadamarkedchangeintheamountofproteinproduced,aswellashowlongitlastsinthemediaduringproduction.Wehavecontinuedthisworkbysilencinggenesresponsibleforunwantedamylolyticactivityinclientproteinproduction.
4. Investigating a Phosphodiesterase Involved in Amoeboid Multicellular Development and Signaling. NickKuburich(Doctoral)*,NirakarAdhikari,JeffHadwiger.OklahomaStateUniversity,Stillwater,Oklahoma
ManyeukaryoticsignalingpathwaysusecAMPasasecondarymessengertoevokespecificresponsestodifferentexternal stimuli. Here, localized levels of cAMP can be controlled by phosphodiesterases,which are sometimesregulatedbyphosphorylation.Dictyosteliumdiscoideumoffersanexcellentmodelsystemtostudytheregulationofphosphodiesterases as it contains relatively few cAMP-specific phosphodiesterases compared tomammals. ThecAMP-specificphosphodiesterase,RegA,regulatesimportantstepsinDictyosteliumdevelopmentandisnegativelyregulated by theMAP kinase, ERK2. This inactivation occurs periodically by external cAMP pulse where a cell-signalingpathwayactivatesERK2.Mammalian studieshave suggested that thecAMP-dependentproteinkinase,PKA,canalsoregulatethephosphodiesteraseactivity.ThisputativeregulationofPKAontheactivityofRegAhasnotbeenfullyinvestigatedinDictyostelium.MassspectrometrywasusedtodetectpotentialphosphorylationsitesonRegA. Two sites of interest have been identified, including a PKA phosphorylation site. Phosphomimic andphosphoablative mutations for the three sites have been constructed. The phenotypes of cells carrying thesemutationshavebeenanalyzedfortheirimpactondevelopmentandcellfate.ThesedatasupportthehypothesisthatRegAisregulatedbymultiplephosphorylationtoregulatesignalingduringmulticellulardevelopment.
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5. ExploringthePotentialRoleofVps1asaFusionProtein.MarielDelgadoCruz(Masters)*,Dr.Kim,Kyoungtae.MissouriStateUniversity,Springfield,MO
Proteintraffickingwithinthecellismorethanjusttheseriesofstepsnecessarytogetcargofromoneendofthecelltoanotherlocation,italsoinvolves,theeasilyoverlooked,fusionstep.Fusionistheverylaststepintraffickingwhenthetransportedcargoispassedonfromthecarriervesicleontothetargetmembrane.ThisstepinintracellulartraffickinghasoftenbeencharacterizedbytheuseofSNAREproteins,however,thereareamultitudeofproteinsinvolvedinthisstep.AmongthemareRabs,Golgins,andMultitetheringproteins.Vps1wassuspectedtoplayaroleinhomotypicGolgifusionafternoticingthatadepletioninthisGTPaseresultedinanincreaseofLateGolginumbers.ThisledtothehypothesisthatVps1isfunctioningasafusionproteinactingtobringtwolateGolgicompartmentstogetherinordertoformalessernumberoflargerGolgicompartments.ThisquestionwasexploredusingyeasttwohybridwhereithasbeenfoundthatVps1interactswithGolgilocalizedSNAREs:Tlg1,Tlg2,Vti1,andSnc1.TofurtherinvestigatethisquestionaGSTpulldownassayandaninvivoyeastmatingexperimentsareallunderway.6. CarPisaCa2+RegulatedPutativePhytasethatisUniquetoPseudomonadsandisHighlyConservedin
ClinicalIsolates.M.M.King(Doctoral)*,S.Mares,andM.Patrauchan.OklahomaStateUniversityStillwater,OK
Pseudomonasaeruginosaisaversatilepathogencausingvariousinfectionsinahumanbody.ItisimportanttoidentifythehostfactorsthatsignaltoP.aeruginosathepatient’simmunocompromisedstatus,triggeringthevirulenceofthepathogen.Ourearlierstudiessuggestedthatelevatedcalcium(Ca2+)isoneofsuchsignals,asitincreasesvirulenceinP.aeruginosa.WehaveidentifiedaproteinCarPthatisimportantforCa2+-regulatedinfectivityinP.aeruginosa.SequenceanalysispredictedphytaseactivityandshowedthatcarPisuniquetoPseudomonads.High-resolutionRNAseqandsequenceanalysessuggestedcomplexregulationofcarPbymultiplehostfactors.PromoteractivityassaysconfirmedtheregulatoryroleofelevatedCa2+,H2O2,andCO2.TocharacterizetheroleofcarPduringinfectionsweaimtodeterminewhethertheencodingandregulatorysequencesofcarPareconservedinclinicalisolates(CI).Theinsilicoanalysisrevealedthesesequencesaremorethan98%identicalin~2,000CIcollectedfrompatientswithburnwounds,upperrespiratoryandurinarytractinfections,andkeratitis.Theidentifiedmutationsdonotalterproteinsequences.ThisworkshowsthatcarPanditsregulatoryelementsarepreservedduringinfectionsandhighlyresponsivetohostenvironment,suggestingtheirimportanceinP.aeruginosapathogenicity.7. MyosinmediatesproteinrecyclingtowardtheGolgi.JaredSmothers(Master’s)*,PaulBallhorn*,Vy
Nguyen*,Dr.KyoungtaeKim*MissouriStateUniversity,Springfield,MissouriMyosinfamilyproteinsareATP-dependentmotorsthatsharethesamebasicpropertiesofactinbinding.ThechemicalenergyprovidedbyATP-hydrolysisintheheaddomaingeneratesthe“powerstroke”necessaryto“walk”alongactinfilaments.Inthisstudy,usingconfocalmicroscopyweassessedthepotentialrolesofallfivemyosin’sinSaccharomycescerevisiae,andtheireffectontherecyclingofSnc1andVps10.MYO1knockoutstrainshadnosignificantdefectinSnc1recycling,butdisplayedseveredefectsinthetraffickingofVps10towardtheGolgi,manifestedbyabnormallocalizationtothelumenofthevacuole.Myo3andMyo5areparalogsthathavelittletonosignificanttrafficdefectwhenknockedoutindividually,butthedoubledeletionofbothMYO3and5ledtoseveredefectsinSnc1andVps10trafficking.TwotemperaturesensitivestrainsofMyo2,myo2-16andmyo2-66,demonstratedtraffickingdefectsatrestrictedtemperatureconditions.Amongallfivemembers,onlyMYO4deletiondidnotaffectproteinrecyclingpathways.Together,ourdataprovidesnovelinsightsintothefunctionofMyo-familyproteinsinproteinrecyclingtrafficsdestinedtowardsthetrans-GolgiNetwork.
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8. SearchingforRecruitmentDomainsandResiduesofVps1toTargetMembrane.JohnC.(Wes)Short(Masters)*,ShivaKumarGoudGadila,KyoungtaeKim.MissouriStateUniversity,Springfield,Missouri.
Intracellularmembranetraffickingrequiresclassicaldynaminsanddynamin-likeproteins(DLPs),ubiquitousthroughouttheeukaryoticdomain.DysfunctionofclassicaldynaminsinhumansislinkedtoAlzheimer’sandotherneuropathies.LossoforthologyeastDLPVps1disruptspathwaysofvesicletrafficking,whichweusetoinvestigategenefunction.InvitroassayshaveshownthatVps1interactsdirectlywithmembranetoexertitsmembrane-remodelingpower.However,themolecularsignalfortherecruitmentofVps1totargetmembraneshasnotbeendescribed.Toinvestigate,geneticvariantsofVps1wereN-terminallyfusedwithmRFPandoverexpressedinwildtype(WT)andvps1∆backgroundwithgenomically-taggedGFPmembrane-associatedproteinmarkers,andexaminedbyconfocalmicroscopyforcolocalization.TheVps1variantsweresetsofdomainfragmentsaloneandincombinations,seriallyshortenedC-terminaltruncations,andselectedpointmutationsatsuggestedbindingsites,whichwouldreestablishorabolishVps1recruitmentandfunction.Resultsshowedthatalltestedvariantsabolishedthenormalphenotype,andnotestedpointmutationscausedabnormality,suggestingthatthefully-formed3Dshapewithitsintactcatalyticandpolymerizationdomainsisessentialtoitsabilitytotargetmembranes.Furtherstructuralanalysiswillnarrowdownpossiblebindingpatchesforfurthertesting.
MedicalMicrobiology/ImmunologyGraduateStudentOralPresentations1. EffectoftheOuterMembranePermeabilizerCompound48/80onIntrinsicResistancetotheHydrophobicBiocide
TriclosaninOpportunisticSerratiaSpecies.1A.Rigsbee(Masters)*,2S.KatzAmburn,3B.King,4K.Boyina,4J.Yang,1W.Sprinkles,3A.A.McDonald,5V.M.Patel,1F.R.Champlin.1OklahomaStateUniversityCenterforHealthSciences,Tulsa,OK;2RogersStateUniversity,Claremore,OK;3NortheasternStateUniversity,BrokenArrow,OK;4OklahomaStateUniversity,Stillwater,OK;5UnionHighSchool,Tulsa,OK.
Unlikemosthydrophobicmolecules,thebiocidetriclosanisabletopenetratethegram-negativebacterialoutermembrane.AtypicalresistancetotriclosaninthenosocomialopportunistsPseudomonasaeruginosaandSerratiamarcescensislargelyduetooutermembranepropertiesthatresultinimpermeabilityforhydrophobiccompounds.ThepresentstudywasundertakentodetermineifthesecellenvelopeimpermeabilitypropertiesareconservedandunderlietriclosanresistanceinotheropportunisticSerratiaspecies.Generalintrinsicresistancetohydrophobiccompoundswasassessedusingthreedisparatebioassaysandonechemicalassay.Batchculturekineticsinthepresenceofcombinationsoftriclosanorthehydrophobicantibioticnovobiocinandtheoutermembranepermeabilizercompound48/80allowedanalysisofoutermembraneinvolvementinintrinsicresistance.Tenindividualspeciesrangedfromgenerallyrefractorytosensitization,asseenwithSerratialiquefaciens,toextremelysusceptive,asseenwithSerratiaodorifera.Moreover,thosespecieswhichexhibitedintrinsicresistancetobothnovobiocinandtriclosanwerereadilysensitizedtodifferentdegreesbychemicaldisruptionoftheiroutermembraneexclusionaryproperties.ThesedatasuggestthatdisparateopportunisticpathogenswithinthegenusSerratiadifferphenotypicallywithregardtothedegreetowhichoutermembraneexclusioncontributestointrinsicresistancetohydrophobicmoleculesingeneral,andtriclosanspecifically.2. ThreeTransketolasesinSalmonellaentericaContributetoDefendingAgainstOxidativeandNitrosativeStresses.
JeffA.Shaw(Doctoral)*andTravisJ.Bourret.CreightonUniversitySchoolofMedicine,Omaha,Nebraska.As a facultative intracellular pathogen, Salmonella enterica serovar Typhimuriummust combat host-derived reactiveoxygenspecies(ROS)andreactivenitrogenspecies(RNS)thatcandamagebacterialDNA,modifyproteinsandenzymes,anddisrupttheredoxstateofinvadingintracellularbacteria.Topowerantioxidantdefensesandovercomethesecytotoxiceffects,S.Typhimuriumutilizes theoxidativebranchof thepentosephosphatepathway (oxPPP) togenerate reducingpowerintheformofNADPH.Inthenon-oxidativebranch(non-oxPPP),enzymesgeneratemetabolicintermediatesthatcanbechanneledtoglycolysis,recycledbacktotheoxPPP,orutilizedasprecursorsorbiosyntheticreactions.Thisstudyinvestigatedthecontributionoftransketolasesinthenon-oxPPPtoresistingROSandRNS.TheS.Typhimuriumgenomecontains genes for three transketolases (TktA, TktB, and STM2340-41), each of which catalyzed the transketolaseenzymaticreaction. WhenexposedtoROSandRNS,mutantstrains lackingall threetransketolasesdemonstratedthegreatest sensitivity in vitro, most of which was attributable to loss of TktA. Furthermore, a ΔtktA ΔtktB mutant hadattenuatedvirulence following intraperitoneal infectionofC57BL/6mice,butvirulencewas restored in iNOS-deficientmice.ThisstudyrevealsanessentialrolefortransketolasesinresistingROSandRNS,andsuggeststhattheymaycontributetotheintracellularsurvivalofS.Typhimurium.
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3. SegmentalAneuploidiesFlankedbyInvertedRepeatsCauseAzoleResistanceintheFungalPathogenCandidaalbicans.RobertTodd(graduate)*,TylerWikoff,ShilpaNair,CurtisFocht,RobertThomas,AnnaSelmecki.CreightonUniversitySchoolofMedicine,Omaha,Nebraska.
Candidaalbicansisthemostprevalentfungalpathogeninimmunocompromisedindividuals.Currently,treatmentofCandidainfectionsreliesheavilyonazoles,afamilyoffungistaticdrugsthatdisruptthebiosynthesisofthefungal-specificsterol,ergosterol.Aneuploidy,amplificationorlossofachromosome,isacommongenomicfeaturefoundin50%ofazole-resistantC.albicans.PreviousworkfromtheSelmeckiLabhasshownthataspecificsegmentalaneuploidy,anamplificationoftheleftarmofchromosome5,canconferazoleresistanceduetotheamplificationoftwodrug-responsegenes.Currently,littleisknownabouthowsegmentalaneuploidiesform.Usingnext-generationsequencingtechnology,chromosomekaryotyping,andlong-rangeDNAsequencingwedescribeseveralnovelsegmentalaneuploidiesfoundinazoleresistantCandidastrainsfromdiversegeneticbackgrounds.Thesesegmentalaneuploidiesconsistofamplifiedregionsofthegenome,someofwhichcanundergocopynumberincreasesofgreaterthan12copies.Theseamplificationscontainimportantdrugresistancegenesandcorrelatewithasignificantincreaseinazoleminimalinhibitoryconcentration.Here,wedescribethesenovelsegmentalaneuploidiesthatconferazoleresistanceandidentifygeneticfeaturesthatelucidateacommonmechanismofformation.4. ROS/RNSInducedChangesinBorreliaburgdorferiOuterSurfaceProteins.AmandaK.Zalud(doctoral)*,TravisJ.
Bourret.CreightonUniversity,Omaha,Nebraska.Borreliaburgdorferi,thecausativeagentofLymedisease,isnaturallymaintainedinvariousmammalianhostsanditstickvectorIxodesscapularis.TheabilityofB.burgdorferitosuccessfullycompleteitsinfectiouscycledependsontheregulationofalargerepertoireoflipoproteins.Lipoproteinexpressioniscontrolledbyalimitednumberoftranscriptionfactorsinresponsetodiverseenvironmentalchallengesencounteredduringinfection,includingchangesinpH,temperature,osmolarity,nutrientavailability,andhost-derivedreactiveoxygenspecies(ROS)andreactivenitrogenspecies(RNS).Inthefollowingstudy,wetestedthehypothesisthatROSandRNSserveasregulatorysignalsforlipoproteinexpressioninB.burgdorferi.Totestthishypothesis,wecomparedtheexpressionofimmunogeniclipoproteinsinB.burgdorferigrowninBSKIIandexposedtohydrogenperoxideorspermineNONOate.AshiftinpHfrom7.6to6.8leadtowidespreadchangesinlipoproteinexpression,whichhasbeenshownpreviously.Surprisingly,sublethalconcentrationsofhydrogenperoxideorthenitricoxidedonorspermineNONOateinhibitedthepH-dependentchangesinlipoproteinexpression.ThesedatasuggestROSandRNScontributetotheregulationoflipoproteinexpressioninB.burgdorferiandmayimpactitsabilitytocompleteitsnaturalinfectiouscycle.5. DeterminationoftheRoleofChlamydialInclusionMembraneProteinsinInclusionGrowthandDevelopmentby
ProximityLabelingofInteractingPartners.MacyG.Olson(doctoralstudent)*,ElizabethA.RucksandScotP.Ouellette.UniversityofNebraskaMedicalCenter,PathologyandMicrobiology,Omaha,Nebraska.
Chlamydiatrachomatis(Ctr),adevelopmentally-regulated,obligateintracellularpathogen,istheleadingcauseofbacterialsexuallytransmittedinfections.Chlamydiaegrowwithinapathogen-specifiedorganelle,termedtheinclusion.Theinclusionmembranemediatesinteractionsbetweenthehostandpathogen.Tosupportchlamydial-hostinteractions,theinclusionmembraneisheavilymodifiedthroughoutthedevelopmentalcyclebychlamydialproteinscalledIncswhichencodetwoormoretransmembranemotifs.WehypothesizethatCtrusestemporalcontrolofIncexpressiontofacilitateinclusiondevelopment,wherebyIncsexpressedearly(e.g.IncF)playanessentialroleinorganization/establishmentoftheinclusionandIncsexpressedlater(e.g.IncA)areinvolvedinnutrientacquisition.Here,wecompareIncFandIncAover-expressiontodeterminetheirrolesininclusiondevelopment,includinghowtheseIncsareinvolvedinprotein-proteininteractionsattheinclusionmembrane.Toexamineourhypothesis,wecreatedCtrL2transformantswithIncF-APEX2,IncATM(transmembranedomain)-APEX2,IncA-APEX2,orAPEX2only.APEX2isanascorbateperoxidasethatbiotinylatesproximalandinteractingproteinsinvivo.PreliminarystudiesshowthatbiotinylatedproteinsidentifiedbymassspectrometryanalysissupportcurrentlyknowndataforIncsandtheireukaryoticproteinbindingpartners.Thesestudiesareimportantforunderstandingmolecularmechanismsinvolvedinestablishmentofthisintracellularpathogenicniche.
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6. TheBorreliaburgdorferibb0168-EncodedDnaKSuppressorEnhancespHDependentLipoproteinExpression.WilliamK.Boyle(doctoral)1*,JeffA.Shaw1,AshleyGroshong2,JonS.Blevins3,FrankC.Gherardini4andTravisJ.Bourret1.CreightonUniversitySchoolofMedicine,Omaha,Nebraska1,UConnHealth,Farmington,Connecticut2,UniversityofArkansasforMedicalSciences,LittleRock,Arkansas3,NationalInstituteofAllergyandInfectiousDiseases,Hamilton,Montana4.
Borreliaburgdorferi,thecausativeagentofLymedisease,mustsensetransmissionfavoringconditionswithintickvectorsandincreasetheexpressionofinfectivityassociatedlipoproteins.Wehypothesizedthataputativetranscriptionalregulator,theDnaKsuppressorprotein(DksA),playsaroleincoordinatingthetranscriptionalresponseofB.burgdorferitoenvironmentalsignalsencounteredduringinfection.Totestthishypothesis,wild-typeanddksA-deficientB.burgdorferistrainsweresubjectedtogrowthconditionsmimickingthetickmidgutduringbloodmealacquisitionbyshiftingmid-logcultures(5x107spirochetes/ml)growninBSKII(pH7.6)toBSKII(pH6.8),andallowingthemtoreachstationaryphase(2x108spirochetes/ml).ImmunoblotsofspirochetelysateswithserumfromB.burgdorferi-infectedmiceindicatethatwild-typespirochetesincreasedtheexpressionofimmunogenicproteinsinresponsetopHshift,whiledksA-deficientstrainsexhibitedaconstitutivelylowexpressionofthesepH-inducibleproteins.Further,weobservedthatdksAisrequiredfortheexpressionofthekeyinfectivityassociatedlipoproteinsDbpAandOspC,alongwiththealternativesigmafactorRpoSinresponsetopHshift.Together,thesedataindicateDksAisaregulatorthat,directlyorindirectly,controlstheRpoS-dependentexpressionofimmunogenicproteinsinresponsetochangesinpH.
UndergraduateorHighSchoolOralPresentations
1. UtilizingGalleriamellonellatoDeterminetheRoleofCalciuminPseudomonasaeruginosaVirulence.LeahKafer(Undergraduate)*1,MichelleKing1,MarietteBarbier2,MariannaPatrauchan1OklahomaStateUniversity,Stillwater,OK1,WestVirginiaUniversity,Morgantown,WV2
PseudomonasaeruginosaisanopportunisticpathogeninfectingthelungsofCysticFibrosispatients,knowntohaveabnormallyhighlevelsofcalcium(Ca2+).OurlabhasshownthatelevatedCa2+increasesplantinfectivityandtheproductionofseveralvirulencefactorsincludingpyocyanin,pyoverdine,andrhamnolipidinP.aeruginosa.Basedontheseobservations,wehypothesizedthatelevatedCa2+enhancesP.aeruginosavirulenceinananimalhostaswell.Totestthishypothesis,Ihaveoptimizedavirulencemodelusingwaxworms,Galleriamellonella.First,weaimedtodeterminethehalflethaldose(LD50)ofP.aeruginosa.Forthis,weinjectedthewormswithPAO1grownin0mMor5mMCa2+.ThelatterdiedingreaterquantitiesandfasterthanthoseinfectedwithPAO1grownwithoutCa2+.TheLD50ofPAO1in0mMCa2+istwo-foldhigherthanthatofPAO1grownin5mMCa2+.Currently,wearedeterminingtheroleinvirulenceofthreeproteins,EfhP,CarP,andCalC,previouslyshowntomediateCa2+regulationofvirulencefactorsproduction.ThisknowledgewillenableidentificationofthecomponentsofCa2+regulatorynetworkcontrollingP.aeruginosavirulence,whichisasteptowardslearningnovelwaystofightP.aeruginosainfections.2. RoleofcentromereheterogeneityinclinicalisolatesofCandidaalbicans.AlisonGuyer(undergraduate)*,
RobertTodd,AnnaSelmeckiCandidaalbicansisafungusnaturallyfoundwithinthehumanmicrobiome.Whenexposedtostress,C.albicanscanrapidlyevolvethrougheffectivemeanssuchasgainorlossofanentirechromosome(aneuploidy).Specifically,whenC.albicansisexposedtotheantifungaldrugfluconazoleitmayacquireasegmentalaneuploidycalledisochromosome5L[i(5L)].i(5L)arisesfromabreakpointinthechromosome5centromere(CEN5),generatingtwocopiesoftheleftarmofchr5.Previousresearchhasdemonstratedi(5L)tobeakeycomponentinfluconazoledrugresistance,butthemechanismdrivingi(5L)formationisunknown.AninterestingfeaturefoundwithinCEN5isaheterozygoussequenceencodingalongterminalrepeat(LTR).Thepurposeofthisstudyistoexplorethepotentialroleofchr5heterogeneityintheformationofi(5L)andacquisitionoffluconazoledrugresistance.WeconductedaninvitroevolutionexperimentinwhichC.albicanswereexposedtofluconazoleandscreenedforchangesinheterozygosityoftheLTRandanotherheterozygouslocusonchr5L,thematingtypelocus(MTL).WefoundthatmostC.albicansclinicalisolatesmaintainedbothheterozygouslociafter100generationsinfluconazole.
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3. AnalyzingProteinInteractionsOfTheHerpesSimplexType1UL34Protein.NathanielB.A.Higdon(Undergraduate)*,SusanL.Bjerke.WashburnUniversity,Topeka,Kansas.
HerpesSimplexVirusType-1(HSV-1)iseasilycommunicableandinfectionscanoccurindifferentlocations.HSV-1proliferateswithinthehostcellnucleus.Oncereplicationiscompletethevirusexitsthenucleus.ViralproteinUL34isessentialfordeparturefromthenucleus.It’sunknownwhichnuclearproteinsUL34isinteractingwithduringthisphase.UL34isahighlyconservedproteininallhumanherpesvirusesandanidealcandidateforfuturedrugtreatments.BlockingUL34functioninHSV-1wouldpreventexitofthenucleusandspreadofinfection.TodetermineinteractionpartnersforUL34,pulldownassayswereperformed.Inourassay,purifiedUL34proteinwasmixedwithHEp-2celllysate;UL34andanybindingpartnerswerethenremovedfromthemixture.OurpastresultsshowedsomepotentialUL34bindingpartners,however,uponexperimentalreplications,inconsistentresultswereobtained.Toobtainsimilarresultsfromourassays,weexpressedanewGSTcontrolproteininE.coli.WewillusethiscontroltocomparetotheproteinsthatarebeingpulleddownbyaGST-taggedUL34protein.WehopeourresultswillagainshowpotentialUL34bindingpartners.Ifmoreconvincingproteininteractionsoccur,isolationexperimentswillbeperformedtoidentifythebindingprotein(s).
4. DeterminingtheLocalizationoftheHypotheticalMembraneProteinCBU_1651fromCoxiellaburnetii
KeeganMcGill(Undergraduate)*BrandonLuedkte,UniversityofNebraskaatKearneyDepartmentofBiology,Kearney,Nebraska
CoxiellaburnetiiisanobligateintracellularpathogenandtheetiologicalagentofQueryFever.Tocausedisease,C.burnetii uses the Type IVB secretion system (T4BSS) to establish a replicative compartment, termed theparasitophorousvacuole(PV),andfromheremanipulatehostcellfunctionsviathereleaseofeffectorproteins.Apotentialeffectorproteinencodedbythegenecbu_1651,whichisuniquetoC.burnetii,isofinterest.Usinginsilicoanalyses,cbu_1651ispredictedtohaveatransmembranedomainandislikelyco-regulatedwiththeT4BSSsinceitislocatedbetweentheT4BSSgenesicmWandicmXandsuggestsapossibleimportantfunctionforCBU_1651duringpathogenesis.TheoverallgoalofthisstudywastocharacterizethelocalizationofCBU_1651duringinvivoandinvitrogrowth.WehavesuccessfullydevelopedpolyclonalantiseraagainstCBU_1651andareusingitforsubsequentassays.WehavefoundbywesternblotthatCBU_1651issecretedinaT4BSSdependentandlipiddependentmannerintothegrowthmedium.Intissueculture,indirectfluorescentantibody(IFA)assaysindicatedCBU_1651tolocalizetothePVlumen.ThisdatasuggeststhatCBU_1651ismediatedbyamechanismthatsensesenvironmentalstimuli.
5. Characterizationoftheroleofredox-activecysteinesintheregulatoryfunctionofDksAintheLymedisease
spirocheteBorreliaburgdorferi.SamKoshy(Undergraduate)*,WilliamBoyle,andTravisJ.Bourret,CreightonUniversitySchoolofMedicine,Omaha,NE
BorreliaburgdorferiisthecausativeagentofLymedisease,andisestimatedtoinfectasmanyas300,000individualsperyearintheUnitedStates.B.burgdorferi’slifecycleincludesthewhite-footedmouse(Peromyscusleucopus),ticksofthegenusIxodes,andhumans.Asitistransmitted,itundergoesavarietyofenvironmentalchangesthatincludeshiftsinpH,temperature,andnutrientavailability.ThisprojectaimstoexploretherelevanceofcysteinesencodingthezincfingerdomaininthetranscriptionfactorDksA(DnaKsuppressorprotein)thatisknowntoplayakeyroleinthestringentresponse.Thezincfingerwillbeassessedbyperformingsite-directedmutagenesisofcysteineswithinaB.burgdorferidksAalleleencodedonplasmids.TheeffectsofthesepointmutationsonDksAfunctionwillbedeterminedbyintroducingthelibraryofmutantplasmidsintodksA-deficientB.burgdorferistrains.Theabilityofthesestrainstocontroltheexpressionofstringentresponsegenes,survivenutrientlimitationandoxidativestress,andinfectpotentialhostswillbeconducted,highlightingtheimportanceofthezincfingermotifwithinDksAinregulatingthestringentresponse,andtogaininsightintothehowB.burgdorferisurvivesandproliferatesinhostileenvironments.
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6. IdentifyingIntracellularTick-borneIllnessesinBison,Bisonbison,viaBloodCellStainingandPCR.Robertson,A.D.(Undergraduate)*,andA.R.Oller.DepartmentofBiologyandAgriculture,UniversityofCentralMissouri.
PrevioustickvectorstudieshaveconfirmedBabesia,Anaplasma,andEhrlichiainCentralMissouriticks.Eachmicroorganismisknowntocausezoonoticcasesofdiseaseinmammalianhostslikeruminantsviaticks.Althoughsometickstudiesutilizedcattle,Bisonstudiesarelacking.Withbison,becominganincreasingfixtureinpasturesduetovariousdietarychangesinmeatconsumptionpractices,thepresenceoftick-borneillnessesincaptivebisonherdsisgreatlyneeded.Theveterinariancollectedapproximately40bloodsamplesviatailveinvenipunctureusingEDTAasananticoagulant,andwasrefrigerated.Samplenumberscontainedgender,age,andweight.Thisprojectlookedatbloodparasitemia(%)ratestoconfirmthepresenceofcirculatingbloodparasites,asitisimportanttoestablishexpectedparasitesandco-infectionswithinbisonherdsinMissouri.BloodsmearsweremadeandstainedwithafilteredWright-Geimsastainthatwouldalsoallowparasitevisualization.Outofatotalof38Bison,fivehavebeenthoroughlyexaminedforpathogens.4thusfarhavesuspectedcasesofAnaplasma,threeEhrlichia,andoneBabesia.Someofthebisonhavecoinfections;furtherexaminationofbloodsmears,alongwithPCRtestingwillyieldmoreconciseresults.IACUCapprovalofUCM#928wasalreadyobtainedandgrantedforthispathogensurvey.
7. GeneratingDeletionandPointMutationstoStudyaNovelPhytase-likeProtein,CarP,That
ProtectsPseudomonasaeruginosafromElevatedCalcium.DanielMcLeod(Undergraduate)*,MichelleKing,Dr.MariannaPatrauchan.OklahomaStateUniversity,Stillwater,OK.
Pseudomonasaeruginosaisanopportunisticpathogenthatinfectswoundsandthelungsofcysticfibrosispatients.Previously,weshowedthatseveralvirulencefactorsinP.aeruginosaareinducedbyelevatedcalcium(Ca2+).Weidentified a putative phytase, CarP, and determined its role in protecting cells against Ca2+ and regulating Ca2+-inducedvirulencefactors.Therefore,wehypothesizedthatthisproteinbindsCa2+andbelongstotheCa2+regulatorynetwork in P. aeruginosa. To study the role of carP in Ca2+ regulation, we used a transposon mutant and acomplementationstrain,wherecarPisclonedunderanarabinose-induciblepromoter.HereweaimtogenerateacleancarPdeletionstrainandacomplementedstrainwherecarPwillberegulatedbyitsnativepromoter.SequenceanalysesshowednoknownCa2+bindingmotifsinCarP,andweaimtoidentifytheresponsibleresidues,whichwilllikelypresentanovelCa2+-bindingmotif.Weareusing inversePCRto replaceE183andE291predicted tobindCa2+with a glutamine,whichwill preventbinding.Wewill thenmeasure theproteins’ ability tobindCa2+ usingisothermalchromatography.Obtainingthesemutantswillalsoenablefuturefunctionalstudies,characterizingtheroleofCarPinP.aeruginosavirulence.
8. SoilMicrobeIsolationSurroundingNativeandInvasiveGrassestoTestforAntimicrobialPropertiesagainstE.S.K.A.P.E.relatives.GeorgieO.Tauber(Undergraduate)*,ClaudiaM.Carvalho,andMitchellJ.GreerDepartmentofBiologicalSciences,FortHaysStateUniversity,Hays,Kansas
Asnon-nativespecies(suchasBothriochloaischaemumandB.blahdii)continuetoinvadenativelands,theyalterthelandscape.Bothriochloaspp.havebeenshowntohaveallelopathiceffects.Theseallelochemicalsneedmoreinvestigationastowhetherornotthesebiochemicalsalterthemicrobesinthesurroundingsoiland/orhaveantimicrobialproperties.Ifso,theseantimicrobialpropertiescouldleadtonewantibiotics.AntibioticandantimicrobialresistancesuchasintheE.S.K.A.P.Epathogens,hasbeguntorunrampantaroundtheglobeandhascausedgreatdilemmatomanyphysicians.TheE.S.K.A.P.E.pathogenspresentarealthreattooursocietytoday.SamplesweretakenfromsoilsurroundingBothriochloaischaemum,B.blahdii,andAndropogongerardii-anativegrass.Afterinitialserialdilutionandselectionofcolonies,theisolatedcoloniesweretestedagainstE.S.K.A.P.E.pathogenrelativesforantimicrobialproperties.Therewerezerozonesofinhibitionfromsamplestakenneartheinvasivespecies,whiletwocoloniesfromAndropogongerardiisamplesshowedzonesofinhibition(bothagainstStaphylococcusepidermidis).Thebacteriawerefoundtobeagram-postive.Furthertestingwillbeperformedtodeterminetheidentityofthebacteria.
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9. LongevityAnalysisofGerm-freeDrosophilamelanogaster.MakaylaNemecek(Undergraduate)*,RebeccaBest,ShelbyPeters,CarliePrososki,LesleyTowery,BradL.Ericson,DarbyJ.Carlson,&Kimberly.A.Carlson,DepartmentofBiology,UniversityofNebraskaatKearney,Kearney,NE68849
Gastrointestinalmicrobiotaandvirusesareakeycomponentincharacterizingguthealthandlongevity.Noravirus,apersistentvirusthatreplicatesinthegutofDrosophilamelanogaster,showsnolethalitytotheorganism.VirusessimilarinnaturetoNoravirusinteractwithgutmicrobiota,andthehealthoftheorganismandlongevitymaybedependent on a persistent viral infection. Our hypothesis is that Nora virus may be needed within thegastrointestinaltractofD.melanogastertomaintainafavorableenvironmentforgutmicrobiotaandincreasethelifespan. GermfreeD.melanogasterweregeneratedwiththeuseofantibioticsanddividedintofourtreatmentgroups: Noraviruspositive/bacteriapositive,Noravirusnegative/bacteriapositive,Noravirusnegative/bacterianegative,andNoravirusnegative/bacterianegative.ThepresenceofNoraviruswasdetectedwiththeuseofRT-PCRandbacterialspecieswasdeterminedbyplatinghomogenizedfliesonLuriaBrothplates.Alongevitystudywasconductedoneachof the treatmentgroupsanddemonstrated thatNoravirusdoesnotenhance longevity,butmicrobiotaisneededwhenvirusispresenttolive.ThisdatasuggeststhatNoravirusinfectionisnotbeneficialtothemicrobialenvironmentwithinthegastrointestinaltract,butfurthertestingisneeded.
10. SilverNanoparticlesonYeastViabilitywithBioinformaticsAnalysis.CullenHorstmann(undergraduate)*,and
KyoungtaeKim.MissouriStateUniversityDepartmentofBiology,Springfield,Missouri.Nanoparticleshavebecomecommoninmanycommerciallyusedproductssuchaszincsunscreenandwaterresistantclothes.Theymayalsobeutilizedinthetargetedtreatmentofcancer,printablemonitoringsystems,andcosteffectivephonesinthefuture.Theeffectsnanoparticleshaveonbiologicalorganismsiscrucialfortheresponsibleuseofthesetechnologies.Weinvestigatedtheeffectsofsilver(Ag)nanoparticlesonbuddingyeast(Saccharomycescerevisiae)usinggrowthassays,FUN-1stainingformetabolicactivity,RNAseq,andRTPCR.OurgrowthassayshowedthatAghasaninhibitoryeffectwithitsconcentrationsabove5µg/ml.HundredsofgenesinAgtreatedcellsweredifferentiallyexpressedaccordingtoourtranscriptomeinvestigation.AlargefractionofupregulatedgenesareidentifiedtoregulateribosomalbiogenesisandRNAprocessing,whereasdownregulatedgenesareknowntoberesponsibleformitochondrialfunctionsbasedonouranalysisofgeneontologyterms.Furthermore,wevalidatedtheRNAseqresultsusinganRTPCRassay.TheresultingexpressionprofileleadsustosuspectthatAgnanoparticleexposurecreatesastressenvironmentinthecell.11. SiteDirectedMutagenesisofKnownVps1UbiquitinationSites.RyanWindish(Undergraduate)*,Kyoungtae
Kim.MissouriStateUniversity,Springfield,Missouri.Ubiquitinationisacellularprocessthatisimportantforproteindegradation.Itoccursthroughaseriesofenzymaticreactionsinwhichubiquitin,akeyfactorubiquitouslyexpressedineukaryoticorganisms,tagsthesubstrateproteinstobedegradedviaproteasomes.VacuolarProteinSorting1(Vps1),whichisyeast’shomologuetomammaliandynamin,hasfiveknownubiquitinationsites.ThisproteiniscrucialtotheretrievalofbothVps10andSnc1,whichplaysignificantrolesforintracellularvesiculartraffickingpathways.WehaveperformedexperimentsbymutatingtheknownubiquitinationsitesofVps1throughsitedirectedmutagenesis.AmutantstrainharboringVps1K561NmutationdisplayedseveredefectsinthetraffickingofSnc1andVps10,suggestingthattheVps1ubiquitinationsitesarepivotalfortheirtraffickingtowardtheGolgiandthatproperturnoverofVps1regulatesthesecargotraffickingprocesses.
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12. InitialCharacterizationoftheTwoClpPIsoformsofChlamydiatrachomatisSuggestsIndependentFunctionalityforEach.NicholasA.Wood(Undergraduate)*1,2,KrystalChung3,NathaliaRodriguesdeAlmeida1,MartinConda-Sheridan1,DerekJ.Fisher3,ScotP.Ouellette11UniversityofNebraskaMedicalCenter,Omaha,Nebraska.2UniversityofSouthDakota,Vermillion,SouthDakota.3SouthernIllinoisUniversity,Carbondale,Illinois.
Chlamydia trachomatis is anobligate intracellular bacterium thatdifferentiatesbetween twodistinct formsduring itsdevelopmentalcycle:elementarybodies(EBs)andreticulatebodies(RBs).TheEBistheelectrondense,infectiousform.Withinthecell,theEBdifferentiatesintotheRB,whichreplicatesanddevelopswithinahostmembranederivedvesicle,termedaninclusion.RBsreplicatewithinthisinclusionandeventuallydifferentiatebackintoEBsbeforereleasefromthehostcell.GiventheuniquefunctionalandmorphologicalformsofChlamydia,weareinterestedinproteomicregulationandturnoverthroughproteindegradation.WehypothesizethattheClpproteasesystemplaysanintegralroleinproteomicturnoverbydegradingspecificproteinsfromonedevelopmentalformortheother.Chlamydiaencodesfiveclpgenes:clpX,clpC,twoclpPparalogs,andclpB.ClpCandClpXarechaperoneproteinsthatunfoldandfeedproteinsintotheClpPproteasetobedegraded,andClpBisadeaggregase.Our initialcharacterizationfocusesonthetwoClpPparalogs.Wedemonstrate theirdevelopmental expression,potential foroligomerization, importanceduring infectionbyantibioticstargeting ClpPs, and the effect of overexpression of inactive ClpPmutant proteins. Together, these data indicate animportantroleforClpPproteinsinchlamydialgrowthandpathogenesis.13. UsinginterpretableneuralnetworkmodelsofPseudomonasaeruginosageneexpressiontorevealpotential
targetsofanunstudiedtranscriptionfactorimplicatedinhighresistancetoanovelantimicrobialpeptide.ChristopherJohnson(Undergraduate)*andDr.DonaldRowen.UniversityofNebraskaatOmaha,Omaha,Nebraska
Antimicrobialpeptides(AMPs)areanintriguingalternativetocurrentlyavailableantibiotictherapies.DASamP2,anAMPdevelopedbytheWanglabattheUniversityofNebraskaMedicalCenter,iseffectiveagainstPseudomonasaeruginosaandStaphylococcusaureus,butthemechanismofactionisnotcurrentlyunderstood.TocharacterizethegeneticfactorsaffectingsusceptibilitytothisAMP,ourlabhaspreviouslyusedtransposonmutagenesistoconstructnumerousmutantstrainsofP.aeruginosademonstratingincreasedresistancetoDASamP2.AnalysisofoneofourmostresistantmutantsrevealedthatupregulationofthetranscriptionfactorPA5189mayberesponsibleforproducingaresistantphenotypecapableofsurvivingover10xMIC.Thoughthisgeneisunstudied,wecanextractbiologicallymeaningfulinsightsfrompubliclyavailablegeneexpressiondatasetsbyusingdenoisingautoencodersordisentangleddeepvariationalautoencoderstoproduceacompressedrepresentationofallgeneexpressiondatasets.Analyzingatrainedneuralnetworkallowsustoconnectrawexpressiondatatohigher-levelbiologicalfeatures.EarlyresultssuggestPA5189maybeinvolvedintheregulationofnumerouseffluxpumps,porins,andothergenes–mostofwhicharealsounstudied,potentiallyindicatingthatwearelookingatanewfacetofhowP.aeruginosaadaptstoantimicrobialassault.
PosterSession2(GraduateStudents)
1. SodiumPyruvateAlterstheImmuneResponsetoInfluenzaAVirusInfectioninMacrophagesHazarAbuSalamah(Graduate)*.Dr.ChristopherLupfer.DepartmentofBiology,MissouriStateUniversity,Springfield,MO65897
Pyruvateistheendproductofglycolysis.ItcaneitherbetransportedintothemitochondriaforuseintheTCAcycleorbeusedtoregenerateNAD+duringaerobicglycolysis.WerecentlydiscoveredthatadditionofsodiumpyruvatetotheculturemediumduringinfectionofmacrophageswithinfluenzaAvirusaffectstheproductionofcytokinesinvolvedinimmunesignaling.Thepurposeofthepresentstudywastodeterminewhethersodiumpyruvate’sroleinenergyproductioninthemacrophagesmayalter the immune response to the infection.While infectionofmacrophageswith influenzaAvirusresulted inhigh levelsofcytokines (IL-6, IL-1β,andTNF-α) in theabsenceofsodiumpyruvate, theadditionofsodiumpyruvatesignificantlyimpairedcytokineproduction.Furthermore,sodiumpyruvatedidnotaffectvirusgrowth,suggestingtheeffectofsodiumpyruvateisontheimmuneresponseproducedbythemacrophagesandnottheviabilityofthevirus.
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2. CharacterizationoftheRoleofPA5189ofPseudomonasaeruginosainResistancetoanAntimicrobialPeptide.ZacharyScott(Masters)*,ChristopherJohnson,andDonaldRowenDepartmentofBiology,UniversityofNebraskaatOmaha,Omaha,Nebraska
Pseudomonasaeruginosaisagramnegativebacillusbacteriumknownforitshighdegreeofantimicrobialresistanceandpathogenicity.Antimicrobialpeptides(AMPs)arepeptides,usuallybetween12and50aminoacidsinlengththatpossessantimicrobialactivity.ThemechanismofactionofonlyafewAMPsisknown.IamworkingwiththesyntheticAMPDASamp2whichhasbeenshowntobeeffectiveagainstP.aeruginosainliquidculturesandinbiofilms.OurlabhasusedtransposonmutagenesisofP.aeruginosatotrytoelucidatethemechanismofactionofDASamp2.Weisolated12mutantswithalteredsusceptibilitytoDASamp2.Ofthoseisolated,theF2-1mutantwasverypromisingbecauseitsMICwasincreased8fold.WefoundthattheF2-1mutanthadatransposoninsertedintothepromoterregionofthegenePA5189.PA5189ispredictedtoencodeatranscriptionfactorofunknownfunction.WehypothesizedthatthetransposonwascausingoverexpressionofPA5189.UsingqRT-PCR,IhaveobservedthatthelevelofPA5189mRNAis7foldhigherintheF2-1mutantthaninwildtypePCR.ThissuggeststhatoverexpressionofthepredictedtranscriptionfactorPA5189canaffectsensitivityofP.aeruginosacellstoanAMP.3. CHARACTERIZATIONOFTWONOVELANTIMICROBIALSAGAINSTAcinetobacterbaumannii. InfenciaXavier
Raj(Masters)*1,AnuradhaRoy2,andIndranilBiswas1.1 Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center,KansasCity,KS66160.2HighThroughputScreeningLab,UniversityofKansas,Lawrence,KS66045
AcinetobacterbaumanniibelongstoagroupofESKAPEpathogens;whichisanopportunisticpathogenprevalentinnosocomialinfections.Thispathogencancauseawiderangeofinfectionsfromminorskinandsoft-tissueinfectionstomoresevereinvasivediseasessuchasbacteremia,meningitisandventilator-associatedpneumonia.A.baumanniihastheabilitytoacquireantibioticresistancecassettesfromtheenvironmentandthistraithasallowedtheorganismtopersistinhealthcaresettingsandalsofacilitatedglobalemergenceofmultidrugresistance(MDR).Theorganismisbecomingresistancetomostofthecommonantibioticsincludingaminoglycosides,β–lactamsandquinolones.TofightagainsttheincreasinginfectionscausedbyESKAPEpathogens,therehasbeenanincreasedeffortinthelastfiveyearstoidentifynovelsmallanti-infectivemoleculesortomodifyexistingcompoundstoincreasetheirpotency.Usingahighthroughputscreeningassay,werecentlyexaminedseveralsmall-moleculelibrariesattheIDADCoreFacility at KU, Lawrence. We identified several potential candidates among ~20000 compounds that eitherspecificallyinhibitedA.baumanniistrainsordisplayedbroad-spectruminhibitoryactivityagainstA.buamanniiandother gram-negative ESKAPE pathogens. The main goal of this study is to characterize two such anti-infectivemoleculestodeterminetheirinhibitoryspectra,modeofaction,andsynergisticactivitieswithotherantibiotics.4. TheHostProteinKinaseCismanipulatedbyChlamydiatrachomatisDuringInfection
PrakashSah1(Doctoral)*,TedHackstadt2,ErikaLutter11Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, OK, USA;2LaboratoryofIntracellularParasites,NIAID,NIHRockyMountainLaboratories,Hamilton,USA
Chlamydia trachomatis is responsible for causing a range of diseases such as blinding trachoma and urogenitalinfectionsleadingtoseriouscomplications.Insideahostcell,C.trachomatislivesinaparasitophorousvacuolecalledaninclusionfromwhereitisabletosecretevariouseffectorstomanipulatehost-cellularfunctionstoitsbenefit.Currently,notmuchisknownaboutChlamydialmanipulationofhostkinasessuchasProteinKinaseC(PKC).PKCsaremembersofAGC familyof kinasesand involved in regulating various cellular functions suchas, growthandproliferation,migration,survivalandapoptosis.WehypothesizethatC.trachomatismanipulatesPKCpathwaystoregulateintracellulardevelopmentinsidethehost,asPKCsareimportant inregulatingvariouscellularfunctions.Indirect immunofluorescenceof infectedcells verified recruitmentofmultiplePKC isoenzymes tomicrodomains(Src-familykinasesrichregions)ontheinclusionofC.trachomatisL2/434.RecruitmentofPKCsubstrates,includingMarcks,wasalsoconfirmed.RecruitmentofPKCsandPKCsubstrateswasfoundtobespeciesspecific.InhibitionofPKCactivitywithStaurosporineandGo6983resulted indecreasedrecoverable infectiousprogeny.WesternblotanalysisrevealeddifferentialactivationofPKCatdifferenttimepointsduringinfection.TheseresultsconfirmPKCsareimportantforintracellulargrowthanddevelopmentofC.trachomatis.
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5. AProkaryoticdCTPDeaminaseintheEukaryoteDictyosteliumdiscoideum.HengLiang(Doctoral)*,PI:CatherineP.Chia,SchoolofBiologicalSciences,UniversityofNebraska-Lincoln,Lincoln,NE,USA.
DNAsynthesisiscrucialinalllivingorganisms.Deoxycytidinetriphosphate(dCTP)deaminase(EC3.5.4.13)isanenzymeinthepathwaywhichconvertsdCTPintodeoxythymidinetriphosphate(dTTP),oneofthebuildingblocksofDNA.Althoughtypicallyfoundonlyingram-negativeprokaryoticcells,twogenesencodingpredicteddCTPdeaminases(dcd1:DDB_G0293580anddcd2:DDB_G0268194)areidentifiedintheannotatedgenomeofDictyosteliumdiscoideum,aeukaryoticsoilamoeba.D.discoideumalsohasgenesfordCMPdeaminaseandthymidylatesynthase,enzymesforpyrimidinebiosynthesisineukaryotes.Weareinvestigatingwhetherdcd1anddcd2areexpressed,andwhethertheactivitiesandlocationoftheirrespectiveproteinsareregulatedeitherbythecellcycleorduringdevelopment.PhylogeneticanalysesofthepredictedproteinsequenceswithprokaryoticdCTPdeaminasesindicatethatdcd1anddcd2havebacterialancestorsthatlikelyenteredtheD.discoideumgenomethroughtwoindependentgenetransferevents.Consistentwithitssuggestedbacterialorigin,theD.discoideumdcd1rescuedtheslowgrowthphenotypeofanE.colidCTPdeaminaseknockout,indicatingthatdcd1encodesanenzymecapableoffunctioningintheprokaryoticpyrimidinebiosynthesispathway.Constructionofadcd1knockoutinD.discoideumisinprogresstoexaminethespecificrolesofdCTPdeaminaseinD.discoideum.6. CharacterizationoftheIS3FamilyofInsertionSequencesintheGenomeofHalanaerobium
hydrogeniformans.RonaldL.Frank,KodyA.Bassett(Masters)*,MelanieR.Mormile.MissouriUniversityofScienceandTechnology,Rolla,Missouri.
Halanaerobiumhydrogeniformansisananaerobic,gram-negative,rod-shaped,haloalkaliphilicbacteriumisolatedfromSoapLakeinWashingtonState.Thegenomesequencewasdeterminedin2011aspartofanefforttorevealsomeoftheadaptationsthatenablethisbacteriumtogrowinahighsalt,highalkalineenvironment.Oneprongofthateffortisagenome-widesurveyofanunusuallyabundantnumberoftransposableelements.Theresultsreportedherearelimitedtooneofninefamiliesofbacterialinsertionsequencesfoundinthis2.6Mbgenome.TheH.hydrogeniformansgenomecontains29locithatharboreitherfunctional,defective,orfossilremnantsoftheIS3familyofbacterialinsertionsequences.Theelementshavebeendividedintofivegroups(ISHahy2,3,4,5,andasolopartialelement).EachofthefivegroupsappearstohaveoriginatedfromanindependentinvasionofthegenomebyauniqueIS3-relatedelement.ThetransposaseoftypicalIS3elementsisencodedbytwooverlappingout-of-frameorfsAandBbyaprogrammedtranslationalframeshiftataslipperysiteupstreamofthestopcodoninorfA.WedescribeherethecharacteristicsofthefivegroupsofIS3-relatedelementsinHalanaerobiumhydrogeniformansastheyrelatetothatmodel.7. TheFattyAcidKinaseFakAShapestheMetabolomeofStaphylococcusaureus.ZacharyDeMars(Doctoral)*
andJeffreyL.Bose.UniversityofKansasMedicalCenter,KansasCity,Kansas.Staphylococcusaureusiscapableofphosphorylatingexogenousfattyacidswhicharethenabletobeincorporatedintothebacteria’smembraneviathefattyacidkinaseFakA.Additionally,FakAplaysasignificantroleinvirulencefactorregulationandskindisease.WepreviouslyshowedthatafakAmutantdisplaysalteredgrowthkineticsinvitro,observedduringlate-exponentialphaseofgrowth.Here,weshowthatthisisbothglucoseandaeration-dependent,indicatinganalteredacetateswitch.Consistentwiththis,acetateproductionwasalteredandthegrowthbenefitwas eliminated with inactivation of the acetate-generating enzyme AckA. Using a mass spectrometry-basedapproach,weidentifiedalteredconcentrationsofTCAcycleintermediatesandbothintracellularandextracellularaminoacids.Together,thesedatademonstratearedirectionofcarbohydratecarbonflowandalteredaminoacidmetabolisminthefakAmutant.WhileATPlevelsweresimilar,inactivationoffakAincreasestheNAD/NADHratio,indicatingamoreoxidizedcellularenvironment.Finally,wesuggestthattheglobalmetabolicregulatoryproteinsCcpAandCodYasbeingimportantregulatorymechanismforthealteredgrowthinafakAmutant.Together,thisdataidentifiesapreviouslyunidentifiedroleforfakAintheglobalphysiologyofS.aureusthatlinksthefattyacidandcentralmetabolism.
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8. CharacterizationoftheNitrogenAssimilationRegulator(glnG)RoleinEscherichiacoliColonizationoftheMammalianIntestines.JustinBowen(Master’s)*,TyrellConway,JerremeJackson.OklahomaStateUniversityMicrobiologyandMolecularGenetics,Stillwater,OK.
Nitrogen,oftenintheformofammonia,isusedbybacteriatogenerateaminoacids.TheglnGgeneinEscherichiacolicodesforthenitrogenassimilationregulatorprotein,NR1,whichactivatesthegenesresponsibleforsurvivalduringammonialimitation.Undernitrogen-limitedgrowthconditionsNR1activatestranscriptionofglutaminesynthetasewhich,alongwithadditionalproteins,facilitatestransportanddegradationofnitrogen-containingcompounds.WhiletheE.colitranscriptionalresponsetonitrogenstarvationinvitrohasbeenstudiedextensively,nitrogenstarvationduringE.colicolonizationofthemammalianintestinehasreceivedlittleattention.Inthisstudy,wewilluseaglnGknockout(DglnG)mutanttostudytheroleofnitrogenmetabolisminMG1655colonizationofthemouseintestine.TotalRNAfromMG1655recoveredfromthemucosalliningofthemouseintestinewillbepreparedandsequencedbydifferentialRNA-seqanalysis,whichwillallowmappingofallglnG-dependentpromoters.WeexpecttheglnGmutantE.colitofailtocolonizetheintestinesiftheglnGgeneisvitaltocolonizationofthemammalianintestine.
9. GENERATIONOFYEAST2-HYBRIDCLONESTOEXAMINETHEROLEOFNUCLEOTIDEOLIGOMERIZATIONAND
BINDINGDOMAIN(NOD)-LIKERECEPTORS.AbbigaleMabary(Masters)*,FacultyAdvisor:Dr.ChristopherLupfer.MissouriStateUniversity.Springfield,Missouri.
NOD-likereceptors(NLRs)areaclassofcytoplasmicproteinsessentialfortheinitiationandregulationofimmuneresponsestoinfectiousdisease,metabolicandcellulardamageandcancer.Thehumangenomeencodesfor22NLRproteins.However,onlyabouthalfofthe22NLRshaveknownfunctions,andthemechanismsbywhichtheyfunctionareevenmoreambiguous.PreviousresearchindicatesthatsomeNLRsactivateinflammation,whileothers,likeNLRP12,functionsasregulatorsofinflammation,thusservingasanegativefeedbackmechanism.NLRP12suppressesinflammationbyinhibitingthetranscriptionfactorNF-κB,whichactivatestranscriptionforcytokinesthatactivatetheimmuneresponsecascade.InhibitionofNF-κBbyNLRP12isimportantinthepreventionofahyper-inflammation,whichisinvolvedinsevereinfectionsaswellascancerdevelopment.AlthoughthegeneralfunctionofNLRP12isknown,howitisactivatedisnotknown.Weare,therefore,embarkingonajourneytofindnovelproteinsthatinteractwithNLRP12todecipherthemechanismsbywhichtheyfunction.Wearegeneratingayeast2-hybridsystemtoexaminetheinteractionofNLRP12withahumancDNAlibrary.Novelinteractionsdiscoveredthroughthis2-hybridscreenshouldprovideinsightintothefunctionofthisNLRproteinandhelpusunderstandtheimmuneresponsetoinfectiousandnon-infectiousdisease.10. Ecology and prevalence of ticks and tick-borne bacterial pathogens in a peri-urban landscape of the
MidwesternU.S. Abrar Alzahrani (Masters)*1, NicholasA.Burnett1, Ram Raghavan2, and Anuradha Ghosh1 1.
Dept.ofBiology,PittsburgStateUniversity(Pittsburg,KS),2.Dept.ofDiagnosticMedicine/Pathobiology,KansasStateUniversity(Manhattan,KS)
Ticks transmit a wide variety of pathogens including viruses, bacteria, protozoa, and helminthes to vertebrates. Their life cycle depends on blood meals from various hosts as well as on environmental conditions such as the temperature and habitat type. The present study proposed to assess the prevalence of various tick species and infection prevalence of bacterial pathogens causing various diseases within the tick community of southeast Kansas and adjacent area. Over 2000 ticks were collected during warmer months of 2016 and 2017 (May-August) from three types of tick habitats (woodland, open grassland and woodland/grassland ecotones) using the flag-drag method. All the ticks (adults and nymphs) were sexed and identified in the laboratory. Majority of these were identified as Amblyomma spp. followed by Dermacentor spp. and rarely Ixodes spp. The ticks were surface-sterilized and total genomic DNA is currently being extracted from the adult ticks; and will be subjected to PCR amplification using bacterial species-specific primers. Microclimate data as well as landscape fragmentation pattern will be analyzed using GIS-based monitoring method. It is comprehensible that a better understanding of the variations in tick-pathogen prevalence is crucial for implementing sound surveillance and management programs and to understand risk for human/animal diseases.