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ARMENISE-HARVARDSYMPOSIUM2002 STRUCTURALBIOLOGYANDMICROBIALPATHOGENESIS ANDTHEHOSTRESPONSE 6thAnnualSymposium June27-29,2002,St.Thomas,U.S.VirginIslands AbouttheSymposium Thethreatofattack,theimportanceofdefense,andtheneedtoknowmoreabouteachhave becomenearuniversalworriessinceSeptember11.Peoplewhoseldomthoughtabouttheseissues inthewakeoftheColdWarnowavidlyfollowthedailynews.Incontrast,attack,defense,and intelligencegatheringarelong-standingconcernsfortheselectgroupofresearcherswho participatedin“StructuralBiologyandMicrobialPathogenesisandtheHostResponse,”the6th AnnualSymposiumoftheGiovanniArmenise-HarvardFoundation.Althoughthesymposium’s themewasdecidedbeforebioterrorismbecamearealitylastFall,thoseeventsgreatlymagnifiedits relevance. Anthrax,AIDS,tuberculosis,andmalariawereamongthegloballyimportantdiseasesexploredin 22invitedlecturesandanequalnumberofposterpresentations.InvestigatorsdescribedusingXraycrystallography,aswellasmicroarraysandotherpost-genomictools,tobetterunderstandthe battlebetweenpathogensandtheirhosts.Thestakesarehigh:speakersemphasizedthatdespite medicalprogress,infectiousdiseasesstillaccountforoneinthreedeathsworldwide–thesametoll theytookonhumankind400yearsago. ThesymposiumwasheldJune27-29atMarriottFrenchman’sReef,St.Thomas,U.S.VirginIslands. AlthoughtheCaribbeanisjustlycelebratedforitsnaturalbeauty,thepeopleoftheseislandslive withendemicmalariaandtuberculosis,andthisregionissecondonlytosub-SaharanAfricainits rateofHIVinfection. Inherclosingremarks,Dr.GiuliaDeLorenzowasoptimisticabouttheimpactofresearchpresented duringthesymposium.Newtechnologiessuchasstructuralbiology,bioinformatics,andfunctional genomicswillhelpgeneratenotonlynewtreatmentsandvaccinesforcombatinghumaninfectious disease,butalsonewstrategiesforprotectingtheglobalfoodsupply.“Understandingthemolecular logicofpathogenesisiscrucialforfutureprogress,”saidDr.DeLorenzo,aprofessorofplantbiology attheUniversita’DiRomaLaSapienzaandamemberoftheprogramcommitteeforthe symposium. Lastyear,FoundationPresidentandCEODanielC.TostesoninauguratedaCareerDevelopment programthatprovidestalentedyoungItalianinvestigatorswithsupportneededtoestablish researchlaboratoriesintheirhomecountry.The2002CareerDevelopmentawardswerepresented toGiampietroSchiavoandRosellaVisintin.Schiavo,whohasbeenstudyingmembranedynamicsat thenerveterminalattheImperialCancerFundinLondon,willbejoiningtheDepartmentof BiologicalChemistryattheUniversityofPadova.Visintinhasbeeninvestigatingkeyregulatorsof mitosisattheMassachusettsInstituteofTechnology,andwillbereturningtoItalytoestablishalab attheEuropeanInstituteofOncologyinMilano.Visintinwasunabletoattendthesymposium. Schiavo,whogavealecture,wastheonlyparticipantaffiliatedwithaU.K.institution;28scientists camefromfiveleadingItalianresearchinstitutionsand48representedthesixbasicscience departmentslocatedontheQuadrangleatHarvardMedicalSchool.Italianparticipantscamefrom theEuropeanInstituteofOncologyinMilano,theUniversityofPadova,theInstituteforCancer ResearchandTreatmentattheUniversityofTorinoSchoolofMedicine,theDipartmentodiRicerca BiologicaeTecnologica(DIBIT)atScientificInstituteSanRaffaeleinMilano,andUniversita’Di RomaLaSapienza. ReverseVaccinology:agenome-basedapproachtovaccinedevelopment </em><strong>RinoRappuoli</strong> IRIS,ChironSpA,Siena <palign=""left"">Historically,thefirststepinvaccinedevelopmentwastogrowtheinfectious agentinthelaboratory.Onceapathogenwasavailableinthelab,itwaskilledorattenuatedfor testingasavaccine,thosebeingtheonlyapproachesknownpriortotheadventofrecombinant DNAtechnology.Anewerabeganwhengeneticengineeringmadeitpossibleforvaccinologiststo scrutinizelaboratory-grownorganismsforindividualantigensthatmightbeusedtoelicita protectiveimmuneresponse–aprocessthatwaslabor-intensiveandoftenquiteslow.</p> <palign=""left"">Now,theabilitytosequencetheentiregenomesofmicroorganismshascleareda newpathtovaccinediscoverythatDr.Rappuolicalled“reversevaccinology.”Toillustratehowthis newapproachworks,hedescribedChiron’sworktowardapreventivevaccineforgroupB meningococcus,asevereandsometimesfataldiseaseofchildrenandadolescents.Insteadof growingandmanipulatinglargequantitiesofthispathogen,companyresearchersfeditsgenetic sequenceintoacomputerprogrammedtoscanforsequencesthatmightencodeantigenswith potentialasvaccines.</p> <palign=""left"">MeningococcusBchallengedreversevaccinologytodemonstratethatitisnot merelyanewtool,butalsoabetterone.After40yearsoftrying,conventionalmethodshadenabled vaccinologiststoidentifybetween15and20potentialgroupBmeningococcusantigens. Unfortunately,testsshowedthatnoneoftheseprovideduniversalprotectionagainstdisease.When Chironscientistsfedtheorganism’sfullsequenceintothecomputer,itpredictedthatsome600 antigensencodedbythebacteriummighthavepromiseforvaccinedevelopment.Thesewere expressedasrecombinantproteinsin<em>Escherichiacoli</em>andnearly350weretestedin mice;about90werenovelsurfaceproteinsand29oftheseappearedtostimulatedesirable immuneresponses.Within18months,reversevaccinologyhadyieldedmorevaccinecandidates thantraditionalapproachesgeneratedinfourdecades,Dr.Rappuolinoted.Thebestofthesenovel antigenshavebeenincorporatedintowhatdevelopershopewillbeauniversallyprotective vaccine,acandidatethatrecentlyenteredPhaseIclinicaltrials.</p> <palign=""left"">Chironisnowapplyingreversevaccinologytothegenomesofotherbacterial pathogens,includingpneumococcus,streptococcus,staphylococcusandtheorganismsresponsible formalariaandtuberculosis.Genome-basedpredictivetechnologyisalsobeingusedtodesign vaccinesagainstHIVandotherviruses.</p> <palign=""left"">Dr.RappuolicharacterizedtheAIDSpandemicas“today’sBlackPlague,”and warnedthatunlesstheeconomicsofvaccinedevelopmentcanbechanged,therewillbelittle incentiveforcompaniestodevelopvaccinesagainstAIDSandotherdiseasesthatmostlykillpeople inthedevelopingworld.Althoughvaccinesarethemostcosteffectivemedicalinterventionever known,theyarelessfinanciallyattractivetocompaniesthantherapeuticdrugs.Heendedhis lecturewithapleathatrichnationsshouldermoreresponsibilityformakingvaccinesthatare desperatelyneededaroundtheworld.</p> <hrnoshade=""noshade""size=""1""width=""400""/> <palign=""left""><strong>OtherPresentations</strong></p> <dl><dt></dt><dd><em>Anthraxtoxinandwaystoinhibitit </em><strong>R.JohnCollier</strong> DepartmentofMicrobiologyandMolecularGenetics HarvardMedicalSchool Email:<a href=""mailto:[email protected]"">[email protected]</a></dd></dl>Theeventsof Fall2001galvanizedpublicinterestinbioterrorism,andaddedtheterm“weaponized”tothe vocabularyofmillions.Althoughthereisnogenericdefenseagainstpathogensthatcanbeturned intoweapons,Dr.Collierisconfidentthatbasicresearchwillyieldstrategiesforbluntingthelethal powerofanthrax,themicrobethathislaboratoryhasstudiedfor15years. AsmostnewspaperreadersbecameawarelastFall,peoplewhocomeintoskincontactwiththe anthraxbacteriumareverylikelytosurviveinfection,whereasinfectionisnearlyalwaysfatalin peoplewiththemisfortunetoinhaleit.Atpresent,theonlydefenseisalimitedsupplyofanotvery goodvaccine;newandbettervaccineswon’tbeavailableforatleastseveralyears,andthereisas yetnopublichealthstrategyformassimmunization.Asaresult,Dr.Colliersaid,thenationis undeniablyvulnerabletoanthraxattack.Thisvulnerabilitycanbereduced,however,ifbetter scientificinsightsintoanthraxvirulencecanbetranslatedintodefensivestratagems. Bacillusanthracisproducesitstoxininthreeparts:ProtectiveAntigen(PA)bindstoareceptoron thehostcellandfacilitatesentryoftwoenzymeeffectorsknownasEdemaFactor(EF)andLethal Factor(LF).Astheirnamessuggest,EFcausesswellingwhereasLFcausesdeath.Originally secretedfromthebacteriaasnontoxicmonomers,orsmallmolecularbuildingblocks,these proteinsassembleonthesurfaceofreceptor-bearingcellstoformtoxicnoncovalentcomplexes.PA bindstocells,coordinatesself-assemblyofthecomplexes,andultimatelydeliversEFandLFtothe cytosol.EFisacalmodulin-dependentadenylatecyclase,andLFisazincmetalloproteasethat cleavesmembersoftheMAPKKfamilyandpossiblyotherintracellularsubstrates. NewfindingsfromDr.Collier’slabandotherresearchteamsindicatethatafterPAcomplexeswith EFandLFonthecellsurface,theresultingstructureschangeshapeseveraltimesastheyinsinuate themselvesthroughthemembraneandintothecell.Afullerunderstandingofthesestructurefunctionrelationshipssuggeststhreepossiblewaystoaborttheirdeadlymission,Dr.Collier reported.Thefirstisadominant-negativeinhibitor,aslightlymutatedformofPAthatappearsto protectinjectedratsagainstexposuretoanthraxtoxin.Anotherpossibilityisapolyvalentinhibitor, asmallpeptidethatpartiallyblocksLFbindingwithPAonthecellsurface;itisalsobeingtestedin animals.Finally,itmaybepossibletomakeasolubleformofthePAreceptorthatwouldattach itselftotheanthraxtoxinbeforeitreachedhostcells.Allthreeareunderactivestudy. <hrnoshade=""noshade""size=""1""width=""400""/> <dl><dt></dt><dd><em>Tetanustoxinanditsjourneyinmotorneurons </em><strong>GiampetroSchiavo</strong> MolecularNeuropathobiologyLaboratory,CancerResearchUK LondonResearchInstitute Email:<a href=""mailto:[email protected]"">[email protected]</a></dd></ dl>Infectionscausedbymembersofthe<em>Clostridia</em>familyhavelongfascinated neuroscientists,inpartbecauseneurotoxinsfromrelatedbacteriausesharedcellularpathwaysto producedramaticallydifferentclinicaleffects.<em>C.tetani</em>causestetanus,markedby lockjaw,thefixedsmileknownas<em>risussardonicus</em>,andrigidspasticparalysisthatcan befatal.<em>C.botulinum</em>,incontrast,causeswidespreadmuscleweaknessthatatworse becomesaflaccidparalysisthatcankill.Neitherissomethingthatadoctorwantstodiscoverina patient.Inthelaboratory,however,neurobiologistshaveusedbothtypesofneurotoxinsastools forunderstandingtransportmechanismsinnervecells. Dr.Schiavofocusesonaxonalretrogradetransport,amechanismnecessaryforneuronalsurvival, inwhichneurotropinsaretransportedfromnerveterminalstothecellbody.Thedistances involvedinaxonaltransportcanbeimpressive:inhumanterms,thedistancefromagiraffe’sbrain toitsspinalmotorneuronswouldbetwicethedistanceoftheTourdeFrance. Organelles,pathogens,neurotrophinsandothersynapticsignalingmoleculesusesimilar mechanismstojourneyfromnerveendingtocellbody.Tetanustoxin(TeNT)isamongthe virulencefactorsthatmakethistrip,whichbeginswhenitbindsattheneuromuscularjunctionand eventuallyleadstothespinalcord.There,TeNT’sinterferencewiththenormalreleaseofinhibitory neurotransmitterresultsinspasticitythatisthehallmarkoftetanusinfection. AlthoughscientistsknowquiteabitabouttheintracellularcatalyticactivityofTeNT,controlofits retrogradetransportislesswellunderstood.Dr.Schiavo’steaminventedatransportassaythat usesanontoxicTeNTfragment(TeNTH<sub>C</sub>)asaprobe.Workingwithlivingmotor neurons,theydeterminedthatTeNTbindstoastructurethatformsonlyinlipid-richmicrodomains inthecellmembrane,andtheysoonrealizedthatsequesteringcholesterolcouldblockentryof TeNT.WhentheypermittedTeNTH<sub>C</sub>tobind,itwasrapidlyendocytosedand transportedintwotypesofvesicularcarriers,oneroundandonetubular,whichtravelatdifferent speedsusingmicrotubulesandtheactincytoskeleton.Theorganellesarenotacidifiedduring axonaltransportandlacktypicalendocyticmarkers.Dr.Schiavo’steamdiscoveredthatoneof thesecompartmentsisthenormalonefortransportingnervegrowthfactor(NGF),whichis apparentlyhijackedbyTeNTH<sub>C</sub>forretrogradeaxonaltransport.WhenDr.Schiavo movestotheUniversityofPadova,heandcollaboratorstherewilluseaproteomicsapproachto characterizethesetransportcompartmentsmoreprecisely. <hrnoshade=""noshade""size=""1""width=""400""/> <dl><dt></dt><dd><em>Geneticdifferencesinthesusceptibilityofthehosttoanthraxlethaltoxin </em><strong>WilliamF.Dietrich</strong> DepartmentofGenetics HarvardMedicalSchool Email:<a href=""mailto:[email protected]"">[email protected]</a></dd ></dl>Therearetwosidestothestoryofeveryinfectiousdisease,onetoldbythepathogenandthe otherbythehost,andDr.Dietrichhasbeenlisteningcloselytothehost.TheLethalFactor(LF) componentofanthraxtoxinkillsmacrophages,keyplayersininnateandacquiredimmunity,by rupturingtheirplasmamembranesandcausingthecell’scontentstospill.AlthoughLFisknownto beazincmetalloproteasethatcleavesmembersoftheMAP(membrane-associatedprotein)kinase family,exactlyhowthesecleavageeventsleadtothedeathofmacrophagesisunclear.Dr.Dietrich’s laboratoryhasbeenexploringthisquestionusinginbredmousestrainswhosemacrophagesexhibit strikingdifferencesinsusceptibilitytotheeffectsofLF.Thesedifferenceshavebeentracedtoa singlegene,locateddownstreamfromtoxinentry,called<em>Ltxs1</em>(lethaltoxinsensitivity 1). <em>Ltxs1</em>hasbeenmappedtomousechromosome11,andDr.Dietrich’steamhas determinedthatitisactuallythesamegeneas<em>Kif1C</em>,whichencodesakinesin-like proteininvolvedinmicrotubuletransportofanunknowncargo.Severallinesofevidencesupport theideathatthetwoareidentical.First,<em>Kif1C</em>istheonlygeneinthe<em>Ltxs1</em> intervalexhibitingpolymorphismsbetweensusceptibleandresistantstrains.Second,multiple allelesof<em>Kif1C</em>determinesusceptibilityorresistanceofculturedmousemacrophages tointoxicationwithLF.Finally,treatmentofresistantmacrophageswithBrefeldin-A(which disrupts<em>Kif1C</em>localization)inducessusceptibilitytoLFintoxication,whileectopic expressionofaresistancealleleof<em>Kif1C</em>insusceptiblemacrophagescausesa4-fold increaseinthenumberofcellssurvivingLFintoxication.Withtheseobservationsinhand,Dr. Dietrichsaidthenextstepsinhisresearchwillinvolveworkingoutthecellbiologyof <em>Kif1C</em>todiscoverwhereitislocalizedandexactlyhowitoperates. <hrnoshade=""noshade""size=""1""width=""400""/> <dl><dt></dt><dd><em>StudiesontheinvolvementofSNAREproteinsinendosomal vacuolizationinducedby</em>Helicobacterpylori<em>VacAtoxin </em><strong>MarinadeBernard</strong> DipartimentodiScienzeBiomedicheSperimentali UniversityofPadova Email:<a href=""mailto:[email protected]"">[email protected]</a></dd></dl><em>Hel icobacterpylori</em>,aGram-negativebacteriuminvolvedinthedevelopmentofchronicgastritis, pepticulcerandgastriclymphoma,producesatoxin,calledVacA,whichinducesvacuoleformation inepithelialcellsinculture.Thesevacuolesoriginatefromlateendocyticcompartments,as demonstratedbythepresenceofthesmallGTPaseRab7intheirlimitingmembrane.Thepresence ofaRabproteinsignifiesthataSNAREproteinontheendosomaltransportcompartmenthasbeen properlymatchedwithacorrespondingSNAREproteinonthetargetmembrane. RecentstudiesshowthatafterVacAbinds,itundergoesconformationalchangesandformsan anion-specificchannelthatthelateendosomalcompartmentpassesthroughbeforeballooninginto avacuolethatmaybe400timeslargerthantheoriginalcompartment.Thisdramaticsizeincrease causedDr.deBernardtoaskwhetherthemembranefromasingleoriginalcompartmentprovides enoughrawmaterialforsuchahugeexpansion,orwhetheradditionalmaterialmustbegathered fromothervesicles.Ifothermaterialsarerequired,theymayderivefromseveralsources: homotypicfusionoflateendosomes,fusionofendosomesandlysosomes,ordepositionintothe membraneofcarriervesiclesderivedfromearlyendosomes.Eachofthesefusionshingesona SNAREhook-upthatcannottakeplacewithoutacytosolicproteincalledα-SNAP. Dr.deBernard’steamtransfectedcellswithadominantnegativemutantofα-SNAP,thenadded VacAextracellularlyorexpresseditwithinthecell.IfvacuolizationwasdependentonSNAREmediatedfusion,thennovacuoleswereexpectedtoform.Infact,theinvestigatorsobservedthat VacAhadnodifficultyinducinghuge,swollenvacuoleseventhoughSNARE-dependentfusionhad beenblocked.ThisfindingledDr.deBernardtohypothesizethatrawmaterialsforvacuole enlargementarealreadypresentintheoriginalendosomalcompartment,andthatRab7maybethe keythatturnsinternalmembranevesicles(orinvaginations)intoanoutermembraneforthe vacuole.SupportcomesfromrecentexperimentsshowingthatcellstransfectedwithadominantnegativeRab7mutantdidnotformvacuoleswhenexposedtoVacA,andfurtherinvestigationsare underway. <hrnoshade=""noshade""size=""1""width=""400""/> <dl><dt></dt><dd><em>Clostridialneurotoxins:mechanismofactionandtherapeuticuse </em><strong>CesareMontecucco</strong> CentroCNRBiomembraneandDipartimentodiScienzeBiomediche UniversityofPadova Email:<a href=""mailto:[email protected]"">[email protected]</a></dd></dl>Clostri dialneurotoxinscompriseonetetanusneurotoxin(TeNT)andsevenbotulinumneurotoxins (BoNT),whichcausetetanusandbotulism,thecontrastingneuroparalyticsyndromesthatDr. Schiavodescribedinhislecture(above).Dr.Montecucco’slaboratoryconductsstructure-function studiesoftheseneurotoxins,andhisfindingsrelatebothtobotulismasclinicaldiseaseandto BoNT’sroleasanincreasinglypopulartherapeuticagent. ThecrystallographicstructuresofBoNT/AandBoNT/Bshowthepresenceofafirstshellof coordinatingresidues(2histidinesand2glutamicacids),andasecondshellofresiduescloseto thecatalyticcenter,whicharedifferentfromanyknownmetalloproteaseandwhicharethoughtto explainthestructures’uniquespectroscopicproperties.WhenDr.Montecucco’steammutated severalsecondshellresiduesinTeNTandBoNT/A,theyfoundparallelsbetweenthespectroscopic andcatalyticpropertiesofthemutantneurotoxins.Theyalsosawthatthemetalloproteolytic activityoftheseneurotoxinsisstronglyactivatedbylipidsthatresemblethoseinsynapticvesicles, afindingconsistentwithDr.Schiavo’sobservationthatTeNTneedscholesteroltoentercells. Physicianshavelongobservedthatpatientswhosurvivetheacutephaseofbotulism,when paralysisaffectsrespiratorymuscles,arelikelytomakeagoodrecovery.Similarly,clinicianswho useBoNTstotreat“everythingfromstroketowriter’scramp”knowthatthebenefitsdon’tlast forever.Inaseriesofexperimentswithrats,Dr.Montecuccofoundthatdamagecausedby introducingBoNT/Aattheneuromuscularjunctiondiminishesasnerveterminalsgeneratenew sproutsovertime.BoNT/Aisthetoxinapprovedforclinicaluse,andtheseanimalexperimentshelp explainwhyrepeatedinjectionsarenecessary.Othertypesofbotulinumtoxinsmayworkbetter. Dr.MontecuccoandhisclinicalcollaboratorshaveshownthatBoNT/Cprovidesmoredurablerelief forseveraldystoniasoffacialmuscles,andtheyarenowevaluatingthetherapeuticpotentialof otherBoNTserotypesaswell. <hrnoshade=""noshade""size=""1""width=""400""/> <dl><dt></dt><dd><em>Comprehensiveidentificationof</em>Mycobacteriumtuberculosis<em> genesrequiredforinfection </em><strong>EricJ.Rubin</strong> DepartmentofImmunologyandInfectiousDiseases HarvardSchoolofPublicHealth Email:<a href=""mailto:[email protected]"">[email protected]</a></dd></dl>Whenthe firstwidespreadepidemicsoftuberculosisstruckEuropeinthe16thCentury,thediseaseswept throughalllevelsofsociety.Aslivingconditionsimprovedforthemiddleandupperclasses, however,thediseasesettledamongtheurbanpoorandthepeopleofunderdevelopedcountries. Morerecently,theAIDSpandemichasgivenTBatremendousboost,manypeoplehavebeen inadequatelymedicated,andferociousdrug-resistantstrainshavesurfacedinfar-flungpartsofthe globe.DespiteTB’sstatusasoneoftheworld’sleadingcausesofdeath,lessisknownaboutitatthe molecularlevelthanaboutotherpathogensthatkillfarfewerpeople.Onereasonforthisrelative lackofinformation,Dr.Rubinsaid,isthatpharmaceuticalcompaniesdonotperceivethe developingworldasaprofitablemarketplacefordrugs. ThefunctionofthousandsofgenesintheM.tuberculosisgenomeremainsunknown.Toaddress thisproblem,hislaboratoryhasinventedanoveltechnique,transposonsitehybridization(TraSH), whichcanbecombinedwithmicroarraytechnologytoidentifyallthegenesexpressedinspecific growthconditions.Dr.Rubin’steamisusingthisapproachtodeterminethefullsetofgenesneeded by<em>M.tuberculosis</em>tocausedisease. Transposonsarenaturallyoccurring,mobilegeneticelementswhosemovementscanbe manipulatedinthelaboratory.TraSHcombineshigh-densitytransposonmutagenesiswithmicro arraymappingofinsertionsitesinpoolsofmutants.Sofar,about100,000mycobacteriamutants havebeencreatedusinga<em>mariner</em>-basedtransposonandefficientphagetransduction. TraSHhasenabledDr.Rubinandhiscolleaguestodefinespecificsetsofgenesrequiredforthe survivalofpathogenicmycobacteriagrownunderavarietyofdifferentconditions.Thistechnique hasallowedthemtoidentifygenesthat<em>M.tuberculosis</em>needstoproceedthrough progressivestagesofinfectioninamousemodel.Notsurprisingly,thegenesrequiredearlyinthe infectiondifferfromthoseneededlateron.Atfirst,arelativelysmallnumberofgenesare essential—mostofthemneededtoacquirenutrients.Butasinfectionprogresses,andthehost fightsback,thepathogenrequiresanincreasingnumberofgenestosurvive.After8weeksof infection,173essentialgeneswereidentified,andscientistshaveyettoidentifyexactlywhat107of themdo.Havingsomanygenesinplayatonce,however,mayexplainwhytheTBpathogenisso adeptatdevelopingresistancetotreatment. <hrnoshade=""noshade""size=""1""width=""400""/> <dl><dt></dt><dd><em>Thelogicofbiosynthesisoftheglycopeptideantibioticvancomycin </em><strong>ChristopherWalsh</strong> DepartmentofBiologicalChemistryandMolecularPharmacology HarvardMedicalSchool Email:<a href=""mailto:[email protected]"">[email protected]</a></dd></dl>V ancomycinwasapprovedbytheFoodandDrugAdministrationin1958,butdidnotcomeinto widespreaduseuntilthe1980s.Itsoongainedareputationamongphysiciansastheantibioticof lastresortforsevereinfectionsinvolvingGram-positivebacteriasuchas<em>Staphylococcus aureus</em>and<em>Enterococcusfaecalis</em>,anditsuseundoubtedlysavedmanylives. Reportsofvancomycin-resistant<em>Enterococcus</em>begantosurfaceasearlyas1988, however,andmorerecentlythesehaveskyrocketed.Scientistsnowknowthatasinglebiochemical changeinthebacteriumcanconferasmuchasathousand-foldincreaseinresistance,Dr.Walsh said. Thereisapressingneedtore-engineervancomycinintoanewantibioticthatwillkilldrugresistantbacteria.Andthewaytodoso,accordingtoDr.Walsh,istoexaminehow <em>Actinomycetes</em>,afamilyoffilamentousbacteria,originallydesignedvancomycinasa weapontowieldagainsttheirenemies.Ifthecreator’soriginallogicisunderstood,thensciencewill knowwheretogonext.Thescientistsarelikeateamofindustrialengineers,analyzingeverystep inanautomobileassemblylinetolearnhowitcanberetooledfornextyear’smodel. Earlierstudiesshowedthatpeptideantibioticssuchasvancomycin,penicillins,andpristinamycins arebiosynthesizedbynonribosomalpeptidesynthetases(NRPS)actingasmultimodularprotein assemblylines.Themanufacturinggenesforthesenonribosomalpeptidesareclusteredtogether, presumablysotheycanregulateandcoordinateoneanother’sactivities.Dr.Walsh’steamhas foundthatvancomycinproductionisdrivenbyaclusterof30genesintheorganism <em>Amycolocaptosusorientalis</em>.Thesegenesencodeenzymesthatmakeaminoacid monomers.Athree-subunitNRPSassemblesthesemonomers,alongwithdeoxyhexose vancosamine,intotheheptapeptidebackboneoftheantibiotic.Thisacyclicheptapeptideisthen oxidativelycrosslinkedbytandemactionofthreehemeproteins,thenglycosylatedbyspecific glycosyltransferasestocreatevancomycin.NowthatDr.Walshandhiscolleagueshave characterizedtheenzymaticstepsneededtobuildvancomycin,thenextstepistoreprogramthe assemblylineandsynthesizenovelantibioticsthatwillside-stepthedefensesofresistant pathogens. <hrnoshade=""noshade""size=""1""width=""400""/> <dl><dt></dt><dd><em>Host-pathogenrecognition:thestructuralbasisoftheinteraction betweenfungalpolygalacturonaseanditsplantinhibitorPGIP </em><strong>BenedettaMattei</strong> DipartimentodiBiologiaVegetale UniversitàdiRoma“LaSapienza” Email:<a href=""mailto:[email protected]"">[email protected]</a></dd></dl>Wh enafungusinitiallyattacksaplant,itmustbatteritswaythroughthecellwallwhiletheplant strugglestoresist.AseriesofstudiesattheUniversitadiRomaLaSapienzahavegradually uncoveredthebattlethatragesbetweenfungalproteins,calledpolygalacturonases(PGs),and correspondingplantrecognitionproteins,knownaspolygalacturonase-inhibitingproteins(PGIPs). Aspartofthisongoingwork,Dr.MatteiandhercolleaguesuseX-raycrystallography,augmented withavarietyofbiophysicalandbiochemicaltechniques,toperformstructuralanalysisof interactionsbetweenspecificPGsandtheircorrespondingPGIPs. Inthecellwall,oneofthefirststepstowarddiseaseisforPGstohydrolyzehomogalacturonan. FightingbackarePGIPs,whichDr.Matteiandhercolleaguespreviouslyfoundtocomprise10 leucinerichrepeats(LRRs)—motifsspecializedforinteractionwithPG.Twoyearsago,hergroup solvedthecrystalstructureofPGfromthephytopathogenicfungus<em>Fusarium moniliforme</em>,describingitasaright-handedparallelβ-helixwith10coils,eachmadeupof threeorfourparallelβ-helicalstrands.Overall,itresemblesasquared-offcoilofspring.These findingsledtomorequestionsaboutwhathappenswhenPGandPGIPinteract. Herrecentexperimentsusedsurfaceplasmonresonanceandmassspectrometrytocharacterize thePG-PGIPinterface,aswellassite-directedmutagenesistolocatethePGresiduesinvolvedin catalysisandsomeoftheresiduesrecognizedbyPGIP.BycloningandcharacterizingPGIPsfrom Arabadopsisandbeanplants,theyhavelearnedthatasingleaminoaciddifferencecandetermine whetherornotaPGIPinhibits<em>F.moniliforme</em>PG.Theyhavealsolearnedthat indigenousplantPGs,neededfornormalprocesses,differslightlyfromfungalPGsandareableto goabouttheirbusinesswithoutbeingrecognizedandattackedbyPGIPs.WhenaPGIPinhibitsthe activityofitscorrespondingfungalPG,itappearstodosobycompetingwiththesubstratefor bindingsitesandcoveringtheactivesitecleft,Dr.Matteisaid.Shepredictedthatstructure-function studiesnowunderwayinherlaboratory,includingarecentcrystalstructureforPGIPfrom <em>Phaseolusvulgaris</em>,willshedadditionallightonhowplantsprotectthemselvesagainst enemies. <hrnoshade=""noshade""size=""1""width=""400""/> <dl><dt></dt><dd><em>Mechanismsofdrugresistanceinprotozoanparasites:awindowinto parasiteevolution </em><strong>DyannWirth</strong> DepartmentofImmunologyandInfectiousDiseases HarvardSchoolofPublicHealth Email:<a href=""mailto:[email protected]"">[email protected]</a></dd></dl>Four hundredyearsago,infectiousdiseasesaccountedforonethirdofglobaldeaths,andthesameis truetoday,Dr.Wirthsaid.Insomecases,decadesofmedicalprogresshavebeenreversedas organismsdevelopedresistancetodrugs.Malariaisavexingexample:<em>Plasmodium falciparum</em>needed16yearstodevelopstrainsresistanttochloroquine,butonly4yearsto beginoutsmartingfansidarandanother4yearsformefloquineresistancetosurface.Strainsthat foiledthenewestanti-malariandrug,atovaquone,showedupafteronly6months. Drugresistanceinmalariaandotherpathogenshasbeentracedtoover-expressionofcarrier proteinsfromtheABCtransportersuperfamily,whicharetypicallyseatedinthecellmembrane andservetopumpdrugsoutoftheorganism.Dr.Wirth’steamhasbeeninvestigatingresistance mechanismsmediatedbyABC-transportersintwoprotozoanparasites,<em>P.falciparum</em> and<em>Leishmaniaenriettii</em>.Ina<em>Leishmaniaenriettii</em>model,vinblastin resistanceiscontrolledbyasinglegene,LeMDRI1.Localizationexperimentsindicatethatthisgene isexpressedintheflagellarpocketandothervesicularcompartments,ratherthanonthesurfaceof theparasite.Dr.Wirthproposesamulti-stepmodelforresistancethatinvolvesdrugsequestration andsubsequentvesiculartransport.Initially,expressionoftheABCtransporterincreasesin responsetodrugtreatment.ParasiteswiththehighestlevelsofABCtransporterproteinshavea survivaladvantage,andgoontodevelopspecificpointmutationswithinthetransportergenethat conferresistance.WhenDr.Wirthandhercolleaguescomparedlaboratorystrainsofdrug-resistant <em>P.falciparum</em>withisolatesfrompatientswhowerefailingtreatmentinthecommunity, pointmutationsweremorecommonintheprimaryisolates. Theseobservationsledthemtoabroaderinvestigationofspecificpointmutationsandsingle nucleotidepolymorphismswithin<em>P.falciparum</em>.Sofar,mostofwhatisknownabout thegeneticsofsurvivalinthisgloballyimportantparasitecomesfromexaminingonegeneata time.The<em>P.falciparum</em>genomeisnearlycomplete,however,andwhenitisfinished thenextstepwillbeusinggenomicstodiscoveralltheplayersindrugresistance.Sofar,Dr.Wirth’s analysisshowsfarlessgeneticvariationin<em>P.falciparum</em>thanexpected,whichsuggests thatitisarelativelyrecentscourge—perhapsonlyabout10,000yearsold.Resistanceand pathogenicitygenesdisplaymorepolymorphismsthanotherpartsofthegenome,however,which probablyhelpsthemevadeattackbydrugsorhostimmuneresponses.Ifmoreofthese advantageouspolymorphismscanbeidentified,somemayturnouttobenewdrugandvaccine targetsin<em>P.falciparum</em>. <hrnoshade=""noshade""size=""1""width=""400""/> <dl><dt></dt><dd><em>Polygalacturonase-inhibitingproteinsinplantdefenseagainst phytopathogenicfungi </em><strong>GiuliaDeLorenzo</strong> DipartimentodiBiologiaVegetale UniversitàdegliStudi“LaSapienza” Email:<a href=""mailto:[email protected]"">[email protected]</a></dd></dl>Like aninvadingforcethatarrivesbyland,sea,andair,pathogensattackplantswithmanyweaponsat once.Thatbeingthecase,it’sagoodthingthatplantshavewhatDr.DeLorenzocalls“impressive redundancyintheirdefensivearsenal.”Centraltothebattlebetweenfungusandplantisthe recognitionsystemintroducedearlierinthesymposiumbyDr.Mattei:polygalacturonase-inhibiting proteins(PGIPs)intheplant’scellwallrecognizefungalendopolygalacturonases(PGs),moveto limitlocalfungaldamage,andformpecticfragmentsthatinitiateachainofdefensiveresponses. Dr.DeLorenzoandhercolleagueshavedoneextensiveresearchonPGIPs.Earlyon,theyrealized thattheversatileleucine-richrepeat(LRR)structureofthesecellwallproteinssuggestedthat plantsmightgenerateaPGIPforeveryPGsecretedbytheirfungalenemies.Thisechoesthehuman immunesystem’scapacityforproducingantibodiesthatrecognizeavastarrayofpathogens.Justas antibodiesareonlyonecomponentofthehumanimmuneresponse,Dr.Lorenzoandother investigatorshavefoundthatrecognitionofpathogenicityfactorsisthefirstofseveralstepstoward diseaseresistance,andthatplantsvaryintheircapacitytoidentifyandresistspecificinvaders. Herlabisnowexploringhowbasicscienceinsightscanbeusedtoimprovetheglobalfoodsupply. PossiblestrategiesforstrengtheningplantdiseaseresistanceincludemakingPGIPsmorealert sentinels,reprogrammingdefensivepathways,ormodifyingthedefenseresponseitself.Thismight involvetransformingcropplantswithdifferent<em>pgip</em>genes.InAfrica,forexample, storedcropseedsareoftenpoisonedandmadeuselessbythefungus<em>Fusarium moniliforme</em>.SomePGIPsfrombeanplantsareexquisitelysensitivetoPGsfrom<em>F. Moniliforme</em>,andthegenesforthesewatchdogproteinsmightbetransferabletosusceptible plants.Inadditiontosharpeningtheplant’sabilitytorecognizePGsdeployedbyitsenemies,Dr.De Lorenzo’sgroupisalsoinvestigatinghowmultipledefensesmightbemobilizedwhenpathogens strikewithseveralweaponsatonce. <hrnoshade=""noshade""size=""1""width=""400""/> <dl><dt></dt><dd><em>Recognitionofbacterialeffectorproteinsbytheplantinnateimmune system </em><strong>BrianStaskawicz</strong> DepartmentofPlantandMicrobialBiology UniversityofCaliforniaatBerkeley Email:<a href=""mailto:[email protected]"">[email protected]</a></dd></dl>Building onthethemesofDr.DeLorenzo’slecture,Dr.Staskawiczdescribedhowpost-genometechnologyis beingusedtocreatedisease-resistantplants.Inhislaboratory,experimentsaredoneusingamodel systemwherethegenomesequencesofbothplantandpestareknown.<em>Pseudomonas syringae</em>isaGram-negativeorganismthatcausesbacterialspeckdiseaseon <em>Arabidopsisthaliana</em>,afavoritemodelplant.Becausebothgenomesequencesare available,abioinformaticsapproachcanbeusedtoidentifyputativeeffectorproteinsin<em>P. syringae</em>. Likeotherpseudomonads,thisplantbacteriumisequippedwithaTypeIIIsecretionsystemthat injectseffectorproteinsinsidehostcells,wheretheycausedisease.Theproductsofresistance genescandefusethesetimebombsiftheyspotthemrightawayandkillcellsaroundtheinfection site,whichkeepsdamagefromspreading.Dr.Staskawicz’teamhasidentifiedarabidopsis resistancegenesthatappeartobepartofatoll-likereceptor(TLR)system.TLRsareahighly conservedpartofinnateimmunesystemsinsimpleanimalmodels,suchasfruitflies,andin mammalsandhumansaswell.Ifscientistsknowthegenesinvolvedinthesepathways,itshouldbe possibletousethesetoblockproteinsencodedbyspecificgenesinthepathogen.Thevalueofthis gene-generesistancestrategyisnowbeingtestedbycollaboratorsofDr.Staskawiczatthe UniversityofFlorida.Agenethatmakespepperplantsresistanttobacterialspotdiseasehasbeen insertedintotomatoplants,whicharenowbeinggrowninthefield.Sofar,Dr.Staskawiczsaysthat thetransgenicplantsappeartosuppressthiscommondiseasequitewell. <hrnoshade=""noshade""size=""1""width=""400""/> <dl><dt></dt><dd><em>Plantdefenseresponsesagainstnecrotrophicfungiareactivatedthrough multiplesignalingpathways </em><strong>SimoneFerrari</strong> DepartmentofMolecularBiology,MassachusettsGeneralHospital DepartmentofGenetics,HarvardMedicalSchool Email:<a href=""mailto:[email protected]"">[email protected]</a></dd></dl >Dr.Ferraribeganhistalkbynotingthatabout15%ofallagriculturalcropsarelosttodisease, whichputstheannualglobalcostofplantinfectionsatabout$500billionperyear.Fungiposea majorthreattocrops,whichiswhylaboratorieslikehisareextremelyinterestedincharacterizing defenseresponsesthatcanfendoffspecifictypesofthesepathogens.Fungicanbedividedintotwo broadcategories:biotrophiconesthatgrowonlyonthelivingtissuesofspecificplants,and necrotrophicpathogensthatkillhostcellsandcancolonizeawiderangeofhosts.Thenecrotrophic fungimacerateplanttissueswithdegradingenzymessuchaspolygalacturonases(PGs),whichwere thetopicofseveralotherpresentationsduringthissymposium. Dr.Ferrari’slabstudiestheinteractionbetweenthemodelplant<em>Arabidopsisthaliana</em> andthenecrotrophicfungus<em>Botrytiscinerea</em>,apre-andpost-harvestpathogenthatis toblameformanyoftherottingvegetablesfoundinhomerefrigerators.Itinfectsmorethan200 plants,andisdifficulttocontrolbecausepesticidesobviouslycan’tbeusedonplantsharvestedas foods.Asanalternativetoagriculturalpoisons,hislabstudieshowselectiveactivationofdefensive responsesmightprotectagainstthispest. Ingeneral,plantsusethreesignalingpathwaystodefendagainstfungi:ethylene(ET),jasmonate (JA),andsalicylicacid(SA).Necrotrophicfungisuchas<em>B.cinerea</em>activatetwoofthese paths,ETandJA,Dr.Ferrarisaid.HisteamhasrecentlycharacterizedtwoArabidopsisgenesthat encodepolygalacturonaseinhibitors(PGIPs)effectiveagainstPGsfrom<em>B.cinerea</em>. AlthoughoverexpressionofeithergenereducesdiseasesymptomsinArabidopsis,Dr.Ferrarihas foundthatduringinfectiontheprotectivegenesAtPGIP1andAtPGIP2arecalledintobattleby separatesignals–muchasArmyandNavyforcesrespondtodifferentcommanders.TheJA pathwayusesonesetofsignalingmoleculestoactivateAtPGIP2,whereasdifferentintermediaries turnonAtPGIP1.Thenextstepwillseewhetherinducingmultipledefenseresponsesthrough separatepathwaysmaybeaneffectivewaytoprotectplantsagainstversatile,wide-rangingfungi suchas<em>B.cinerea</em>. <hrnoshade=""noshade""size=""1""width=""400""/> <dl><dt></dt><dd><em>Genomescienceinbacterialpathogenbiologyandevolution </em><strong>JohnJ.Mekalanos</strong> DepartmentofMicrobiologyandMolecularGenetics HarvardMedicalSchool Email:<a href=""mailto:[email protected]"">[email protected]</a></dd> </dl>Nearly70bacterialgenomeshavebeenfullysequencedandanother160areinprogress,a developmentthatisrevolutionizingthestudyofmicrobialpathogens.Onceanorganism’scomplete genomesequenceisavailable,researcherscanlearnmuchaboutitsbiologybyusingsequence homologytoassignorguessatthefunctionsofitsgenes.Inaddition,variouspost-genome technologiesenableresearcherstoanalyzeextremelylargenumbersofgenessimultaneously.InDr. Mekalanos’lab,thesenewmethodshaveyieldedimportantinsightsintotheevolutionof<em>V. cholerae</em>strainsresponsibleforaseriesofcholerapandemics,aswellasdetailsaboutthe molecularbasisfor<em>V.cholerae</em>pathogenesis. WorkingwithcollaboratorsatTheInstituteforGeneticsResearch,Dr.Mekalanos’teamsequenced a<em>V.cholerae</em>straincalledN16961.Theydevelopedmicroarrayscontainingatleast 93%ofallopenpredictedreadingframes(potentialgenes)forthisstrain,andusedthesearraysto comparethegenomesofseveral<em>V.cholerae</em>strains.Theyfoundasetofgenescommon toallpandemicbiotypes,aswellasgenesthatsettheso-calledclassicalstrain,whichisblamedfor sixearlierpandemics,apartfromitssuccessor,the7<sup>th</sup>pandemicorElTorbiotype. Only22genesdifferentiateElTorfromtheclassicalstrainandfromotherstrainsthatfizzledbefore causingglobaldisease;nowthechallengeistodetermineexactlywhichgeneproductsmaketheEl Torstrainsuchathreattopublichealth. AlreadyDr.Mekalanos’teamhasidentifiedseveralgenesthatmaketheElTorstrainwhatitisand whichmightbegoodantibiotictargetsbecausetheirproteinsareessentialforbacterialreplication. Anotherprojecthasfocusedonquorumsensing,themechanismthatenablesbacteriacolonizing theintestinaltracttosenseandregulatetheirowndensity.Thispopulationcontrolmeasuremight beusedagainstthemicrobes,ifadrugcouldsignal“enoughbacteriaalready”wheninfactthereare onlyafew.Oneofthequorumsensinggenesalsoappearstohelpregulateexpressionofimportant <em>V.cholerae</em>virulencegenes.Theinvestigatorsarenowprofiling<em>V. cholerae</em>geneexpressioninarangeofanimalandhumanhostsaswellasdifferent laboratorygrowthconditions,withthegoaloflearningmoreabouthow<em>V.cholerae</em> sensesandrespondstohostsignals. Eventually,Dr.Mekalanoshopestheseinvestigationswillcontributetobettertreatments,a preventivevaccine,andenvironmentalcontrolstrategiesthatcanpreventthousandsofcasesat once,ratherthancombatingcholeraonepatientatatime. <hrnoshade=""noshade""size=""1""width=""400""/> <dl><dt></dt><dd><em>Searchingforthefunctionof</em>Neisseriameningitidis<em>B proteinsidentifiedonagenomicbase </em><strong>EmanuelePapini</strong> DepartmentofBiomedicalSciences UniversityofPadua Email:<ahref=""mailto:[email protected]"">[email protected]</a></dd></dl>For themostpart,theGram-negativebacteria<em>N.meningitidis</em>dwellswithoutincidentin thehumannoseandthroat.Everysooften,however,itinvadestheepitheliumanddiffusesintothe bloodstream.Whenthishappens,“nootherinfectionsoquicklyslays,”inthewordsofanearly20th Centuryphysician.Meningitisorfatalmeningococcalsepsisisespeciallylikelyinchildren,and althoughthesurvivalrateisabout90%forcasesidentifiedearly,thetreatmentwindowisvery narrowandoutcomesareespeciallypoorwhendiagnosisisdelayed. Althoughpreventivevaccinesareavailableforseveralpathogenicserotypesof<em>N. meningitides</em>,nonehasbeendevelopedforserogroupB.Byanalyzingthegenomeofthe MC58strainof<em>N.meningitidis</em>B,Dr.Papiniandhiscolleagueshaveidentifiedpotential virulencefactorsincludingextracellularproteinsthatinteractwithhostmucosaandblood.They haveused<em>E.coli</em>toexpresshighlypurifiedpreparationsoftheseproteins,someof whichappearpromisingforvaccinedevelopment.Sofar,themostinterestingoftheseisalollypopshapedproteinonthe<em>N.meningitidis</em>surfacecalledNadA,whichisendocytosedby humanmonocytesandcanactatthelevelofthebloodstreamaswellasatthemucosa.Inthe laboratory,ithasahighaffinityformucosa-likeChangcells,suggestingthatitisanadhesionfactor. NadAalsotriggersreleaseofcytokinesthatmightinitiateorworsensepticshockinpatients.But themostinterestingpossibility,whichDr.Papini’slabiscurrentlypursuing,isthatitmightbea novelvaccinecandidate. <hrnoshade=""noshade""size=""1""width=""400""/> <dl><dt></dt><dd><em>Structuralandbiochemicalcharacterizationofthepolioviruscellentry pathway </em><strong>JamesM.Hogle</strong> DepartmentofBiologicalChemistryandMolecularPharmacology HarvardMedicalSchool Email:<a href=""mailto:[email protected]"">[email protected]</a></dd></dl>M orethan15yearsago,Dr.Hogledeterminedtheatomicstructureofpoliovirus.Sincethen,his researchhasrevealedthatwhenthevirusentersatargetcell,itresemblesaMarslandingmodule thatsettlesintoplace,drillsintothesurface,thenpoursgeneticmaterialintothehole.Structural, kinetic,biochemical,andgeneticstudiesallcontributedtothisunderstandingofhowthevirus breachescells. Thedramabeginsintheextracellularenvironment:asthematureviriontravelsfromcelltocellor hosttohost,amaturationcleavageofacapsidprotein,VP0,locksitinameta-stablestate.Thevirus remainsrigidlystableuntilitencountersVpr,itsmatchingreceptorontargetcells.Uponcontact, thereceptoractsasacatalystthatovercomesanenergybarrierandfreesupthemeta-stablestate. Aseriesofconformationalchanges,whichDr.Hogel’steamhasdocumentedovertheyears,allows thevirustoattachtomembranes,formapore,andreleaseitsRNAgenomeintothecytoplasm.This sequenceofeventsappearstobeaprototypeforthematurationandcellentryofmorecomplex envelopedviruses,suchasinfluenzaandHIV. Nowthattheyhavethebigpicture,Dr.Hogleandhiscolleagueshavedevelopedasimple,liposomebasedmodelsystemforcarryingoutdetailedstructuralandbiochemicalstudiesofpoliovirus’ entryintomembranes.Liposomesdecoratedwithpoliovirusreceptorsareusedtocapturethe virus,thenpurifiedforelectronmicroscopy.Theresearchershavedemonstratedthatreceptordecoratedliposomesinduceconformationalchangesthatenablepoliovirustoattachdirectlyto membranes,andpreliminarydatasuggestthatthePv-decoratedliposomesallowRNA translocation.Thesestudiesareongoing. Dr.Hogleintroducedhispresentationbynotingthathisscientificcareerhasbeendevotedto studyingapathogenwhich,iftheWorldHealthOrganization’s2005eradicationgoalismet,will soonbeeliminated.Heeagerlyanticipatesthismoment,andremarkedthathealwaysknewthat eventuallytheurgencyforstudyingthisorganismwoulddiminish. <hrnoshade=""noshade""size=""1""width=""400""/> <dl><dt></dt><dd><em>Underconstruction:buildingareovirusfactoryfromviralandcellular components </em><strong>MaxL.Nibert</strong> DepartmentofMicrobiologyandMolecularGenetics HarvardMedicalSchool Email:<a href=""mailto:[email protected]"">[email protected]</a></dd></dl>Thefamily <em>Reoviridae</em>includesgenerawhosemembersinfectplants,invertebrates,and vertebratesandcausediseaseinnon-humanhostsrangingfromfishtohorses.Thereovirusesof greatestclinicalimportanceinhumansarethegroupBrotaviruses,whichcausediarrhealdisease. Reovirus-infectedcellsaredistinguishedbythepresenceofstructurescalledviralinclusions,which insomecasesserveasfactorieswheretheviralRNAgenomeisreplicatedandlarge,non-enveloped viralparticlesarebuilt. Muchasengineersmightanalyzetheworkingsofanautomobilefactory,Dr.Nibertandhis colleaguessetouttodeterminehowthesevirusfactoriesareformed,whererawmaterialscome from,andhowtheyareorganized.Thepurposeofthisinvestigationistofigureouthowantiviral drugsmightsomedaydisruptthisviralassemblyline.Theoutputofthesefactoriesisanonenvelopedviruswithtwocapsidproteins,10genomicsegments,adoublestrandoflinearRNAin itscore,andawell-definedthree-dimensionalstructure.Areovirusispartlydisassembleduponcell entry,genomicsegmentsaretranscribedinthecytoplasm,andtheresultingproteinsarefedinto thefactory. Dr.Niberthypothesizedthatsomethingasneatlyconstructedasareovirusmustbebuiltinahighly orderedintracellularenvironment.Byusingimmunofluorescencemicroscopyandapanelof antibodiestoviralandcellularproteins,histeamfoundthatreovirusfactoriescomeintwoshapes, globularandfilamentous,withthefilamentousshapeprevailingin22of24strainstested.They determinedthatthisshapeoccurswhenthefactoriesareassociatedwithcellularmicrotubules,a linkageaccomplishedbyareoviruscoreproteincalledµ2,anNTPaseinvolvedinviralRNA synthesis.Theroleofµ2wasconfirmedwhentheinvestigatorsintroducedapointmutationandthe factoriesbecameglobular.Dr.Nibert’sgrouphassincestudiedhowrawmaterialsforvirion constructionarerecruitedintothefactories.Experimentsshowthatµ2alsoplaysarolehere,by recruitingamajorreovirusnonstructuralprotein,µNS,thoughttobindRNA,tofilamentous factories.Currentstudiesareaddressingotherprotein–proteinandprotein–RNAinteractions,as wellasconsideringhowcomponentsofthecellularubiquitin–proteasomesystemareassociated withthefactories. <hrnoshade=""noshade""size=""1""width=""400""/> <dl><dt></dt><dd><em>Down-regulationofMHCclassImoleculesbyhumancytomegalovirus </em><strong>DomenicoTortorella</strong> DepartmentofPathology HarvardMedicalSchool Email:<a href=""mailto:[email protected]"">[email protected]</a></dd></dl>Maj orhistocompatibilitycomplex(MHC)moleculesplayanimportantroleinhumanimmunityby restrictingT-cellresponsestoproteinantigens.AkeyfunctionofMHCclassImoleculesistobind peptidesgeneratedbyinvadingvirusesorbacteria,andtopresentthemonthesurfaceofinfected cellssothatCD8+Tcellswillattackanddestroythem.EverystepintheformationofMHC-antigen complexesisvulnerabletointerference,andDr.Tortorellahasbeenstudyingastrategythathuman cytomegalovirus(HCMV)usestothwartthisantigenpresentationsystem.Theevasionstrategy usedbythisvirushasbroadmedicalimplicationsbecauseitparallelseventsthatoccurincystic fibrosis,andcouldpointthewaytobettertreatmentsforthissevere,inheriteddisorder. HCMVgeneratestwouniquegeneproducts,US2andUS11,thatsubvertnormalstepsinMHCclassI antigenpresentation.Forstarters,US2andUS11speedthebreakdownofclassIheavychains, whichinterfereswithantigenbindingbythetransmembranetailoftheMHCmolecule.Theheavy chainsareextractedfromtheendoplasmicreticulum(ER)andrelocatedtothecytosol,wherethey areshuttledtotheproteasome–thecellularequivalentofagarbagedisposal–wheretheyare groundupandthrownaway.Thisisanormalwasteremovalmethodthatcellsusetorid themselvesofmisfoldedproteinsandothertrash,Dr.Tortorellanoted,whichUS2exploitsby taggingnormalMHCclassImoleculesforprematuredegradation.ThebreakdownofclassIheavy chainsisstrikinglysimilartothedegradationofamutantformofcysticfibrosistransmembrane conductanceregulator(CFTR)whichhasbeenimplicatedinprogressionofCF.Bothseemtobe four-stepprocesses,Dr.Tortorellasaid,involvingrecognition,recruitment,relocation,and degradation. HisgroupisusingseveralmethodstoshedlightonadditionalfunctionsofUS2andUS11.A mutagenesisstudyusingdeletionandchimericmutantsofUS2,US11andclassIheavychains suggeststhatthecellemploysdifferentcomplexestoextractproteinsfromtheER.Aninvitro degradationsystemwasusedtoidentifyERandcytosolicproteinsinvolvedmarkingMHCclassI moleculesfordestruction.Morerecently,high-throughputscreeningwasusedtoexaminemore than16,000chemicalstoseeifanywouldinterferewithUS2-andUS11-mediateddestructionof classIheavychains.Fourinhibitorswereidentified,Dr.Tortorellasaid,andheiscertainthesewill bevaluabletoolsforstudyingthedegradationreaction.Itisevenpossiblethatoneofthesemay eventuallyturnouttobeaviabledrugforcysticfibrosisorotherdiseasesthatinvolvedegradation ofmisfolded,butpotentiallyfunctionalproteins. <hrnoshade=""noshade""size=""1""width=""400""/> <dl><dt></dt><dd><em>Three-dimensionalstructureofiron-bindingproteinsfrom </em>Helicobacterpylori<em>and</em>Bacillusanthracis<em> </em><strong>GiuseppeZanotti</strong> DipartimentodiChmicaorganicaeCentroCNRBiopolimeri UniversitàdiPadova Email:<ahref=""mailto:[email protected]"">[email protected]</a></dd></dl>At firstglance,onemightnotexpectahigh-profilebiologicalwarfareagentandtheleadingcauseof ulcerstohavemuchincommon.Whattheyturnouttoshare,however,isthatbothneedironto growproperly.<em>Bacillusanthracis</em>hasbeeninthenewssinceitwasdeployedasa bioterrorismagentlastFall,andthereisintenseinterestindevelopingnovel,second-generation preventivevaccinesthataresaferthanthecurrentone,whichisnotoriousforitsadverseeffects. Thepathtoanewkindofvaccinemightbeilluminatedbycomparisonsofanthrax’siron-binding proteinswiththosefoundin<em>Helicobacterpylori</em>. Inthe<em>B.anthracis</em>genome,Dr.Zanottiandhiscolleagueshaveidentifiedtwogenes thatencodeferritins,proteinsthatstoreironandreduceoxidativestress.CalledDlp-1and-2,these aredodecamershomologoustotheDNAbindingproteinof<em>Escherichiacoli</em>,calledDps. Dps-likeproteinsaregenerallyhighlyimmunogenic.<em>Helicobacterpylori</em>,whichisbest knownforitsassociationwithulcerdisease,hasaniron-bindingproteinknownasHP-NAP,which hastheunusualabilitytoactivateneutrophils.Becauseithasprovedhighlyimmunogenicin humansandmice,ithasbeenincorporatedintoanexperimental<em>H.pylori</em>vaccine beingdevelopedbyChiron,Dr.Zanottinoted. HislabhasrecentlysolvedthecrystalstructuresofDlp-1,Dlp-2andHP-NAPgrownin<em>E. coli</em>.Dr.Zanotti’steamshowedthateachhasaquaternarystructuresimilartothatofthe dodecamericbacterialferritinsintheDps-likefamily:theyaresphere-likeproteinswith32-facets andaninternalcavity.FunctionalstudiesconfirmthatDlp-1andDlp-2areinvolvedinironuptake andregulation,fundamentalactivitiesduringbacterialgrowth.HP-NAPhasadifferentdistribution ofsurface-potentialcharges,duetothepresenceofmanypositivelychargedresidues,andDr. ZanottispeculatesthatthismayexplainwhyitcanactivatehumanleukocyteswhileDlp-1and-2 cannot.HP-NAPisapromisingvaccinecomponent,andalthoughithasfeaturesthatDlp-1and-2do not,neverthelessthesimilaritiesamongtheseferritinsmayaugerwellfordevelopmentoffuture anthraxvaccines. <hrnoshade=""noshade""size=""1""width=""400""/> <dl><dt></dt><dd><em>Bacterial/cytolysin-mediateddeliveryofproteintothecytosolof mammaliancells </em><strong>DarrenHiggins</strong> DepartmentofMicrobiologyandMolecularGenetics HarvardMedicalSchool Email:<a href=""mailto:[email protected]"">[email protected]</a></dd></dl><em>List eriamonocytogenes</em>isacommonbacteriumthatpreysmainlyoninfants,elderlypeople,and pregnantwomen.Itisamongthemostlethaloffood-bornepathogens,withacasefatalityrateof 25%to60%.Becauseithasingeniousmechanismsforgettingintomammaliancellsandspreading fromonecelltothenext,Dr.Higginsandhiscolleaguesfind<em>L.monocytogenes</em>auseful modelforstudyingintracellularbacterialpathogenesis,mechanismsofgrowthandcelltocell spread,andbacterialdeterminantsofthehostT-cellresponse.Inworkpartlysupportedbya1999 ArmeniseFoundationFellowshipGrant,Dr.Higginsdevelopedaversatilesystemthatincorporates abacterialcytolysinintoavectorfordeliveringproteinsofinteresttohostphagocytes. Dr.Higgins’teamstartedwithanespeciallyharmlessstrainof<em>Escherichiacoli</em>,called K-12,andexpressedinitcytoplasmicrecombinantlisteriolysinO(LLO),whichefficientlytargets proteinstothecytosolofmacrophagesanddendriticcells,so-calledprofessionalphagocyticcells. LLOisapore-formingcytolysinwhosenaturalbiologicalroleistoexplode<em>L. monocytogenes</em>vacuoles,sothatbacteriacanescapeintothecytosolandreplicate.They usedthisvectortodeliverseveraldifferentproteinstoprofessionalphagocyticcells. Fortheirnextseriesofexperiments,theinvestigatorsusedanovelpackagingmethodcalleda diaminopimelate(dap)auxotrophtoexpressLLOand<em>Yersiniapseudotuberculosis</em> invasinin<em>E.coli</em>K-12.Theresultingbacterialvectorcoulddeliveraproteincargoto thecytosolofnon-professionalphagocyticcells.Followinginvasin-mediatedentryintomammalian cells,the<em>E.coli</em>remainedwithinvacuolesyetunderwentlysisbecausedapauxotrophs arestampedwithakindofbiologicalexpirationdate.LLO-mediatedperforationofthevacuolethen allowedreleaseofcargoproteinsintothecytosol. UsingT-cellactivationassays,Dr.Higgins’teamdemonstratedthattheinvasive/autolyticE. coli/LLOstraincoulddeliverantigenicproteintothecytosolicmajorhistocompatibilitycomplex (MHC)classIprocessingpathway,whichsubsequentlypresentedproteinfragmentstocytotoxicTcells.Intheory,thissystemcouldbeusedtodeliveranyantigenicproteinforpresentationonMHC classImolecules,whichcouldproveveryusefulforprimingprotectiveT-cellresponses,Dr.Higgins said.Inthelongrun,thiscouldbeaplatformtechnologyforlaunchingnewvaccines. <hrnoshade=""noshade""size=""1""width=""400""/> <dl><dt></dt><dd><em>Electronmicroscopicstudiesonintegrins:activationandligandbinding </em><strong>ThomasWalz</strong> DepartmentofCellBiology HarvardMedicalSchool Email:<a href=""mailto:[email protected]"">[email protected]</a></dd></dl>Integrinsare celladhesionreceptorsthatmediatethebindinganddetachmentofligandsinvolvedincell-celland cell-pathogeninteractions.In2001,Dr.Walzandcolleaguesdeterminedthecrystalstructureofthe extracellulardomainofαVβ3integrin.Theirstudiesrevealedanextraordinaryheaddomain,which theyobservedineithera“clasped”or“unclasped”position.Todeterminewhichpositionbound ligandsmoreeffectively,theyusedmolecularelectronmicroscopytovisualizethestructural changesinvolvedinactivationandligandbinding. ThecrystalstructureofαVβ3’sextracellulardomainrevealedabentconformation,inwhichthe ligand-bindingheadpieceisfoldedbackontothetailsofthemolecule,sothat<em>insitu</em> theligand-bindingsitewouldbeclosetothemembranesurface.Thisconformationwasthoughtto beanartifactofX-raycrystallographywithnophysiologicalsignificance,Dr.Walzsaid.Tofindout, theyexposedtheintegrintocalciumormanganese,observedwhathappened,thenaddedligandto themix.Acombinationofelectronmicroscopicstudiesandligand-bindingassaysrevealedthatthe bentconformation,seeninthepresenceofCa<sup>2+</sup>,actuallyrepresentstheinactivestate ofthemolecule. Incontrast,whenintegrinαVβ3wasincubatedwithMn<sup>2+</sup>,boththeclaspedand unclaspedformswereactivatedandtheirtailsfullyextended.Theseobservationssuggesta “switchblade”-likemotionofαVβ3duringtheactivationprocessthatinvolvesthestraighteningof thebentreceptor.Whenligandwasadded,bothassumedthebindingposition.Dr.Walz’steamhas nowmovedontoanalyzingchangesofshapeinadifferentintegrin,α5β1,broughtaboutbyligand binding. <hrnoshade=""noshade""size=""1""width=""400""/> <dl><dt></dt><dd><em>Requirementforap38MAPkinasesignalingpathwayin </em>Caenorhabditiselegans<em>innateimmunity </em><strong>FrederickM.Ausubel</strong> DepartmentofGenetics,HarvardMedicalSchool DepartmentofMolecularBiology,MassachusettsGeneralHospital Email:<a href=""mailto:[email protected]"">[email protected]</a></dd></dl >Theinnateimmunesystemisthebody’sfirstlineofdefenseagainstthreatsfromtheoutside world.Itreliesonphysicalandchemicalbarriersandtherapiddeploymentofspecializedcellsthat torightoffinvadersuntiltheacquiredimmunesystemcangenerateantibodiesandTcells. Importantinsightsintomammalianinnateimmunityhaveemergedfromstudiesin <em>Drosophila</em>,wheregeneticistshaveidentifiedkeygenesthatareimportantnotonlyin fliesbutinhumansaswell.Morerecently,developmentofexperimentalhost-pathogenmodels involvingbacterialinfectionof<em>Caenorhabditiselegans</em>providestheopportunityto exploreevolutionarilyconservedpathwaysofinnateimmunityinthegeneticallytractable nematodehost.Dr.AusubelandhiscolleaguesatMassachusettsGeneralHospitalhaveputthis modeltoespeciallygooduse. IntheAusubellaboratory,a<em>C.elegans-Pseudomonasaeruginosa</em>pathogenesismodel wasusedtoscreenfor<em>C.elegans</em>mutantswithenhancedsusceptibilitytopathogens. Thescreenidentifiedtwogenesrequiredforhostdefense:<em>esp-2</em>and<em>esp8</em>,encodingMAPkinasekinaseandMAPkinasekinasekinasecomponentsofap38MAP kinasesignalingcassettein<em>C.elegans</em>.Whentheesp8genewasknockedout,the nematodeswereeasilykilledbypathogens,Dr.Ausubelsaid.ThedefensesystemwasalsoshortcircuitedwhenRNAinhibitionwasusedtotemporarilyturnoffthep38MAPkinasestepinthe pathway.Inseparateexperimentsusing<em>Salmonellaenterica</em>asthepathogen, programmedcelldeathinthe<em>C.elegans</em>gonad(previouslyshowntoprotectagainst <em>S.enterica</em>-mediatedkilling)wasfoundtobedownstreamofthep38MAPcascadeand dependentonlipopolysaccharide,acomponentoftheGram-negativebacterialcellwall. Thesefindingsfitintoaglobalpictureofinnateimmunitythathasemergedoverthepastdecade. Dr.AusubelsummarizedevidenceforaMAPkinasesignalingpathwayinplants,whichparallels self-defensemechanismsinanimalsassimpleas<em>C.elegans</em>andascomplexashumans. Recentfindingsin<em>C.elegans</em>validatetheuseofthisorganismasamodelforstudying innateimmunity.Moreimportantly,theseexperimentsaddweighttotheideathattheMAPkinase cascadeis“themostancientandconservedcomponentininnateimmunity,”Dr.Ausubel said.";"Symposium2002:St.Thomas,U.S.VirginIslands";"";publish;open;open;"";symposium-2002st-thomas-u-s-virgin-islands;"";"";2013-02-1415:39:03;2013-02-14 15:39:03;"";0;http://armeniseharvard.sypdevelopment.com/?p=258;0;post;"";0